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Chapter 4
Most successful pathogens are not highly virulent because the immediate killing of the host cello prevents the spread of
the pathogen.

Classification of organisms
All living organisms are either prokaryotic or eukaryotic.
Prokaryotes are bacteria and archaea. These are less complex lack a nucleus and organelles, whereas eukaryotes do
contain those.

Bacteria
Size, shape, and multicell arrangement
Bacteria are the smallest organisms, in a rang from 0.1-10 um and can have multiple
different shapes like;
- Cocci: spherical and ovoid
- Bacilli: straight rods
- Coccobacilli: very short rods, often mistaken for cocci
- Fusiform bacilli: rod-shaped with tapered ends
- Spirilla: rigid spiral
- Spirochetes: flexible and undulating spiral

Besides different shapes there are also different multicell arrangement, dependent on
the degree of stickiness.
- Diplococci: cocci form two-cell arrangements (Streptococcus pneumoniae and
Neisseria gonorrhoeae)
- Streptococci: cocci form chains (streptococcus pyogenes)
- Staphylococci: cocci form clusters
These arrangements can also be formed by rods instead of cocci.

Keep in mind
• All living organisms can be divided into prokaryotes and eukaryotes
• Prokaryotes do not contain a nucleus or membrane-enclosed structures like those found in eukaryotes
• Bacteria can be classified by genus and species, just like all other organisms
• Further classification of bacteria can be based on size, shape, and arrangement.

Staining
For classification, shape and multicell arrangement is not enough so staining is needed because they are colorless.
Basic days contain cations (positively charged molecules) that are attracted to the negatively charged bacteria and color
those.
Acidic dyes contain anions (negatively charged molecules) that are repelled by the negatively charged bacteria and color
the background.
Basic dyes are used in two kinds of procedures;
- Simple stains consist of one dye and are used for identifying shape and multicell arrangement. For example
Methylene blue, carbolfuchsin, safranin and Crystel violet
- Differential stains consist of two or more dyes and are used to distinguish between two or more organisms or
parts of organisms. For example the Gram (call wall), negative (capsula), Zhiel-Neelsen acid-fast and the
endospore stain.

The gram stain

The gram stain divides bacteria in four groups; Gram-positive, -negative, -variable and -nonreactive, using differences in
the cell walls.
Steps;
- Crystal violet, taken up by all bacteria
- Treated with iodine as a mordant (sets color inside the cell and makes is permanent) for the gram-positive cells
to retain the color
- Adding alcohol so the gram-negative bacteria lose the color while the gram-positive bacteria retain it.
- Add red dye safranin to color the now colorless gram-negative bacteria
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So, gram-positive bacteria get the violet dye, -negative get the red safranin dye, -variable stain unevenly and -
nonreactive do not stain and need another staining method to be seen.
Old cells do not always stain properly because their cell wall gets leaky.

The negative (capsule) stain

The negative stain is used to identify shapes, in particular spirochetes, and to identify the presence of a capsule. The
capsule facilitates adherence limits access of antibiotics and inhibits phagocytosis, thereby undermine defense
mechanisms of the host cells. So, it is important for bacteria that infect humans.
The dyes nigrosin or India ink are used to color the background of encapsulated bacteria, making the capsule visible
(without coloring the capsule itself). Sometime a second dye is used to color the inside of the bacteria inside the capsule.

The flagella stain

Bacteria that have a flagella are motile, motility is important for invasion. They can’t be seen under the microscope so
the flagella stain is used to coat the surface with dye or metal molecules like silver to make them visible. This staining
method is not popular because it is very difficult and time consuming.

The Ziehl-Neelsen Acid-Fast stain

The Ziehl-Neelsen Acid-Fast stain is used to identify Myobacterium tuberculosis which causes tuberculosis and
Myobacterium leprae which causes leprosy. Because both bacteria have a cell wall that contains mycolic acid and lipids,
what makes it hard to penetrate, heat is needed to break this mycolic acid down and let the stain enter the cell.
When bacteria don’t lose their stain after being washed with acid thy are classified as acid-fast. Only bacteria that
contain mycolic acid in their cell wall are acid-fast.
After adding the dye, an acid-alcohol solution is added to remove the dye from the cells that don’t have mycolic acid in
their cell wall. To make the now colorless not acid-fast cells visible methylene blue is added.

The endospore stain

Some types of bacteria can undergo sporulation, the process in which endospores are formed. Endospores are small,
tough dormant structures that contain a copy of the genetic information and some chemicals. They are really resistant to
antibiotics etc., so bacteria that are able to do sporulation are hard to neutralize.
In the endospore stain the sample is heated and colored with malachite green. After this the sample is washed with
water what removes the dye from each cell but not for the endospores. Then safranin is added to color the non-
endospore parts of the cells. Without using malachite green first, the endospore would now be colorless.

Keep in mind
• Staining is used to make organisms visible under the microscope.
• There are two major types of stains; simple stains which only use one dye and differential stains which use more
than one dye.
• The gram stain is used to classify bacteria based on their cell wall structure.
• Several stains, such as the capsule, flagella and endospore stains, are used to identify structures associated with
the bacterial cell.
• The acid-fast stain is used to confirm the identity of Myobacterium species.
• Cell morphology and staining are often used for diagnostic testing.

Host-pathogen relationships
Pathogens are organisms that can infect humans and cause illness. Infectious diseases involve host-pathogen
interactions. These interactions vary, depending on the pathogen’s ability to;
- overcome/evade host defenses
- increase in number
- establish infection
- transmit the infection to new hosts
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The ability of a pathogen to infect depends on the host’s susceptibility.

Every cell that is exposed to the outside interacts with multiple microorganisms, of which most are harmless or even
useful by providing protection from pathogens producing vitamins and helping in digesting food. These are mutualistic
microorganisms which depend on the human body for their survival and humans benefit from their presence.
Some mutualistic microorganisms can become opportunistic pathogens, organism that take advantage of the host’s
increased susceptibility and thereby cause disease. In contrast to primary pathogens, which cause disease in healthy
individuals, they are not pathogenic under normal circumstances.
Infection requires that the pathogen;
- is able to multiply in sufficient numbers to secure establishment in the host
- be transmissible to new hosts
Often signs of disease help in transmission, for example coughing and diarrhea.

For pathogens, the death of many infected cells is unfavorable because then transmission and spreading is not possible.
In most cases the pathogen causes damage but no disease.

Keep in mind
• Pathogens are organisms that cause disease in humans.

• Infectious disease is a complex process that involves both the pathogen and the host.
• Pathogens can be classified as opportunistic (causing disease if the host’s defenses are compromised in some
way) or primary (causing disease even if the host’s defenses are intact).

Bacterial pathogenicity and virulence

Pathogens must be able to;
- Adhere to, penetrate and persist in the host cell
- Avoid or compromise the host defense mechanisms
- Damage the host and permit spread of the infection
- Able to exit from on host and infect another host
Virulence is how fit the pathogen is in fighting the host, how harmless it is. The virulence depends on virulence factors
which are genetically encoded.
Bacteria have to be able to survive in and outside the host cell and on which one it is present determines which genes are
active.
Virulence factors are carried on the chromosome or on plasmids which can be transferred between bacteria so it can
cause harmless bacteria to become pathogenic.
Clusters of virulence genes are called pathogenicity islands of which most are regulated by quorum sensing.

Quorum sensing
In quorum sensing proteins relay information to other proteins which regulate genes involved in the transcription of
virulence genes.
This method is based on cell population densities. Some genes are only expressed with sufficient cell population density.
Bacteria only start producing toxins until with they are with many, to hide from the host’s defenses because these
defenses can easily deal with small numbers of pathogens.
When auto-induction takes place, so when bacteria start secreting small diffusible molecules in the host’s circulation that
can be sensed by other bacteria, quorum sensing occurs.

Quorum sensing plays a different role in the genetic regulation in different kinds of pathogens. In opportunistic
pathogens, quorum sensing controls only 5-20% of the virulence genes and in primary pathogens all of them.

Biofilms
Bacteria adhere and grow as biofilms, of which growth is subject to quorum sensing. Biofilms can prevent the entry of
antimicrobial agents which are toxic to bacteria, and they are very hard to remove. Because biofilms are able to capture
nutrients, the bacteria ar able to increase in number.
The biofilm attracts the host’s defenses, but this results in frustrated phagocytosis, causing the formation of gigantic cells
that form a tough collagen capsule. This capsule prevents wound healing and the formation of new blood vessels.
However, sometimes biofilms can also facilitate wound healing because it triggers an immune response in wound
healing.
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When protheses are implemented, a protein film will be produced to coat it, consisting of fibronectin, fibrinogen,
albumin, immunoglobulins etc. All these proteins serve as binding sites for bacteria so bacteria will be accumulating on
the protein film, forming a biofilm. Several things can happen;
- Bacteria accumulate on the device
- Bacteria detach from the device and move into the surrounding
- The biofilm forms a tenacious gelatinous mass that is resistant to host defenses
- Pieces of the biofilm detach from the device and spread to other locations. If large enough they can cause fatal
thromboembolisms.
Zie plaatje p.58

Keep in mind
• Infection by a pathogen requires the pathogen to come in, stay in, defeat the host defense, damage the host,
and be transmissible.
• Virulence refers to how fit a pathogen is to survive in the host and thus how harmful it is.
• There are virulence genes that are carried on the bacterial chromosome or on plasmids.
• Virulence gene expression can be regulated by quorum sensing.
• A biofilm can assist in the infectious process by inhibiting exposure of bacteria to the host defense, antibiotics
and other molecules toxic to bacteria.

The host cell

The eukaryotic cell is the primary target for infectious diseases because they contain structures involved in the infectious
process.

Plasma membrane
Both eukaryotic and prokaryotic cells contain a plasma membrane but the composition is different as in eukaryotes it
contains cholesterol but ergosterol in prokaryote because they also have a cell wall. Both components contribute to
strength and support.
The eukaryotic membrane is made up hydrophobic lipids that form a barrier between the outside and the inside of the
cell. Because interaction between the cell and its environment is needed, lipids are bound to phosphate-ions which are
hydrophilic so the lipid chains are facing inwards and the phosphate groups outward.
Besides this the membrane contains proteins for communication, transport and connection to the cytoskeleton. Besides
taking up signals, receptors can also be site for virus attachment.
The proteins are not stuck, they can move freely in the membrane because it has a fluid mosaic model.
Besides as barrier for the outside, this membrane structure is also seen on membrane-enclosed structure inside the cell
which allows their interaction with the plasma membrane.

The role of the plasma membrane in infection

The plasma membrane should be damaged for microorganisms to entry the cell. Some host cells have receptors for viral
particles on their surface. If this is the case, a viral particle can attach and thereby gain entry. For example, the influenza
virus with ICAM-1 receptors on cells of the respiratory system.
When the particle leaves the host cell it is wrapped by a piece of the plasma membrane, the viral envelope. This plays a
role in infecting a new host and protection against the host’s defense.

Cytoplasm
The cytoplasm is all the volume inside the plasma membrane and outside the nucleus. It consists of the cytosol,
organelles and not membrane-enclosed structures.
Eukaryotic cells are larger than prokaryotic cells, but they contain less cytosol because it has to share the volume with
organelles which are not present in prokaryotic cells.

The role of the cytoplasm in cytoplasm

During infections, the structures are taken over by the virus and in the cytosol the viral particles are assembled.

Non-membrane-enclosed structures
Eukaryotic cells have 3 types of non-membrane-enclosed structures the cytoskeleton, cilia and ribosomes.

Cytoskeleton
The cytoskeleton gives the cell structural integrity and determine how cells are joined together in a tissue. It consists of
three components; microfilaments, intermediate filaments and microtubules
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Microfilaments are thin and made up of actin molecules. These are solid and cause the cytoplasm to get a gel-like
consistency. Intermediate filaments are way larger and consists of the protein keratins. These give extra strength and
stability and are involved in positioning cells along each other. Microtubules are the largest. It are hollow tubes made up
of the protein tubulin. Not every type of eukaryotic cells contains microtubules because they are found in cilia and
flagella so they are involved in movement of structures inside the cytoplasm, in particular of chromosomes during
mitosis/meiosis.

The role of the cytoskeleton in infection

Pathogens can take advantage of the cytoskeleton. An example is the Shigella bacterium that destroy the lining of the
intestinal tract. As the infection spreads Shigella moves from one cell to the next using a protruding structure made up of
microfilaments.
Another example is Listeria monocytogenes which uses actin of the host cell for its movement.
Besides this the cytoskeleton is involved in phagocytosis, one of the host’s defense mechanisms.

Cilia
Cilia are only found in eukaryotic cells and are made up of microtubules. In one cilium, 9 pairs of microtubules form a ring
with 2 microtubules in the middle. Their movement causes liquids to move across the surface of the membrane.

The role of cilia in infection

Together with mucus-secreting cells, ciliated cells work to remove foreign material from the respiratory tract. This
prevent microorganisms from staying in, what is needed for successful infection.

Flagella
Flagella are made up of microtubules so contain the protein tubulin. In humans, sperm cells are the only cells that
contain flagella. A flagellum rotates at the anchor point, making a whip-like motion.
Flagella play a role in infection by allowing pathogens to other locations in the body of the host.

Ribosomes
Both eukaryotic and prokaryotic cells contain ribosomes, which are responsible for the production of proteins. They
either float around freely in the cytosol or are attached to the ER. Ribosomes in eukaryotic cells differ from those in
prokaryotic cells but both types are made up of protein and ribosomal RNA.

The role of eukaryotic ribosomes in infection

Eukaryotic ribosomes are involved in viral infections because they are taken over by the virus to make new virus
particles. Ribosomes are not involved in bacterial infections but they do play a role in their treatment because bacterial
ribosomes are targets of antibiotics. Many antibiotics target subunits of bacterial ribosomes and not of eukaryotic
ribosomes so the bacterium can no longer make proteins and dies. The eukaryotic ribosomes are not affected while the
bacterial ones are is because they are structurally different, selective toxicity, so the bacteria are killed and the cell is not.

Membrane-enclosed structures
Structures that are enclosed by a membrane are called organelles. The membranes are the same as the plasma
membrane what makes fusion possible.

Mitochondria
Mitochondria produce ATP. Working cells contain many whereas resting cells only got a few.
The mitochondria contain two membranes of which the inner membranes have many folds, named cristae, on which the
ATP is produced.
They have their own ribosomes and DNA that replicated independently of the host cell.
Mitochondria share multiple characteristics with bacteria;
- Independent replication
- A single circular chromosome
- Same kind of ribosomes which is different then in the rest of the cell
- Same kind of DNA and RNA polymerase
- Same mechanism of ATP production
Because of all these similarities the endosymbiotic theory was developed which describes a symbiotic relationship
between bacteria and eukaryotes in which bacteria over time would have been integrated into eukaryotic cells as
mitochondria.

Endoplasmic reticulum and Golgi apparatus

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Both the ER and the Golgi apparatus are membrane systems of flattened sacks and platlike structures. They are not
present in prokaryotes.
In the ER cellular components are synthesized. If ribosomes are attached to it, it is called the rough endoplasmic
reticulum and without it is called the smooth endoplasmic reticulum. In the rough ER proteins are produced and in the
smooth ER non-proteins, among which are lipids.
The synthesized material goes to the Golgi apparatus for the finishing steps, or it goes directly to the membrane for
transport. Golgi has three functions;
- Modifying and packaging products coming from the ER.
- Renewing the plasma membrane
- Production of lysosomes
The ER and Golgi are surrounded by the same membrane, so they are able to interact and fuse. In this way products can
come from the ER to Golgi.

The role of the ER in infection

In the ER, biosynthesis and assembly of viruses takes place during viral infections and it is associated with the adaptive
immune response.

Lysosomes
Lysosomes destroy evading microorganisms and foreign material in the cell. It are vesicles with powerful enzymes and
are produced by the Golgi apparatus. The lysosomes recycle host cell components that are not needed or not working.
Inhibition of lysosomal activity is lethal for the cell.

The role of lysosomes in infection

Lysosomes play a role in phagocytosis, a part of the innate immune response.

Proteasomes
Proteasomes are composed of ring structures and their function is the degradation of proteins. They recognize proteins
that are tagged with ubiquitin after which these proteins are bound by regulatory proteins of the proteosome. Then the
protein enters the core of rings where it is degraded. Often the fragments are further degraded and components are
reused.

The role of the proteosome in infection

Proteins that are associated with pathogens are degraded in the proteosome, then go to the ER where they are
combined with self-proteins. After this the combination of self- and pathogenic proteins move to the cell surface and
trigger the immune system to attack the pathogens.

Perixomes
Perixomes are responsible for the degradation of fatty acids. Most of the by-products are poisonous to the cell and the
perixomes should get rid of these.

Nucleus
The nucleus is only found in eukaryotic cells and not in prokaryotes. It is enclosed by the nuclear membrane and contains
nucleoplasm with the nucleoli in it where ribosomal RNA is made. The DNA is stored in the nucleus which is in the form
of chromatin (hair-like structure) when the cell is not dividing and it forms pairs of chromosomes when it starts dividing.
Here transcription takes place so an RNA transcript is made which leaves the nucleus via pores in the nuclear membrane.

Endocytosis and exocytosis

Endocytosis is the process in which a cell takes up materials and exocytosis in which unneeded materials are expelled.
There are three ways in which endocytosis can occur; pinocytosis, phagocytosis and receptor-mediated endocytosis.
In pinocytosis, the membrane rolls over anything that’s comes in contact with it, making a small vesicle. This is usually
seen with small molecules.
In phagocytosis the membrane is pushed outward to form a pseudopodium which surrounds material to bring it into the
cell, so forming a vesicle. This is part of the host’s defense.
In receptor-mediated endocytosis extracellular material binds receptors on the surface of the cell after which the
membrane starts to sink into the cell and a vesicle is formed.

Exocytosis is the reverse of endocytosis. In this process vesicles are made by organelles that then move to the plasma
membrane, fuse with it and release their components in the extracellular fluids.
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Keep in mind
• The structure pf the prokaryotic cell is distinctly different from that of the eukaryotic cell.
• Many of the structures of the eukaryotic cell have important roles in the infectious disease process.

Summary
- All living organisms can divided into prokaryotic eukaryotes.
- Prokaryotes are less complex cells that do not contain a nucleus or cytoplasmic membrane-enclosed organelles
like those seen in eukaryotic cells .
- Bacteria are classified by genus and species and have distinct sizes, shapes and arrangements.
- There are several staining techniques that can be used to classify and characterize different bacteria.
- Pathogens are organisms that cause disease in humans.
- Infection is a complex process that involves both the pathogen and the host.
- Pathogens can be primary/obligate (causing disease even though the host’s defenses are intact) or opportunistic
(causing disease when the host’s defenses are diminished).
- Many of the structures of the eukaryotic host cell have important roles in the infection process.

Chapter 5
Portals of entry
Points where pathogens can enter the body are called portals of entry and these can be divided in three groups; mucous
membranes, skin and parenteral routes.
Skin and mucous membranes are directly in contact with the external environment but for pathogens to enter via the
parenteral route there should be breaks in the body’s barriers.

Mucous membranes
Mucous membranes are located adjacent to the external environment, and they are present in the respiratory,
gastrointestinal and genitourinary tract.
The mucous membrane can be seen as a barrier and is part of the immune system, so it has defenses that prevent entry
even though they are in direct contact with pathogens.

The respiratory tract

Pathogens can enter the respiratory tract through breathing. It is the most favorable of all portals of entry because, for
example, you can stop eating stopping entry via de gastrointestinal tract but you cannot stop breathing to stop it via de
respiratory tract.
The pathogens are found in droplets and dust particles and like that enter the body via breathing.
Besides a portal of entry the respiratory tract is also a productive portal of exit, a point were pathogens leave the body.

The gastrointestinal tract

With the gastrointestinal tract microorganisms enter the body via foods and liquids you eat or drink. One if its defenses is
the production of acid and bile, required for normal digestion. Although many microorganisms die under these
circumstances, some pathogens actually prefer them.
Helicobacter pylori uses the respiratory tract as portal of entry and is present in one out of two people.
Infection with H. pylori is a risk factor for gastroduodenal ulcers and cancer.
A virulence factor of this bacterium is production of the enzyme urease that hydrolyzes urea into carbon dioxide and
ammonia. Because of this the acidic pH is neutralized, protecting the bacterium. When going through the stomach, H.
pylori nestles in the mucosa where it is protected can start the infection. This infection destructs the tissue and causes
formation of ulcers. Depending on other factors eventually cancer can develop.
Vie feces, the gastrointestinal tract is also a portal of exit. The fecal-oral route of contamination plays a role in many
infections.

The genitourinary tract

Infections in the genitourinary tract are more common in women than in men because their urethra and anus are much
closer to each other. Because of this, bacteria from the feces can easily come into the urinary tract and because of the
short urethra in women the pathogens can also go to the bladder.
An example of an bacterium is Proteus mirabilis, which has urease as a virulence factor that makes the environment
more alkaline so it can grow.
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This portal of entry is used by pathogens that cause diseases of the reproductive tract that are sexually transmitted. It
can also be used as a portal of exit.

Skin
The skin is the largest organ of the body. It is covered with microorganisms, so no breathing or eating/drinking is needed
for their association because they are already there. Fortunately, most are harmless.
The skin is impenetrable, so microorganisms need to find an opening to gain entry, for example via something like a hair
follicle or sweat gland.

The parenteral route

The parenteral route depends on breaks in the barrier like with injection and insect bites (vector transmission). The
severity of the infection depends on the severity of the break. The skin is made up of two layers, the dermis and the
epidermis. The epidermis consists of death and dying cells and there is no access to blood whereas there is in the dermis.
This means that only cuts that extend to the dermis cause systematic infection instead of just at the site of just locally.
This is also seen in surgery where microorganisms constantly gain entry to deeper tissues.

Some organisms have preferred portals of entry and only cause infection when they enter via this portal. This is because
of presence of a suitable environment to grow.

Establishment
After entry, the microorganism has to be able to stay but this can be difficult due to;
- Physical obstacles
- No adherence to tissue
- Vast arrays of defense in destroying the organism
Some organisms are not infectious without a capsule for adherence and for inhibiting host’s defenses. All pathogens have
a way of adherence, for example E. coli has fimbriae, many other bacteria use adhesins and some use sticky
glycoproteins on host cells.
An example is Streptococcus mutans. Fluids produced by oral tissues form a dental pellicle, a protein film that coats the
teeth. S. mutans adheres to the pellicle and starts producing the enzyme glycosyltransferase which acts on sugar in food.
This results other organisms to be able to bind S. mutans, forming a plaque, a biofilm. The combination of proteins and
enzymes they produce cause formation of a cavity in the teeth.

Increasing the numbers

Because it provides safety and success, some bacteria have high reproductive rates. This needs the growth environment
to be ideal, which is almost often the case in the human body.
Different pathogens have different number required for success. The lethal dose 50% (LD50) is the number of
microorganisms needed to kill 50% of the hosts and the infectious dose 50% (ID50) for 50% of the population to show
signs of infection. The most virulent pathogens have a low LD50 and ID50. When a bacterium has both a low LD50 and a
high doubling time it can be very dangerous because the rapid increase can overwhelm the patient. Many antibiotics
work by attacking the bacteria that grow rapidly to prevent its negative outcomes.

Defeat the hosts defenses

The host has multiple defenses against pathogens that gained entry and managed to stay in, but these pathogens can
have two things that make them successful;
- Structure of the pathogen cell (passive) for example being capsulated
- Attacking the hosts defenses (active)

Capsule and cell wall protection strategies (passive)

By encapsulating themselves, pathogens protect themselves from phagocytosis, the first line defense of the innate
immune system, because the capsule prevents adherence of the phagocyte to the surface of the bacterium.
The adaptive immune response is the production of antibodies against this capsule. These antibodies bind, opsonization,
so phagocytes can adhere to them and phagocytosis is possible.
Besides the capsule, also the cell wall serves as a structural defense, protecting the bacteria from environmental pressure
and increasing the virulence with components of the wall. For example;
- M proteins: Streptococcus pyogenes has M proteins in its cell wall which increase the virulence by increasing
adherence, causing resistance against heat and acidic environments, inhibiting phagocytosis and causing toxic
shock.
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- Mycolic acid: Mycobacterium tuberculosis has mycolic acid in its cell wall which protects against inactivated
phagocytes and antibiotics etc.

Enzyme protection strategies (active)

Bacteria produce extracellular enzymes so the infection can spread and they can kill host defense cells. All the following
enzymes are exotoxins, used against host defenses;
- Leukocidins: destroy white blood cells which are part of both the innate and adaptive immune system. There are
two kind of white blood cells, namely neutrophils and macrophages. Lymphocytes, the kind of white blood cells
responsible for the adaptive immune response, are destroyed by leukocidins, produced by streptococcal and
staphylococcal pathogens.
- Hemolysins; are toxins that damage the membrane of red and white blood cells and cause them to lyse and is
produced by a variety of bacteria. When hemolysis is produced by a streptococcal pathogen it is called
streptolysin which can be divided different types that differ in the destruction they cause.
- Coagulase: causes clot formation, what can be used by both the host and the pathogen. It can be beneficial for
hosts because clotting in vessels around site of infection closes the pathogens in, thereby preventing their
spread.
- Staphylokinase and streptokinase: start a cascade that results in the breakdown of fibrin and dissolves clots,
ensuring the spread.
- Hyaluronidase and collagenase: break down connective tissue and collagen so infection can spread.
Besides this, there might be a genetic predisposition that is involved in whether infections are minor or major.

Besides protection by structural components or production of enzymes, pathogens can defend themselves by hiding
because in the long run, the innate and adaptive immune system will catch up. The best defense is hiding inside a host
cell, here the humoral response cannot find them. This is easy for obligate (able to survive in only one environment so
inside the host cell for viruses) intracellular parasites but for bacteria it isn’t. bacteria use their cytoskeleton to come into
the cell and move around in it.
When Salmonella comes in contact with the host cell it changes its configuration because salmonella produces the
invasion changes the structure of actin filaments of the cytoskeleton. Because of this change, the bacterium comes into
the cell and inside it can use the actin filaments to move around. Besides this the pathogen can also use to host’s
cadherin to move from one cell to another without being exposed to immune defenses.

Keep in mind
 There are five requirements for a successful infection: get in, stay in, defeat the host defenses, damage the host,
and be transmissible.

 Places at which pathogens enter the body are called portals of entry.
 The major portals of entry are the mucous membranes, the skin, and parenteral routes.
 Mucous membrane portals of entry are associated with the respiratory, digestive, and genitourinary tracts of
the body.
 Establishment (the requirement of staying in) can be accomplished using adhesin molecules, which are
glycolipids or lipoproteins. In addition, some pathogens take advantage of structures such as fimbriae to adhere
to tissues.
 Virulence for a given pathogen can be gauged by the LD50 and ID50 of that organism.
 Pathogens can defeat a host’s defenses in two ways: passively (by using structures such as the capsule) and
actively (by attacking the host defense directly through the production of enzymes).

Damaging the host

Damaged caused by infection can be divided in two groups; damage that occurs when the bacteria are present and
damage that is a byproduct of the hosts response. The damage that occurs in presence of bacteria can either be direct or
indirect;
- Direct: host cells and tissues at the site of infection are destructed. The hosts defense limits the damage.
- Indirect: indirect damage is way more dangerous than direct damage because it is mostly seen in systemic
disease. It is caused by the production of bacterial toxins which are soluble and can easily move through blood
and lymph.
Besides being fatal, toxins have some common characteristics like fever, shock, diarrhea and cardiac and neurological
trauma. There are two types of toxins; exotoxins and endotoxins.

Exotoxins
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Exotoxins are secreted by specific pathogens so they can act in absence of the pathogen and enter host cells.
Genes coding for exotoxins often lay on plasmids or prophages, which can be transferred to other bacteria, making them
even more dangerous. There are different types of exotoxins;
- Cytotoxins: kill cells that they come in contact with
- Neurotoxins: interfere with neurological signal transmission
- Enterotoxins: affect the lining of the digestive system.
Some examples of dangerous exotoxins are Anthrax, Diphtheria, Botulinum, Tetanus and Cholera.

Anthrax toxin
Bacillus anthracis is a gram-positive rod that produces the cytotoxin anthrax. This toxin consists of three parts; an edema
factor, a protective antigen and a lethal factor. These parts are produced separately and when assembled on the
bacterial membrane it is not yet toxic. When it leaves the bacterium and attaches to the host cell it is endocytosed where
the acidic environment causes a conformational change, turning it into its toxic form. The protective antigen part then
forms a pore in the vesicle membrane after which the edema and lethal factor change their shape to be able to squeeze
out into the cytoplasm.
Anthrax disrupts the signaling capabilities of both macrophages and dendritic cells and even kills the macrophages.
Although they are not killed, the dendritic can no longer participate in the hosts defense.

Diphtheria toxin
Corynebacterium diphtheriae produces the cytotoxin Diphteria. As like Anthrax this is produced in an inactive form. It
becomes enzymatically active after secretion after which it is that potent a single molecule is able to kill a cell.
The toxin consists of and alfa and a beta polypeptide chains. The beta chain binds to the target cell and the alfa chain is
transported across the membrane. Inside the host cell, the alfa chain inhibits protein synthesis but without the beta
chain the toxin is harmless because no entry to the host cell is possible.

Botulinum toxin
Clostridium botulinum is a gram-positive anaerobic rod that produces the neurotoxin botulinum. There are seven
different types of this toxin but all inhibit the release of the neurotransmitter acetylcholine by using snare proteins,
resulting in paralysis. Snare proteins are present in vesicle and plasma membranes and are normally responsible for the
fusion of vesicles with the plasma membrane for exocytosis. The paralysis can result in death because muscles required
for respiration are affected.

Tetanus toxin
Clostridium tetani is a gram-positive, obligate anaerobic rod that produces the tetanus toxin which is related to the
Botulinum toxin. Tetanus blocks the relaxation impulse, thereby causing a loss of skeletal muscle control, resulting in
uncontrollable muscle contractions. In lockjaw, these contractions happen in the facial muscles.

Cholera toxin
Vibrio cholera produces the enterotoxin cholera. This toxin consists of a beta chain that binds to receptors on the target
cell and an alfa chain that induces epithelial cells of the intestine to release electrolytes. This results in diarrhea and
vomiting and can be lethal. Because of the extreme water loss, rice-water stool happens where the diarrhea is mainly
liquid with bits of mucus.

Other exotoxins
Some toxins are superantigens, which cause a major immune response that damages the host itself.
Streptococcus pyogenes produces a cytotoxin that is a superantigen destroys blood capillaries and can cause heart
damage.
Also, staphylococcus aureus produces a superantigen, the toxic shock syndrome toxin. Toxic shock causes a great loss in
body liquids. This can lead to low blood volume and low blood pressure what eventually can result in death.

Exotoxins are very antigenic so they stimulate the host to make antibodies. Because of this it is easy to make vaccines
with these antibodies against the toxins.
For example, the DTaP vaccine that is derived from exotoxins from Corynebacterium diphtheria and Clostridium tetanus
in combination antigenic compounds of Bordetella pertussis. These toxins are inactivated so they lose their toxicity but
not their antigenicity, now they are called toxoids.

Endotoxins
Endotoxins are compounds of the outer membrane of gram-negative bacteria. They become active after being released
in the bloodstream during cell division or once the bacterium died.
11

There are many differences between exotoxins and endotoxins. Endotoxins;

- are part of gram-negative bacteria, exotoxins are produced by gram-positive bacteria
- are not nearly as toxic
- do not have specific targets in the host
- are a normal part of the bacterial cell wall

The outer layer of gram-negative bacteria is made up of lipoproteins, phospholipids and lip0polysacharides (LPS).
Lipopolysaccharides contain Lipid A which has endotoxin properties.

All endotoxins cause the same symptoms; fever, chills, muscle weakness and aches. When large amounts of endotoxins
are present the symptoms may be worse like shock and disseminated intravascular coagulation (DIC) with minor blood
clotting through the whole body what can lead to multiorgan failure. Besides this the clotting systems are then used up
and cannot be used for serious blood loss anymore.

Endotoxins are not very antigenic so they can cause antibody production but this immune response is very weak.
Because of this and the fact that endotoxins are stable and independent of viable cells they can contaminate hospital
materials and stay toxic for a long time. Because of this and of that they are still able to cause damage after a successful
antibiotics treatment, they are likely to be transferred to patients. Limulus amebocyte lysate assay (LAL) is used to
identify the possible endotoxin contamination. This test used white blood cells of the horseshoe crab which clot into a
gel-like matrix in the presence of endotoxins that becomes turbid. The degree of turbidity is used as a measure for the
amount of endotoxin contamination.

Keep in mind
 Damage to the host can be either direct or indirect. Direct damage is usually localized, and indirect damage is
usually through the production of toxins.

 Exotoxins are extremely lethal substances produced by living cells (usually Gram-positive) and in most cases are
proteins.
 Exotoxins can be cytotoxins (which kill cells), neurotoxins (which interfere with neurological signaling), or
enterotoxins (which affect the lining of the digestive system).
 Exotoxins can cause the production of antibody.
 Endotoxins are contained inside the bacterial cell wall and are released on the death of the organism.
 Endotoxins are products of Gram-negative organisms, do not effectively cause the generation of antibody, and
are less toxic than exotoxins.
 Lipid A, which is part of the Gram-negative phospholipid outer layer of the bacterial cell wall, has endotoxin
properties.

Viral pathogenic effects

Cell damage and cell death caused by viruses is the cytopathy effect (CPE), this can happen in three ways;
- Viral overload: the virus explodes out of the host cell, infecting and lysing the surrounding host cells
- Cytocidal effect: host defense mechanisms identify and kill virally infected cells
- Non-cytocidal effect: the virus stops synthesis of DNA, RNA and proteins by the host, thereby forcing the host
cell to put all its effort into virus production
Inclusion bodies are a sign of cytopathology, for example Negri bodies seen in rabies infection. Other sings of viral
infection are syncytia (gigantic cells) and production of hormones and interferons.
Also parasites damage the body. They do this by moving in the body, taking up space and causing immune responses.

Summary
 The requirements for infection include entry, establishment, avoiding host defenses, damaging the host, and
exiting from the host.
 Portals of entry for pathogens include skin, parenteral routes, and the mu- cous membranes of the respiratory,
gastrointestinal, and genitourinary tracts.
 Pathogens use virulence factors such as adhesins to establish themselves in the host.
 To avoid being killed by the host’s defenses, pathogens use an array of virulence factors, including capsules, M
proteins, mycolic acids, leukocidins, hemolysin, coagulase, kinases, hyaluronidase and collagenase.

 Some bacterial pathogens cause damage to host cells by releasing exotoxins; these include specific types such as
cytotoxins, neurotoxins, and enterotoxins.
12

 Endotoxins on the outer membrane or cell wall of Gram-negative bacteria cause damage to the host and are
released and disseminated when the bacterial cell dies or during division.

Chapter 6
Transmission of infection
For the transmission of infection we look at reservoirs and mechanisms of transmission.

Reservoirs of pathogens
Reservoirs are places where pathogens can grow and accumulate. There are three; humans, animals and non-living
reservoirs.
In humans, sometimes infectious diseases are transmitted before the individual gets symptoms so it can difficult to
determine whether someone is a reservoir.
Zoonotic diseases can be transferred from animals to humans, for example Rabies and Lyme. Often the transference is
the disease happens by direct contact but it can also happen indirect via waste material etc.
Non-living reservoirs include for example water, food and soil, of which water is the most dangerous.

Keep in mind
- Transmission is the final requirement of a succesful infection.
- Reserbpirs are places where pathogens grow and accumulate.
- There are three potential reseroirs of infection: humans, other animals and non-living reservoirs.
- Human reservoirs can be infected people who are asymptomatic carriers.
- Animal-to -human infections are referred to as zoonotic disease.
- Non-living reservoirs include water soil and food.

Mechanisms of transmission
For the transfer of infectious organisms there are three mechanisms; contact, vehicle and vector transmission.

Contact transmission
Contact transmission takes place when a healthy person comes in contact with a pathogen by touching or being close to
someone else who is infected. There are three subtypes;
- Direct: there is no intermediary between the infected and uninfected person so for example with touching,
kissing and sexual interaction. Disease transmitted in this way are smallpox, hepatitis A, staphylococcal infection
and sexually transmitted. disease. Besides these, this mechanisms can also be used in zoonotic disease when the
infected person is an animal.
- Indirect: there is an intermediary, called formites, usually non-living Iike tissues, towels and contaminated
needles.
- Droplet: transfer of respiratory diseases via coughing, sneezing and laughing. The distances are short but size of
the droplet is important because large ones will fall on the ground more quickly. The smaller the droplet the
more dangerous it is as agent of disease.

Vehicle transmission
In vehicle transmission, pathogens are riding along with components that should be clean like food, water, air and drugs.
Water can play a role in vehicle transmission but also serve as a reservoir. When food transmits microorganisms this is
often seen as food poisoning. The contamination of food is caused by bad preparation and refrigeration.
The air can contain many microorganisms which then travel through the air from host to host. Sometimes pathogens are
transformed into an aerosol form for optimal transmission.

Vector transmission
In vector transmission, pathogens are transmitted to a healthy person by a vector, usually arthropods (flies, mosquitoes,
fleas, ticks and lice.
This can happen in two ways;
- Mechanical: body parts of the vector are contaminated. The infectious organisms are then brushed off the
vector and come onto the host. For example house flies often get pathogens form fecal material on them
- Biological: occurs through biting
13

Factors affecting disease transmission

Several factors can influence the disease process;
- Age: affects the overall health. It causes a decline in defensive capability which results in an increase in disease
with age.
- Gender: urinary tract infections are more seen in women and respiratory infections more in men.
- Lifestyle
- Occupation
- Emotional state: decision making, risk taking and immune status have a role in susceptibility.
- Climate: there are three ways in which climate can play a role
Colder climates show higher incidence in respiratory infections
Warmer climates have longer periods of when the vector can be present
Climate affects the variety of organisms present on a certain location

Portals of exit
Most of the time, portals of entry and exit are the same. Pathogens often via secretions like saliva, sputum and nasal
secretion but also via blood, semen, urine and feces.

Keep in mind
- Mechanisms of transmission are ways on which organisms move from place to place. There are three
mechanisms: contact, vehicle and vector transmission.
- Contact transmission can be direct, indirect or droplet transmission.
- There can be predisposing factors for disease such as the host’s age, gender, lifestyle, occupation and emotional
state.
- Organisms leave the host by portals of exit, which are essentially the same as the portals of entry.

The compromised host

The relationship between the host’s defense and infectious disease is illustrated bu the acquired immune deficiency
syndrome (AIDS) in which loss in host defense leads to opportunistic infections (infections that were harmless but not
can cause death).
Besides AIDS there are many other situations in which the host’s defense is compromised and specific types of
compromise are associated with specific risks for infectious disease.
Some groups have a compromised immune system and are therefore at increased risk of infection;
- People with AIDS
- People with genetic immunodeficiency diseases
- People undergoing chemotherapy
- People taking broad-spectrum antibiotics
- Transplant patients
- Burn patients
- Premature infants
- Newborn infants
- The elderly
- Surgical patients
- Patients on artificial ventilators, with catheters of cannulas

Neutropenia
In neutropenia there are lower-than-normal number of neutrophils in the blood. neutrophils are important components
of the innate immune system.
Neutropenia is seen is cytotoxic chemotherapy and in diseases and infections that damage the bone marrow or use up
neutrophils quicker than they are produced. Cytotoxic chemotherapy kills rapidly growing cells like tumor cells but they
make the patient immunoincompetent which increases the risk of getting infections during administration of these drugs.
Infections that are associated with neutropenia are bacterial or fungal. Bacterial infections start as soon as the neutrophil
levels are decreased whereas fungal infections only occur when there has been neutropenia for a longer time.
Gram-negative bacteria septicemias and gram-positive bacteria that originate in the blood.

Organ transplantation
There are always immunological differences between the patient and the transplanted organ, unless the organ comes
from an identical twin. This causes a reaction that intends to destroy the transplanted organ so the organ is being
rejected. To decrease the immune response and prevent rejection, patients get drugs that diminish the patient’s immune
14

capability. This also causes the patient to be more susceptible for infections. These infections need broad-spectrum
antibiotics but an overuse of these leads to superinfections and increased resistance to antibiotics.

Burn patients
Burn patients are more susceptible for infection because they have lost a part of the primary physical barrier. The loss of
skin increases the chance of septicemia. Burn patients are very susceptible for fungal infection and for Pseudomonas
auruginosa which is very resistant.

Opportunistic infections
Many bacteria are residents of the body that provide protection against pathogens, for example by preventing inhabiting
areas by other microorganisms. Sometimes there is production of bacteriocins, chemicals that are similar to antibiotics
and inhibit growth of the bacteria that produced them.
Under the right circumstances, many bacterial residents can become pathogenic so this can happen when they move to a
place in the body where they are not normally found, for example when E. coli travels from the large intestine where it is
part of the normal flora to the urethra where it is pathogenic.
The ability for resident bacteria to move to other places is caused by the impropriate use of antibiotics.

Nosocomial infections
A nosocomial infection is acquired in the hospital. They often occur during medical treatment so both the patient and the
medical worker can be affected. Anything that allows entry of pathogens into the body can cause nosocomial infections.
The hospital must consider;
- Source of infection
- Mode of transmission
- Susceptibility of the patient
- Prevention and control
The sources of infection are often the environment, other patients, staff, visitors, insects , instruments like catheters and
the use of toilet facilities. Patients are often immunocompromised, which in combination with opportunistic pathigens
increases the chance of an infection.

Universal precautions
There are universal requirements for guidelines in patient care.
These do apply to: blood, semen, vaginal secretion, tissue specimens and cerebrospinal, synovial, plural, peritoneal,
pericardial and amniotic fluids.
These do not apply to: feces, nasal secretions, sputum, sweat, tears, vomit, urine (as long as they don’t contain blood.

Preventing and controlling Nosocomial infections

Because of the nosocomial infections some hospitals follow programs which involve;
- Surveillance of nosocomial infections in patients and staff
- On-site microbiology laboratory plus standardized isolation procedures.
- Standardized procedures for use of catheters and hospital equipment
- Proper decontamination and sanitary procedures
- Mandatory nosocomial-disease education programs
- In some cases, infection-control specialist on staff
-
Keep in Mind
• One of the most important parts of the infectious process is the host defense.
• Compromise of the host defense can be due to a variety of problems
such as infection with HIV resulting in AIDS, congenital immunodeficiency, transplantation, chemotherapy, or
other conditions that debilitate the patient.
• Some infections are caused by opportunistic pathogens, which are normally harmless but can be pathogenic if
the right conditions exist.
• Infections that occur in hospitals are referred to as nosocomial infections.
• Universal precautions are specific guidelines for patient care, which provide proper procedures for dealing with
blood, semen, vaginal secretions, and other samples of tissue and body fluids.

Epidemiology
Epidemiology is the study of the factors and mechanisms involved in the frequency and spread of diseases and other
health-related problems. Besides for studying disease it can be used for making methods for control and prevention.
15

Incidence and prevalence

The incidence is the number of new cases contracted within a set population in a specific period. This provides
information about the spread of the disease.
The prevalence is the total number of people infected within a population at any given time. This are new and already
existing cases, and this provides information about how seriously and how long a disease affects the population.

Morbidity and mortality rates

The morbidity rate is the number of individuals affected during a set period, divided by the total population.
The mortality rate is the number of deaths due to the disease during a period, divided by the total population.

Diseases can be classified as;

- Sporadic: occur in a random and unpredictable manner. They are not a threat for the public health.
- Endemic: is constantly present in the population but with too low number to be a problem for the public health,
for example having a cold.
- Epidemic: occurs when the incidence becomes higher than expected, it causes the morbidity and mortality rates
to increases and it becomes a problem for the public health. Several factors contribute to the risk on developing
an epidemic. For example, large cities allow a rapid spread, an aging population has higher number of
immunologically compromised people, lack of her immunity and the ability to travel (allowing spreading disease
all over the world).
- Pandemic: occurs when the disease occurs in epidemic proportions all over the world.

Epidemics are affected by the type of pathogen and the mode of transmission. There are two types of endemics;
- Common-source outbreak: arises from contact with contaminated substances so it usually occurs when water is
contaminated with fecal material and when food is prepared improperly. Large numbers of people are affected
but after finding the source of infection it is quickly resolved.
- Propagated epidemic: there is amplification of the number of infected individuals via person-to-person contact.
This stays in the population for a long period and is difficult to deal with.

Herd immunity
Herd immunity can arise from vaccination or after infection. Whit smallpox, after a sufficient part of the population was
vaccinated, the population as a whole became immune and the disease disappeared. This is because there weren’t
enough susceptible individuals anymore for transmission. After disappearance of the disease, they stopped vaccinating,
what means that if the same pathogen would emerge the infection would be able to spread again.

Types of epidemiological studies

There are two types of epidemiological studies: descriptive and analytical.
Descriptive epidemiological studies are involved in physical aspects of patients and in the spread of the disease. These
include numbers of cases, location and time of infection, age, gender, race, marital status and occupation. With this
information the index case could be identified, the first person that was infected. Sometimes this is impossible, for
example because the index case is recovered, moved away or died.
Analytical epidemiological studies are involved in establishing a cause-and-effect relationship and always use a control
group. In a retrospective analytical study already available information is used of patients that already have the disease,
to identify factors preceding the epidemic. This can be used to do predictions and to take preventative measures.
A prospective analytical study collects date and does not use hindsight, so it looks at factors that occur as the epidemic
proceeds.

Diseases that are nationally notifiable are important to control and must be reported to national centers.

Keep in mind
• Epidemiology is the study of factors and mechanisms involved in the frequency or spread of diseases or health-
related problems.

• Incidence describes the number of new cases contracted within a set population in a specific period.
• Prevalence is the total number of people infected within a population at any given time.
• The morbidity rate of a disease is the percentage of individuals affected by the disease during a set period.
• The mortality rate is the percentage of deaths due to a specific disease during a specific period.
• Diseases can be sporadic (occurring only occasionally), endemic (constantly in the population), epidemic (a
higher-than-normal incidence of a disease), or pandemic (a worldwide epidemic).
• Herd immunity can limit the spread of infection.
16

• There are two types of epidemiological study: descriptive and analytical.

• Analytical epidemiological studies always contain a control group and can be retrospective or prospective.

Diagnosing infections
There are two key criteria in any diagnostic method: specificity and sensitivity.
When a test is specific it is able to identify a pathogen reliably and correctly. When a test is sensitive it is able to detect
small amounts of the pathogen or the antibodies against it.
Samples should be taken in or near site of infections because here is the highest pathogen load and when taking a
sample, the normal flora must be circumvented
Common samples are blood, urine, feces, sputum, swabs and biopsies.

Growth-based diagnostics
Growth-based diagnostics are based on the fact that microorganisms grow differently on different media. The
microorganisms can grow on solid or liquid media that contain all nutrients. The circumstances in the host are mimicked.
Some media can be used for the growth of many bacteria but there are also other kind of media;
- Selective: suppresses the growth of some organisms
- Elective: only supports the growth of the organisms of interest
- Differential: show differences between organisms in mixed samples
When the mixture of organisms has been grown on a sample, the pathogen of interest must be isolated in pure cultures.
Cultures that only contain the pathogen colony morphology can be assessed.
Besides the morphology, in growth-based diagnostics also physiology can be analyzed. In this test various media are
made that differ in one component, for example the carbon source and by determining where the pathogen can grow
helps with identification.

Biotechnology and health

Most cells of different cell types have the same basic properties;
- Need energy to function
- Energy is supplied in chemical form
- Synthesize proteins from a template
These provide opportunities for external manipulation.
One example of a technique used for biotechnology is by using recombinant DNA.

Recombinant DNA technique

To make a recombinant DNA, a piece of DNA is cut out and inserted in DNA of another organism, a vector, usually a
plasmid. To be amplified this is introduced into a host cell and the protein that the gene (that is inserted in the plasmid)
encodes for is produced.
Nowadays, DNA can be amplified by the polymerase chain reaction (PCR), in which the DNA segment to be amplified is
mixed with primers, nucleotides and DNA polymerase to produce many copies.

Monoclonal antibodies
Monoclonal antibodies have the same specificities and binding abilities and are all produced from a single cell called the
clonal cell. The monoclonal antibodies can bind antigens and other non-self molecules like drugs and viral/bacterial
products.
Antibody producing cells have a short lifespan whereas tumor cells are immortal but incapable of producing antibodies.
When these cells are fused you get a hybridoma (with a hybrid of the nuclei of the cells) that can be used for producing
monoclonal antibodies. These can be used for diagnostics, therapies, to attack the immune system of an organ-
transplanted patient (leaving the rest of the immune system alone so it can still fight in the rest of the body) and they can
be attached to toxins or drugs to serve as delivery vehicles, etc.
Antigen-binding fragments can be made in bacterial systems. Then the information for the antibody component is
inserted into the phage genome, joined to DNA that codes for a protein on the outside of the phage so when the protein
is produced it is the antibody fragment joined to the viral protein. Then, new phages display the antibody to their
environment, phage display.
This is not done with one antibody fragment but with a library, to see which binds the best to the antigen. When the
fragment has been selected, it is taken out of the phage vector and inserted into another vector for production of this
fragment only. The genetic information of the fragment gen be fused with fluorescent proteins.

Serology-based diagnostic methods

Serology-based diagnostics is based on antigens of the pathogen. Isolated or manufactured antibodies are used to detect
the antigens or host’s antibodies. Usually, blood is used as a sample.
17

The antigen-antibody reaction is optimal if both are present in the same concentrations. For succesful detection, samples
are usually dilated in a range of concentrations, serial dilution, and the lowest concentration at which a reaction cam be
detected is called titer. to be able to see the reaction with the naked eye, large volumes are used. When antigen and
antibody bind they become insoluble and are then visible.
In a Gruber reaction, an unknown antigen is detected by using a known antibody.
In a Widal reaction a known antigen is used to detect a unknown antibody
When using smaller amount of sample a signal should be produced like fluorescence to measure binding.
So, the method depends on the amount of sample;
- Precipitation: detects 0.1-1 mg of antigen
- Immunofluorescence: 50 ug (least sensitive method)
- Active agglutination: 2.5 ug
- Passive agglutination: 5 times as sensitive as active agglutination
- Radio- and enzyme immunoassay: 0.1-1 ng

Precipitation
Precipitation is performed in test tubes and a reaction can be seen as a white ring. The antigen sample is put in a well in a
gel matrix with antibody. Electric current can be added to speed up the movement in the gel, electro immunodiffusion.

Agglutination
in agglutination, one of the reaction components (so the antibody or the antigen) is coupled to a bulky component like
erythrocytes or beads. When there is no reaction happening, the solution is milky and when there is a reaction the beads
will agglutinate.

Immunoblotting
In immunoblotting a mixture of antigens or antibodies can be analyzed. They are first separated by gel electrophoresis,
then transferred on a membrane and incubated in a solution with antibody. This is Western Blot. The antibodie sin the
solution bind the antigen on the membrane and this binding can be detected and measured, for example when it is
conjugated to a fluorescent molecule. Sometimes not the antibody itself is labeled but a second antibody that binds the
first antibody.
Direct immunofluorescence: the use of fluorescent antibodies in serology-based diagnostic
Indirect immunofluorescence: using a secondary fluorescent antibody
Sometimes antigens from a patient sample are put on the membrane and antibodies are used for detection. However,
more often there are known antigens on the membrane and antibodies from the patient sample are used. Then the
results are confirmed using ELISA.

ELISA (enzyme-linked immunosorbent assay)

In ELISA a microtiter plate is used, and the antigen-antibody binding is in a well instead of on a membrane.
Direct: the assay detects the antigen in the sample and the wells are coated with a known antibody
Indirect: the assay detects antibodies in the sample and the wells are coated with antigen
For the detection, the antibody should be labeled or a further labeled antibody should be involved.

Diagnostic methods based on nucleic acids

In this method, the pathogen is identified based on the nucleic acid sequence that is resent in the patient sample.

PCR
In PCR, primers are needed to amplify a DNA fragment. When the primers are complementary to a specific pathogen
gene, the PCR product will only be nucleic acid of the pathogen in the sample. The PCR reaction mixture analyzed with
gel electrophoresis. When there is a band for a segment of the correct size, the patient has been exposed to the
pathogen.
When the pathogen has RNA instead in DNA, reverse transcriptase PCR, real time PCR (RT-PCR), is done. This starts with
reverse transcriptase and does not need gel electrophoresis, measurement is done with a fluorescent signal. A
oligonucleotide probe, which is complementary to the pathogen DNA, is added in the reaction mixture and binds to
product.

Hybridization
Hybridization is the binding of the probe. This can be done with Southern blotting for DNA and Nottern blotting for RNA.
For detection of hybridization a label should be used.
18

Microarray analysis is used for the analysis of small DNA samples containing many genes and is based on hybridization.
With this technique you can determine whether a certain DNA sequence is included in the sample. It is often used for
diagnosis by comparing healthy and diseased samples.

Zie boek

Chapter 7
Disease is any negative change in a person’s health. Etiology is the cause of the disease. So, microorganisms cause a
decline in health, the disease, and these microorganisms are the etiological agents of the disease.

The relationship between the human host and microorganisms

The normal flora in humans is also called microbiota.
Humans are superorganisms and the inhabiting microbes the microbiome.
There are three types of relationships between host and bacteria in the host;
- Commensalism: one of the partners is benefited and one is unaffected. This relationship is seen between
humans and the microbial flora.
- Mutualism: both of the partners provide each other benefits. Humans provide the bacteria nutrients and a place
to stay and the bacteria provide the host with vitamin K and B.
- Parasitism: one of the partners benefits at the expense of the other. This is seen with pathogens.
The microbial flora can protect humans from disease by microbial antagonism. Bacteria that live in certain areas of the
body fight to prevent outsider taking up residence. In this way inhibits our microbial flora the growth of pathogens by
using all available nutrients and oxygen, acidify the region and some produce bacteriocins (which works as antibiotics so
they kill invading organism but don’t affect bacteria that produce them).

After many tests was found out there actually is no normal microbiome. There is much variation between persons in the
human microbiome composition. Every human has a individual microbial fingerprint and even this changes over time.
The different microbial communities do have the same metabolic tasks.
The fluctuations in microbiome are caused by changes in diet, season, hormones and exposure to antibiotics. Besides this
the mother microbiome can affect a child’s health and the child’s microbiome influences the way of delivery.
The microbiome can prevention but it can also cause susceptibility for infections and contribute to diseases like diabetes,
obesity, atherosclerosis, autism, allergies and chronic gastrointestinal diseases (like Crohn’s and celiac), and it can
influence the response to drug treatment. Because if this the composition can be use for early diagnosis and health
improvement by changing it.
Also, the microbiome affects the choice of partner because mating preference depends on body odor, to which
microorganisms contribute.

A healthy microbiome is stable which means there is little changes under stress and if there are it quickly returns to the
previous composition.

Etiology of disease
Koch’s postulates;
- The same pathogen must be present in every case of the disease.
- The pathogen must be isolated from the sick host and purified.
- The pure pathogen must cause the same disease when given to uninfected hosts.
- The pathogen must be re-isolated from these newly infected hosts.
There are exceptions;
- some microorganisms cannot be researched with the postulates, for example because they cannot grow on
artificial media. Viruses always require cells to be produced so then the presence of the pathogen’s genetic
information in the sample is used.
- Sometimes a person carries the pathogen but does not have any symptoms
- Some organisms can cause a variety of diseases.

Keep in mind
• The cause of a disease is referred to as its etiology.
• The body contains normal microbial flora made up of bacteria that are beneficial to the host.
• There are three types of relationship between bacteria and their hosts: commensalism, mutualism and
parasitism.
19

• Koch’s postulates are an important way of evaluating the etiology of a disease.

Development of disease
The development of disease takes place in five periods;
- Incubation period: time between initial infection and the first symptoms. The length of this period depends on
the virulence of the pathogen, the lower the virulence the longer the period.
- Prodromal period: mild unspecific symptoms appear
- Period of illness: major symptoms appear
- Period of decline: the symptoms subside. During this period the infection goes to the end but also a secondary
infection can occur.
- Period of convalescence: regaining of strength, proceeding full recovery.

Communicable and contagious disease

If a disease is communicable, it is able to spread from one person to another, for example tuberculosis. Communicable
diseases that spread very easily are contagious, they are communicable on contact with an infected person. For control
of communicable and contagious diseases there are three methods;
- Isolation: preventing an infected person from having contact with the general population. There are seven kinds
of isolation; strict isolation, respiratory isolation, protective isolation, enteric precautions, wound and skin
precautions, discharge precautions and blood precautions. Isolation is hard before diagnosis has taken place.
- Quarantine: healthy individuals who may have been exposed to a communicable disease are separated from the
general population. This last as long as the prevented incubation time and is lifted if there are no symptoms
during this period. It can be hard because of the difficulty in enforcing it.
- Vector control: destruction of vector habitats and inhibition of vector breeding. A vector is any organism that
carries pathogens from one host to another.

Keep in mind
• The development of a disease can be broken down into five periods: incubation, prodromal, illness, decline and
convalescence.
• Communicable diseases can be spread from one person to another.
• Contagious diseases are communicable on contact with an infected individual.
• Methods for the control of communicable and contagious diseases include isolation, quarantine and vector
control.

Duration of disease
The duration of disease is variable and this can be used to make four categories;
- Acute diseases: develop quickly but last only for a short time. E.g., smallpox and measles.
- Chronic disease: develop slowly but remain for longer periods. E.g., hepatitis.
- Subacute: has an insidious onset of 6-12 months and can be fatal. E.g., subacute sclerosing panencephalitis
(SSPE).
- Latent: the pathogen remains in the host after signs of symptoms disappear and can become reactivated
because of stress in the host. E.g., chickenpox.

Persistent bacterial infection

Some infections are persistent, the pathogens maintain infection even in presence of a good immune response.
Sometimes persistent infection cause no symptoms and two of these kind of infections are tuberculosis and typhoid
fever.
Tuberculosis starts in the lungs and can move through the lung via dendritic cells of the host defense. Often the infection
is cleared after onset of the adaptive immune response but sometimes they can stay for life and be reactivated later in
life when the person has become immunocompromised, for example due to aging.
In tuberculosis, the bacterium stays in granulomas, bodies made up of host cells like macrophages, T-/B-cells, DC’s,
neutrophils, fibroblasts and matrix components. These granulomas are formed when activated macrophages aggregate
into gigantic cells, similar tio syncytia in viral infections. But how does the bacterium survive contact with a cell that
normally would phagocytose and kill it? When the M. tuberculosis is in a phagosome it remodels the structure,
preventing development of an acidic hydrolytic environment. The pathogen inhibits fusion of the phagosome with
lysosomes.
The bacterium S. enterica serovar Typhi can cause many problems in the intestinal tract but when the pathogen becomes
systemic it can cause typhoid fever. This starts with infection, usually in connection with Peyer’s patches in the intestine.
It infects phagocytes in the lamina propria, gaining access to blood and lymph via which it can spread.
20

Some of the persons infected with S. enterica serovar Typhi become carriers that shed pathogens in their stool/urine for
the rest of their lives but never get symptoms. The bacteria survive the host’s defense because they get phagocytosed
but the phagosomes don’t fuse with lysosomes.
There several other mechanisms bacteria can use to evade the host’s defenses and cause persistent infection;
- Evade the innate immune response by destroying the antimicrobial toxin nitric oxide.
- Form megasomes inside macrophages by fusing with many phagosomes. Even though they are inside a
macrophage they can protect the pathogens.
- Subvert the adaptive immune response by blocking T-cell activation.
- Take advantage of genetic diversity to confuse the adaptive immune response.

The scope of infection

Local infection are the easiest to deal with. When the organisms start to move from the original location, the focus of
infection, you get a systemic infection which is far more dangerous. This usually happens when the pathogens gain
excess to the blood or lymph because these go everywhere in the body.
It is called bacteremia when bacteria are present in the blood, septicemia when they are growing in the blood and
toxemia when bacterial toxins are moving through the blood. viruses being present in the blood is called viremia.

Infections can be;

- Primary: causing the initial acute onset of symptoms
- Subclinical: there is an infection, but no symptoms appear. The infected persons are carriers who can infect
others.
- Secondary: develops in a host that is weakened by a primary infection. Because these infections take advantage
of the weakened host they are more dangerous than primary infections.

Toxic shock and sepsis

Toxic shock and sepsis both arise as a result of infection but are really different.
In toxic shock there is a great leakage of plasma from the circulation and the blood pressure decreases. It is caused by
the activation of neutrophils, which takes place these come in contact with bacterial surface proteins like M proteins on
streptococcal species. Toxic shock is fetal in 30-70% of patients in which it occurs.
When Streptococcus pyogenes reproduces in the blood it can cause streptococcal toxic shock syndrome (STSS). While S.
pyogenes grows in the blood its M proteins are shed after which they bind the plasma protein fibrinogen. M protein-
fibrinogen complexes bind and activate neutrophils which then release inflammatory molecules, heparin-binding
proteins. These induce changes in vascular permeability causing a rapid loss in fluids, decrease in blood pressure and
difficulty in ventilating the lungs.
Because of this M protein rich strains are much more virulent than those containing small amounts.
In sepsis there is presence of pathogens or their toxins in the blood. There are two kinds of sepsis; severe and acute.
Severe sepsis has systemic inflammation and organ dysfunction, and abnormal temperature, heart rate, respiratory rate
and leukocyte count, elevated levels of liver enzymes and altered cerebral function. Patients get killed slowly over several
weeks and autopsy shows limited necrosis and tissue inflammation.
Acute septic shock occurs suddenly, and patients die within 24-48 hours. The autopsy shows much tissue inflammation
and cell damage.
As in toxic shock there is a decrease in blood pressure but with sepsis the cause is not related to the activation of
neutrophils.

Keep in mind
• Disease can be acute, chronic or subacute
• In latent disease, pathogens remain in the host after signs and symptoms have disappeared but can be
reactivated after longer periods.
• Pathogens such as Mycobacterium tuberculosis can cause persistent disease in which infections continue even
though the host has a working immune defense.
• Infection can be localized or systemic and can be classified as primary with acute initial symptoms, subclinical
without symptoms or secondary occurring after a primary infection.
• Infection can result in toxic shock or sepsis.

Summary
• Normal microbial flora helps to protect against opportunistic infections.
• Etiology is defined as the cause of a disease.
• Koch’s postulates can be used to evaluate and identify the etiology of a dis- ease.
• Disease can be acute, chronic, sub-acute, or latent.
21

• Infection can be local or systemic.

• Relationships between bacteria and hosts can be described as commensal, mutual, or parasitic.
• There are five periods in the development of an infection: incubation period, prodromal period, period of
disease, period of illness, and period of convalescence.
• Communicable diseases can be spread from one person to another.
• Contagious diseases are spread on contact with an infected person.
• Infection can cause septic shock or sepsis.

Chapter 8
Emerging infectious diseases
Diseases whose incidence has increased in the pas t 30 years of which threatens to increase, are called emerging
infectious diseases. Often these diseases were unseen or rarely seen in humans before, or they had been recognized but
their cause as unknown.
Re-emerging infectious diseases were previously controlled but now have returned.
Emerging and re-emerging are both caused by changes in pathogen, host or environment. Drivers are;
- Biological factors likem immune status of the host and the virulence of the pathogen.
- Social factors such as trade, travel, migration, sexual practices and food consumption
- Political factors like treatments, education, technology and communication infrastructure.
- Economic factors like research and development budget, etc.

Emerging diseases have had four transitions;

- First transition, the crowd transition: occurred when people began living close to each other and places became
more crowded, allowing easy transmission zoonotic disease (transmitted from animals to humans).
- Second transition: occurred when European civilization came in contact with other societies which allowed the
exchange of pools of infections and vectors.
- Third transition: caused by worldwide exploration and colonization. Because these pathogens were never
encountered before many people had zo defense against them.
- Fourth transition, global urbanization: caused by the increase of dense populations, poverty, air travel,
technological development, climate change, that cause emergence and re-emergence of diseases that now have
more potential to spread quickly than before.

Environment and infectious disease

When humans intrude new environments they come in contacts with wild animals, increasing the risk of emcountering
unknown pathogens. This is also influenced by climate change because vectors are able to spread in new areas because
of raised temperatures.

Foodborne infections
EHEC causes severe foodborne infections. It can grow between 7-50 degrees and in acidic foods, so infection ca be
prevented by cooking food thoroughly. Person-to-person transmission happens via the oral-fecal route. EHEC produces
the Shiga toxin, a virulence factor it got from a bacteriophage.
The population is growing so there is more pressure on the production of meat, leading to infections from farm animal to
humans.

Globalization and transmission

There are two ways in which ecology and changing behavior can contribute to emergence of infectious diseases;
- Increased opportunity to animal-to human infection
- Increased opportunity for transmission from one human to another once one person is infected

In re-assortment genes rearrange themselves and cause changes in characteristics. This can also happen between
different pathogens, what could lead to new strains of pathogens which can spread more rapidly.

Most infections have long incubation periods, so once the disease is reconized the pathogen probably already has been
spread further and because of air travel infections can even go worldwide in only a few days.
Immunodeficiency and increase in compromised hosts, amplifies this because of the increasing number of potential
targets.
22

Hurdles to interspecies transfer

For a pathogen to be able to replicate in the human host it must overcome two major hurdles;
- It must adapt in a way that it can replicate in human cells
- It must be able to configure itself so it can be easily transmitted between humans.
To accomplish both, pathogens need to undergo genetic changes. This is easier for viruses because these are prone to
mutations.

Emerging viral diseases

Most emerging disease are caused by viruses. Viral replication requires specific factors and absence, or foreignness of
these factors prevents the viruses from replicating in the host. When adjustment to this absence or foreignness only
requires a minor genetic change in the virus there is an increased risk in jumping to a new host. RNA viruses have to
biggest chance of making the jump because these are adapted the best to the tree mechanisms that can overcome the
problem; mutation, re-assortment and recombination.
Some emerging viral diseases are SARS, West Nile Fever, Dengue Fever and Viral Hemorrhage Fever.
Ziek boek van verschillende ziektes p155-161

Keep in mind
• Emerging infectious diseases are those whose incidence in humans has increased in the past 30 years or
threatens to increase in the near future.
• The environment can contribute to emergence of disease when humans come into contact with previously
unknown pathogens.
• Genetic mutation, re-assortment and recombination can give rise to more dangerous pathogens.
• Pathogens that can affect humans must first be able to adapt to humans and then be easily transmitted from
one human to another.
• Most emerging infectious diseases are caused by viruses.
• SARS, West Nile virus and viral hemorrhagic fevers are examples of emerging infectious diseases.

Re-emerging infectious diseases

Tuberculosis
One third of the world’s population is at the moment infected with Mycobacterium tuberculosis, of which only 10% will
actually develop tuberculosis and it in one fourth of people living with HIV it causes death.
Symptoms are cough, chest pain, weakness, weight loss, fever and night sweats. After development of effective
antibiotics death rates dropped but different factors led to a new rise in incidence;
- Immune status and fitness of the host: people who are particularly vulnerable for M. tuberculosis are people
with HIV and elderly. Poverty and homelessness contributes to the spread of tuberculosis in shelters and
prisons, where weakened people live (due to drug addiction, alcohol, tobacco, insufficient nutrients etc.).
- Virulence and fitness of the pathogen: treatment of tuberculosis consists of four antimicrobial drugs for 6
months. Many patient fail taking these because of disappearing symptoms and having to put up with side
effects. When patients don’t complete the treatment they remain infectious for a longer period, so they can
spread even more, and can cause development of resistant strains. Inappropriate treatment is the primary
cause of multi-drug resistant tuberculosis (MDR-TB). For MDR-TB first-line drugs are not effective but second-
line drugs are. However, further resistance can develop, extensively-drug resistance tuberculosis (XDR-TB). After
this even more resistance can develop, extremely drug-resistant (XXDR-TB) and totally drug resistant
tuberculosis (TDR-TB).
Initial signs of TB are similar to those of ither respiratory diseases so fir detection we should look at fever,
fatigue, weight loss, chest pain, shortness of breath and coughing.

Influenza
The outcome of infection with the influenza virus depends on the virus and on the host’s health status. A previous
infection can lead to partial protection.
The virus contains gene constellations, clusters of genes determining the virulence and single mutations in these
constellations can affect this virulence.
Influenza is not caused by a stable virus, it is a RNA virus that constantly undergoes mutations changing the
characteristics. Because of its high mutation rate and the fact the virus has a stable reservoir in aquatic birds, epidemics
and pandemics are more likely to occur and eradication is difficult to achieve.
The virus contains 8 pieces of RNA and has two glycoproteins, hemagglutinin and neuraminidase, on its surface. There
are 17 different types of hemagglutinin and 10 different types of neuraminidases, of which H1/H2/H3 and N1/N2 are
linked to human infections, and all others in mostly birds.
23

Changes in these glycoproteins have led to epidemics and pandemics in humans and animals. There have been three
major pandemics in humans;
- Spanish influenza: caused by a virus containing H1 and N1 and was the most highly infectious of the three. It
mainly affected young adults
- Asian influenza: caused by a virus containing H2 and N2, these are different from those in the Spanish so there
wasn’t herd immunity

………

Chapter 9

The bacterial cell wall

To handle changes in osmotic pressure, bacteria have a cell wall with peptidoglycan as its major component.
Peptidoglycan consists of NAG (N-acetylglucosamine) and NAM (N-acetylmuramic acid) which bind covanlently, giving
strength.

Building the cell wall

There are three phases in peptidoglycan assembly;
- The cytoplasmic phase: NAG and NAM are produced in the cytoplasm by enzymes encoded by murA-F gnes.
These enzymes attach amino acids to the NAM. The cell wall is constantly refurbished, especially when dividing,
what requires the peptidoglycan layers to be laid down both transversely and vertically.
This phase is a potential target for antibiotic therapy.
- The membrane-associated phase: NAG and NAM are linked with a lipid portion of the bacterial membrane. The
insertion of NAG and NAM in the membrane happens via the lipid carrier cycle of which the first step is
formation of a bond between the peptidoglycan and the side of the membrane facing the cytoplasm.
- The extracytoplasmic phase: the cell wall grows outward from the cell so new subunits (NAG and NAM) have to
be moved from the inner to the outer side of the membrane. When arrived at the outer lipid layer they interact
with membrane-bound enzymes what allows their incorporation in the growing cell wall.
Also, these enzymes are potential targets for antibiotic therapy.
After incorporation of the building blocks, the peptidoglycan layers need to be joined together to form a
meshwork. These connections give the wall increased strength.
Gram-negative and -positive bacteria differ in the amount of peptidoglycan in their cell wall, gram-positive have many
layers whereas gram-negative only have a few.

Keep in mind
- Many of the structures associated with the bacterial cell wall are involved in the infection process.

- The cell wall is the protective outer structure of the bacterial cell.
- The cell wall is made of peptidoglycan, which is a series of repeating disaccharides and polypeptide pieces.
- The wall is thicker in Gram-positive bacteria than in Gram-negative ones.
- Peptidoglycan is constructed inside the cell and transported to the outside of the cell.
- The peptidoglycan forms a meshwork foundation for the cell wall and is a target for antibiotics.

Additional cell wall components

The Gram-staining effects is based on the components of the cell walls

Wall components in Gram-positive bacteria

Gram-positive bacteria contain many layers of peptidoglycan, but it also contains teichoic acid molecules, repeating
subunits of sugar-phosphate molecules to which other sugars and amino acids are attached. There is two types of
teichoic acid; wall teichoic acids that go partly through and lipoteichoic acids that go completely through the
peptidoglycan and link the plasma membrane. Both types protrude above the peptidoglycan layer.

Wall components in Gram-negative bacteria

24

Cell wall is gram-negative bacteria are much more complex. They only have a thin layer of peptidoglycan and because of
that they need extra protection of an outer membrane which is a lipopolysaccharide layer (LPS) of lipids, proteins and
polysaccharides. The lipoprotein molecules fasten the outer membrane to the peptidoglycan.
The outer membrane is a lipid bilayer but has a unique outer layer composed of lipopolysaccharides instead of stand
phospholipids. The layer contains porins and translocation protein systems, most found in the periplasmic space which is
located between the plasma membrane and the outer membrane and contains a gel-like material with proteins secreted
by the cell.

Some bacteria don’t have a cell wall at all what makes them difficult to culture and non-responsive to antibiotics.

Keep in mind
• Gram-positive cells have many layers of peptidoglycan and also contain teichoic acid.
• Teichoic acid is a glycoprotein that can be found in two forms: wall teichoic acid and lipoteichoic acid.
• Gram-negative organisms have no teichoic acid and very little peptidoglycan but have an outer membrane layer
made up of phospholipids and lipopolysaccharides.

Clinical significance of the bacterial cell wall

Gram-positive bacteria:
Staphylococcus aureus causes nosocomial infections, and these are the worst respiratory infections. The bacterium
colonizes the nasal passages, mediated by teichoic acid, an essential element for the infection. The colonization can be
inhibited by the antibiotic mupirocin, but resistance is rising.
The Tag O gene codes for teichoic acid and can be used as a target.
Streptococcus pyogenes causes strep throat and necrotizing fasciitis. It incorporates virulence factors in the cell wall,
among which M proteins that protrude from the cell wall. Antibodies against the M proteins can inhibit the infection, so
this protein is essential for the infection. It is a major virulence factor because it inhibits phagocytosis but it is very
susceptible for mutations so there are different variants that need different antibodies. This variance limits the
effectiveness of the adaptive immune response.
Mycobacterium tuberculosis and leprae make mycolic acid, a waxy lipid that is incorporated into the cell wall and causes
resistance to environmental stresses, antibiotics and host defenses.
Gram-negative bacteria:
The outer membrane of gram-negative bacteria protects against antibiotics so an infection with these bacteria is less
sensitive. The large antibiotics cannot go through the porins, but smaller ones can. When administering antibiotics, porin
encoding genes can mutate to get smaller opening to also inhibit entry of the smaller molecules.
LPS of the outer membrane is an endotoxin. The two parts of the outer membrane with clinical relevance are;
- Lipid A|: attaches the LPS to the phospholipid bilayer. When it is released it acts as an endotoxin and when too
much is present, the immune responses can overreact, causing adverse effects and shock.
- O polysaccharide: are carbohydrate chains on the side of the membrane facing the extracellular fluid. They vary
between species and are recognized by the immune system. They can be used for the identification of bacteria.

Keep in mind
• The bacterial cell wall has clinical significance.
• Gram-positive teichoic acid is involved with the production of inflammation.
• Some Gram-positive bacteria incorporate virulence factors known as M proteins in their cell walls.
• Mycobacterium species are Gram-positive bacteria that incorporate mycolic acid (a waxy substance) in their cell
walls, which makes them resistant to antibiotics.
• In Gram-negative organisms the outer membrane layer protects against disinfectants and antibiotics.
• The outer layer of Gram-negative organisms functions as an endotoxin.
•
Structures outside the bacterial cell wall
There are 5 structures that can be found on the cell wall but not all five are present on all bacteria. The glycocalyx,
fimbriae and pili are involved in adherence and the flagellum, axial filaments and pili in motility.

The glycocalyx
When glycocalyx is in the surrounding of cells it gives colonies a wet and shiny look. It is a sticky substance that contains
polysaccharides and polypeptides, which are produced in the cytoplasm and are secreted. After secretion they bind to
the outside of the cell. They can bind to the cell in two ways. When molecules are loosely attached this glycocalyx is the
slime layer whereas when it is a highly organized structure that adheres tightly to the wall it is called a capsule. Both
types give the ability to adhere to surfaces, protects from desiccation, and can be used as source of nutrition in
desperate times.
25

Clinical significance of the glycocalyx, slime layer and capsule

When invading bacteria gain entry in the urinary or respiratory tract there is a chance they get swept away by
mechanisms of the host. The glycocalyx plays an important role in this with adherence.
The slime layer plays a role in the formation of biofilms. The slime layer permits organisms to bind to the tooth surface
after which many other organisms also adhere, forming a 3. Eventually the tooth surface will be broken down.
Some bacteria are only infectious when being encapsulated, for example Streptococcus pneumoniae, Bacillus anthracis
and Klebsiella pneumoniae. This is because the capsule inhibits phagocytosis, because this inhibits binding of the
phagocyte to the bacterium.

Fimbriae and pili

Fimbriae and pili are involved in adherence and pili also in transfer of genetic material via conjugation. They are found in
gram-negative bacteria and their number variate between species. Both are composed of pilin protein subunits.

Clinical significance or Fimbriae and Pili

With their role in adherence, fimbriae and pili are important for the bacterium to stay in, for example Neisseria
gonorrhoeae is only infectious when fimbriae are present. Especially for organisms that infect the urinary tract they are
important because otherwise the bacteria would be flushed away. This is also the case of E. coli in the gastrointestinal
tract.
The binding ability of pili is a potential target for vaccine development because when antibodies bind pili they block the
binding to host cells.
By retraction of the pili, the bacterium makes a twitching and gliding movement. When the bacterium binds a host cell
the pili retracts what allows what makes it able for the bacterium to move across the surface. The movements are made
by detachment of pili subunits what pulls the cell forward, followed by reformation and elongation.
This motility allows the formation of biofilms, helping bacteria to become resistant to antibiotics.
Besides adherence and motility, pili can be used for immune escape by;
- Phase variation, decreasing the number of pili after initial infection to decrease the number of targets for
antibodies.
- Antigenic variation and post-translational modification in which the structures are changed or masked so they
are no longer recognized by antibodies.
- Secretion of fragments of pili, S pili, that bind antibodies and inactivate them.
- Facilitate transfer of genetic material from one bacterium to another, called conjugation. In this way there can
be genetic modifications, leading to production of dangerous toxins and transfer of genes that are responsible
for resistance to antibiotics.

Axial filaments
Axial filaments wrap around bacterium and make them motile. They are found on spirochetes between the plasma
membrane and the outer membrane and produce rotational movements causing the entire organism to rotate in a
corkscrew manner.

Clinical significance of axial filaments

Because of the rotational movements of the axial filament, the bacterium is able to bore through tissue. This is seen in
Treponema pallidum causing syphilis and in Borrelia burgdorferi causing Lyme disease where movement of the organism
is from a external location through tissue into the blood and back into the tissue.

Flagella
A flagella is used for motility and it is structured in a way that maximizes its function, reducing wastage of energy. They
extend far from the cell wall.

Structure of the bacterial flagellum

The flagellum on bacteria consists of three parts; a filament, hook and basal body. The filament is made of flagellin
molecules which attach to each other, forming a chain with a helical structure so. The core is hollow. The hook links the
filament to the basal body that anchors the whole structure in the cell.
The flagellum makes a rotational movement with 100.000 rotations/min, moving the bacterium 20 cell lengths in
seconds. Because of this speed, there is stress on the connection between de filament and the cell, so where the basal
body is found. The basal body is a rod with ring attached to. Depending on the kind of bacterium (gram-negative or
positive) the cell has one or two pairs of rings. Gram-positive cells have a thick layer of peptidoglycan, and one pair of
rings are attached to the plasma membrane, and the flagellum extends through the whole layer of peptidoglycan
Because gram-negative cells have only a thin layer of peptidoglycan, they need something for reinforcement at the
connection of the flagellum and the cell and therefor they have a second pair of rings.
26

Flagellar configurations
In bacteria there are four distinct patterns of flagella arrangement;
- Monotrichous: the bacterium has one flagellum at one end of the cell, this is the most common.
- Amphitrichous: the bacterium has two flagella, one at each end of the cell.
- Lophotrichous: the bacterium has two or more flagella, all located at one end of the cell.
- Peritrichous: the entire bacterium is surrounded by flagella.

Clinical significance of flagella

Helicobacter pylori causes gastric ulcers, lives in folds of the stomach and used its flagellum to go the mucus layer.
The movement of bacteria to other places can lead to opportunistic infections.
When invading organisms enter a place that is already occupied by the normal flora, these resident bacteria inhibit the
growth of the invading bacteria. However, for example after an overuse of antibiotics, the resident bacteria are
decreased in number, motile invading organisms can cause an opportunistic infection. Besides this, all organism that are
able to move from tissue to lymph or blood can cause a systemic infection.

Intracellular movement
Some pathogens, for example Salmonella and Shigella, use cytoskeletal structures to gain access to the host cell, for their
intracellular movement within the host cell, and to move from one cell to the next. Shigella is associated with dysentery,
it is nonmotile but using these cytoskeletal structures it is able to move.

Keep in mind
• Structures found outside the cell wall are all involved in the infection process.

• The glycocalyx can be found in either the capsule or slime-layer form and can satisfy the requirement of “staying
in” as a result of their adherence to host cells.
• In many cases, an organism is not pathogenic unless it has a capsule.
• Fimbriae also satisfy the “stay in” requirement for infection by adhering to host cells.
• Pili can be used to adhere to host cells but also serve the function of allowing the transfer of genetic information
between bacteria in the process known as conjugation.
• Axial filaments allow organisms to penetrate tissues by means of rotational movement.
• Flagella permit movement, which helps organisms stay in.
• There are four forms of flagella seen in bacteria: monotrichous, amphitrichous, lophotrichous, and peritrichous.
• The host cell’s cytoskeleton can be used for intracellular movement during the infection.

Structures inside the bacterial cell wall

Structures inside the cell wall that are involved in intrinsic cellular functions are; the plasma membrane, the nuclear
region, plasmids, ribosomes, inclusion bodies and endospores.

The plasma membrane

A characteristic of both the bacterial and eukaryotic plasma membrane is selective permeability, only certain molecules
can pass through easily.
Both the bacterial membrane and the cell wall that surrounds it provides a barrier between the outside and inside of the
cell, but unlike the cell wall the bacterial membrane is also involved in physiological functions like transport, secretion,
energy generation and replication.

Structure of the plasma membrane

The bacterial membrane consists of a phospholipid bilayer, with the hydrophobic tails facing inwards. In the bilayer float
many different proteins. There are two basic types of membrane proteins; peripheral and integral. Peripheral proteins
are found in the inside or outside of the membrane whereas integral proteins penetrate the whole wall. Sometimes
these are pores that connect the interior to the external environment.

Energy production
Each metabolic functions needs ATP, for example in movement of the flagellum each rotations needs energy. Eukaryotic
cells have mitochondria to produce all this energy but bacteria use the membrane and thereby uses the electron
transport chain, a series of membrane proteins generation ATP by the transfer of electrons and proton/H+ from protein
to protein. This process causes a gradient across the membrane, creating a proton motive force which is a form of energy
that can be used immediately or can be stored as ATP by the enzyme ATP synthase.
27

Membrane transport
The cell wall and membrane play a role in coping with environmental stresses. The cell wall consists of a meshwork
structure which allows things to go through whereas the membrane regulates what goes in and out.
To get rid of waste and to get access to nutrients, transport is needed. Mechanisms of transport have three functions;
osmosis, passive transport and active transport.
Because the membrane is selectively permeable, small molecules can move across it. Osmosis is the movement if water
across the membrane from low to high solute concentration. When the concentration of solutes is lower inside the cell,
water moves out so the cell will shrink, plasmolysis. When the concentration of solutes is higher at the outside, water will
come and what causes cell to expand and sometimes lyse.
A hypertonic solution has a solute concentration that is higher than inside the cell and can cause shrinking or plasmolysis.
A hypotonic solution has a solute concentration that is lower than inside the cell and can cause lysis.
In an isotonic environment, the solute concentrations inside and outside the cell are the same and water moves equally
into and out of the cell.

Passive transport
There are two types of passive transport; simple and facilitated diffusion. Both don’t require energy.
Simple diffusion depends on concentration gradients and the higher this gradient, the faster the solutes will move from
the place with higher concentration to the place with the lower concentration. It only occurs with solute molecules that
are soluble in lipid or with molecules small enough to pass through.
During simple diffusion, the concentration gradient will become smaller so the diffusion will get slower, but it continues
until the solute concentrations are equal and there is no gradient anymore.
In facilitated diffusion move across the membrane through permeases, transport proteins in the membrane whose
function depends on their 3D-shape. When a solute binds on the site of the permease facing the external environment its
3D-shape is changed such that to opening on the external side closes and on the interior side opens so the solute can
move into the cytoplasm. After this the permease gets its original structure back.
When solute molecule is too big to fit into the binding site of permease, the cell secretes enzymes into the external
environment to break the solute into smaller pieces to be able to bind the permease.

Active transport
In active transport, solutes are carrier over the membrane against the concentration gradient, requiring energy from ATP
or proton motive force. Carrier proteins in the plasma membrane are found.
There are three types of active transport; efflux pumping, ABC transport systems and groups translocation.
In efflux pumping a revolving door mechanism is used in which pumps bring in molecules and bring out other at the same
time. For energy no ATP is used, only a proton motive force directly coming from the electron transport chain. This is
seen in both prokaryotes and eukaryotes.
ABC (ATP binding cassette) transport systems are a complex of multiple proteins. During the process, the molecule to be
transported forms a complex with a binding protein on the outside of the plasma membrane after which it goes to a
complex of proteins located in the membrane which transport the molecule into the cytoplasm
Group translocation only happens in bacteria and involves making sure the transported molecules stay inside. For
example, once inside the cell, glucose needs to be kept inside. As the concentration inside the cell increases, a gradient is
formed which allows glucose molecules inside the cell to move back across the membrane by simple diffusion. To
prevent this, phosphotransferase enzyme attaches a phosphate group to the glucose after entering the cell, removing it
from the diffusion equilibrium reaction so it stays inside and can be used as an energy source. For this binding of
phosphate to glucose, energy is needed. This energy comes from phosphoenolpyruvate (PEP).

Secretion
In secretion several plasma membrane proteins act in a specific sequence. Molecules designed to be moved out are
recognized through a signal sequence of 20 amino acids. This sequence helps interaction with the plasma membrane and
is removed when the molecules crosses the membrane and exits the cell.

Clinical significance of the plasma protein

Damage to the membrane inhibits the transport of essential components, produce energy and replicate DNA. The
outside can come into the cell and the inside can go out.
The membrane is used as a target for antibiotics. However, because of many similarities with eukaryotic membranes,
these antibiotics can have major side effects.

Keep in mind
• The plasma membrane surrounds the cytoplasm in bacterial cells.
• This membrane is composed of a phospholipid bilayer, which is selectively permeable.
28

• The membrane contains many different types of protein that are either localized on the inner or outer side of
the membrane (peripheral proteins) or span the entire membrane (integral proteins).
• The membrane in bacteria is involved with energy production (ATP is formed here) and also in the replication of
the bacterial chromosome.
• Membrane transport can involve osmosis and passive transport (which require no energy) or active transport
(which requires ATP).

The nuclear region

Bacteria have no nucleus so the region of the cell where the DNA is located is called the nuclear region.
There is one circular chromosome which is supercoiled and associated with positively charged proteins for stabilization.

Plasmids
Plasmids are extrachromosomal pieces of DNA that are separated from the chromosome. Plasmids are between 0.1.10%
of the chromosome, carry genes for toxins and resistance to antibiotics and can be transferred between bacteria through
pili by coagulation.

Clinical significance of DNA and plasmids

DNA is one of the five targets for antibiotics because any damage to it can already be lethal. Unlike the plasma
membrane this target is not selectively toxic because the DNA of the pathogens is similar to that of the host. However,
there are therapies used for viral infections that are reactive to viral nucleic acids and not toxic to the host.
Plasmids play a role as a therapeutic target and in antibiotic resistance because they make the toxins that cause the
symptoms of an infection, and they carry all genes of resistance.

Ribosomes
Ribosomes are non-membrane-enclosed structures that play a role in the synthesis of proteins. A ribosome is composed
of two subunits that are apart from each other in the cytoplasm until a mRNA comes by. Then, the subunits fold around
this mRNA and translation takes place. The bacterial ribosome subunits can be distinguished from eukaryotic ribosome
subunits because they differ in size.

Clinical significance of ribosomes

Bacterial ribosomes are one of the five targets for antibiotics because is synthesis of proteins is required for bacteria to
live and inhibition of this process can be lethal. Different antibiotics target different parts of the ribosomes.

Inclusion bodies
In inclusion bodies, material the cell may require are stored. There are different types, of which one are inclusion bodies
for glycogen, a form of glucose. Metachromatic granules are inclusion bodies that store phosphates.

Endospores
Some bacteria, have the ability to do sporulation. When a spore-forming bacteria is exposed to too much environmental
stress, and endospore is formed. Mainly gram-positive rods, for example Bacillus and Clostridium, do sporulation but also
the highly pathogenic gram-negative Coxiella burnetiid can form endospore. An endospore causes dormancy to the cell
and through the process of germination a cell can return to its vegetative (growing) state.
Endospores are resistant to heat , desiccation, toxic chemicals, antibiotics, ultraviolet irradiation and long periods of
boiling.
Sporogenesis consists of several steps;
- Replication of the bacterial chromosome
- Sequestering of the copy along with the cytosol
- Volume of cytosol and the copy becomes cut off from the rest of the cell when a septum forms
- The larger part of the cell wraps itself around the smaller volume, forming a forespore composed of two
membranes
- Large amounts of peptidoglycan deposit between the two membranes of the forespore
- The rest of the original cell degrades, leaving the bacterial genetic information protected inside the endospore
Germination occurs when the environmental stress has decreased or after chemical cues. Then water comes in what
causes the endospore to swell and crack. The water activates metabolic reactions with dipicolinic acid and calcium (two
chemicals packed in the endospore).

Clinical significance of sporogenesis

29

Because the endospores are really resistant, the organisms that produce them can withstand treatment and continue the
infection. For example Clostridium botulinum produces a endospore that’s survives food processing procedures and
when they germinate this results in cells that produce the botulinum toxin, causing damage.
Because endospores are resistant to heat, when sterilizing instrument heat and high pressure have to be combined to
deal with both C. botulinum cells and endospores.
Unfortunately, endospores can also be used in weapons, for example in the anthrax attacks in which endospores of
Bacillus anthracis were manipulated into powder that remained airborne.

Keep in mind
• There is no nucleus in bacteria: the DNA is found freely floating in the cytoplasm in an area referred to as the
nuclear region.
• Bacteria can contain extrachromosomal structures known as plasmids.
• Plasmids are very important in clinical disease because they can carry genes for antibiotic resistance as well as
genes for the production of toxins.
• Ribosomes of bacteria are different from those found in eukaryotic cells and are consequently a selective target
for antibiotic therapy.
• Endospore formation can protect bacteria from environmental pressures and are also very resistant to
antibiotics and disinfectants.
• After environmental pressures subside, endospores can undergo germination into the original bacteria.
• Endospore formation makes pathogens extremely difficult to deal with.

Summary
• Many of the structures associated with the bacterial cell wall are involved in the infection process.

• The cell wall is a meshwork made up of layers of peptidoglycan. There are many layers in Gram-positive
organisms and few in Gram-negative organ- isms.
• The cell wall is a primary target for attack by antibiotics.
• Some Gram-positive bacteria incorporate M protein or mycolic acids, which are virulence factors.
• Gram-negative cells contain an outer layer made up of lipoproteins, lipopoly- saccharides, and phospholipids
that protect against antibiotics and function as an endotoxin.
• The five structures found outside the cell wall — capsules, fimbriae, flagella, axial filaments, and pili — are
involved in the infection process.
• The plasma membrane surrounds the cytoplasm and is the place where the replication of DNA and the
production of ATP take place. It is also a target of antibiotics.
• Bacteria have no nucleus, and the DNA floats freely in the cytoplasm.
• Plasmids are extrachromosomal structures made of DNA that contain the genes for toxins and antibiotic
resistance. These plasmids can be transferred from one bacterial cell to another.
• Endospore formation protects bacteria from environmental pressure and also from antibiotics and disinfectants.
This structure can have a major role in clinical settings.

Chapter 11
The structure of DNA and RNA
Deoxyribonucleic acid (DNA)
The DNA is the blueprint for components of the cell that are passed on the next generations of cells and this ability is
allowed by its structure. This structure makes to process of replication easier and makes gene expression more efficient
and almost error-free.
In the helix, the bases are facing inward but because of a minor and a major groove between the twists they are still
accessible.
Nucleotides are composed of a phosphate, a deoxyribose and a base and these three bind in a very specific way with a
precise orientation that permits building of the strands. Nucleotides are joined by formation of a phosphodiester bond
between the 3’ hydroxyl group of the sugar and the 5’ phosphate group of the next nucleotide.
There are two types of bases; the double-ringed purines adenine and guanine and the single-ringed pyrimidines thymine
and cytosine. Between adenine and thymine there are always two hydrogen bonds whereas there are three between
cytosine and guanine, This binding and the fact that the strands lay anti-parallel affect the helical structure.

Ribonucleic acid
RNA and DNA differ from each other in;
30

- RNA contains ribose instead of deoxyribose

- RNA has uracil as a base instead of thymine
- RNA is usually found on single-stranded, but sometimes it folds on itself to form double-stranded areas
There are three types of RNA;
- Messenger RNA: contains information derived from the DNA and is used for the synthesis of proteins. In
eukaryotes one mRNA molecule is an RNA copy if a single gene. Prokaryotes contain polycistronic mRNA
molecules which encodes for multiple proteins
- Transfer RNA: carries amino acids to the ribosome for the construction of proteins
- Ribosomal RNA: contributes in maintaining the shape of the ribosome and the orientation of the protein that is
being synthesized.
If there are complementary bases close to each other in the RNA strand they can pair, forming a loop. In addition, the
RNA molecule can form a 3D-structure, which is seen in tRNA and rRNA.

Keep in mind
• DNA stands for deoxyribonucleic acid.
• DNA is the informational molecule of the cell and is a double-stranded molecule made up of nucleotides.
• A nucleotide is composed of a phosphate, a sugar, and one of the four nucleotide bases (adenine, thymine,
guanine, or cytosine).
• RNA stands for ribonucleic acid and is made by copying one strand of DNA.
• In RNA, thymine is replaced by uracil.
• There are three types of RNA: messenger, transfer, and ribosomal.

DNA replication
DNA separation and supercoiling
Double-helical molecules sometimes twist around itself, supercoiling, which allows very small cells to contain al lot of
information. For replication and transcription to take place, this supercoiling has to be undone. This is done by the
enzyme topoisomerase which breaks the DNA chain, relaxing the supercoils, and reseals the strand again. Then the
enzyme helicase separates the chains. After separation of the helix there are two requirements for replication to take
place;
- Enough of each for nucleotides
- A primer-template junction, the point where a primer, a short segment of RNA, binds a template.

DNA polymerase
The DNA polymerase can add a new nucleotide at the RNA primer. The new DNA strand can only be made in direction of
3’ to 5’ and this is the same for transcription.
When a new phosphodiester bond is formed, a two-phosphate molecule is released which is cleaved into two individual
phosphate molecules, releasing energy. This energy is used for the elongation of the strand. The growth of the strand
happens really fast and the longer the enzyme is attached to the DNA the faster the addition of nucleotides occurs.
There are different types of DNA polymerase, differing In the number of nucleotides they can add.

Proofreading by DNA polymerase

The observed error for replication is one in every 1010 pairings whereas experiments show this would once every 105
pairings. This difference is caused by the proofreading capability of DNA polymerase. DNA polymerase contains a
exonuclease that can remove the growing 3’ of the strand. The activity of the exonuclease is attracted to nucleotides that
are incorporated wrong and removes these.
When a wrong nucleotide tries to attach the activity of DNA polymerase slows down. An incorrect nucleotide changes the
configuration of the growing strand in the DNA polymerase, moving it to the exonuclease site and increasing its activity.
After removal of the wrong nucleotide, the strand shifts back to the polymerase active site.
A benefit of proofreading is the fact that DNA polymerase does not have to detach and attach again which would have
been very time consuming.

The replication fork

The replication fork is the location on the helix where the replication is taking place. It moves along the DNA and at this
place the strands are separated. Both strands are replicated at the same time.
The two strands are antiparallel but because nucleotides are added to the 3’, this makes it impossible to replicate the
strand simultaneously in the same direction. This means one strand lays in the good position, is the leading strand, and
the other one is not, the lagging strand. At the leading strand DNA polymerase is chasing the fork whereas it is moving
away from it at the lagging strand. Because it is moving away the lagging strand is replicated in pieces, Okazaki
31

fragments. Because polymerase needs a free 3’end, each Okazaki fragment needs its own RNA primer, so polymerase
needs to wait for the fork to expose a segment of the template.
The primer is made by primase, a type of RNA polymerase that can synthesize RNA without an available 3’end. When the
fork moves, this primase attaches to the strand to synthesize the RNA primer, using the lagging strand as a template.
After the RNA primer has been synthesized the DNA polymerase can add nucleotides until it reached the end of the
previous Okazaki fragment. After this, the RNA primer is removed by the enzyme RNAase H, leaving a gap that is then
filled by DNA polymerase. The ends of the pieces are linked by the enzyme DNA ligase.

Initiation and termination of replication

The initiation of replication starts at the origin of replication and is controlled by the replicator sequence and the initiator
protein. The replicator sequence is a set of sequences with many A-T basepairs which means there are less hydrogen
bonds, and this part is opened more easily. The initiator protein recognizes the replicator sequence, binds to it and
unwinds to the strands adjacent to this sequence.
When the entire chromosome has been copied the replication will terminate and the replication components dissociate
from the DNA. At this point the chromosomes are still linked but in cell division they have to separate which is done by
the enzyme topoisomerase (also involved in the relaxation of the supercoils).

Keep in mind
• DNA is faithfully replicated so that the same genetic information is passed on from generation to generation.

• The enzymes topoisomerase and helicase unwind and separate the strands of DNA to be replicated.
• DNA polymerase copies each of the parental strands so that each daughter cell will contain a chromosome
made up of a parental and daughter strand.
• DNA polymerase has a proofreading capability to prevent mistakes during replication, and it replicates in only
one direction (from the 3' end of the strand).
• The replication fork is the site at which replication is occurring.
• At the replication fork there is a leading strand, which is replicated continuously, and a lagging strand, which is
replicated in pieces known as Okazaki fragments.
• Replication is initiated at a site on the DNA called the origin of replication and proceeds until the entire
chromosome has been copied.

The genetic code

With the 4 different nucleotides 65 different codons can be made while there are only 20 different amino acids. This
means there is redundancy, degenerate, which has possibly evolved to minimize the effects of mutations. The last letter
of the codon does not change the corresponding amino acid and is insignificant, wobble hypothesis.
Besides codons for amino acid there are also three stop codons and a start codon. In the use of codons there are three
rules;
- The codon sequence is always read in the direction from 5’ to 3’.
- There is translation of a reading frame.
- There is no overlap or gap.

Gene expression
A gene codes for a functional product and gene expression is the production of that product.
Mutations that occur during replication are carried to the next generations and can cause pathogens to become more or
less virulent.
Mechanisms of expression involve specific interactions between proteins, RNA and DNA, and are highly regulated.
Gene expression consists of transcription and translation.

Transcription
Both in transcription and in DNA replication, the DNA is used as a template, but there are some differences in
transcription in comparison to replication;
- Only strand of DNA serves as a template.
- No primer is required.
- RNA does not remain base-paired with the template once the process is completed.
- RNA polymerase does not as good in proofreading what makes transcription less accurate.
- Transcription copies only certain pieces of the DNA strand whereas in replication all of it is copied.
32

There are three steps in transcription: initiation, elongation and termination. During initiation, the RNA polymerase binds
to the promotor, causing a conformation. In front of the complex the strands separate, making a bubble in which the
RNA is made. The bubble is only as large as needed and right after the RN polymerase the strands are rejoined.
After the addition of 10 bases, the elongation phase will start in which the RNA polymerase has to multitask. It unwinds
the DNA, adds bases to the growing RNA strand and re-anneals the DNA and simultaneously it does some proofreading.
In the termination step the RNA polymerase reaces a signal to stop and detaches from the DNA.

Translation
Translation is an expensive process, a lot of energy is used. All three types of RNA are needed.

mRNA in translation
mRNA has a protein-coding region with an open reading frame (ORF). Translation starts at start codon in this ORF, goes
from 5’ to 3’ and stops at the stop codon.
Prokaryotic mRNA molecules contain a binding site for a ribosome where they bind complementary to get the mRNA
properly orientated.

tRNA in translation
tRNA molecules bring the amino acids to the mRNA and ribosomes to be added to the growing chain, it attaches to a
specific amino acid and a specific codon on the mRNA. Every tRNA molecule is about 75-95 nucleotides long and ends
with 3’-CCA-5’, the site at which the enzyme aminoacyl-tRNA synthetase couples amino acids. The fact that every tRNA is
specific for only one amino acid is very important because otherwise it could carry just any amino acid what would result
in many mistakes as the ribosome is unable to discriminate between right and wrong amino acids it only needs the
proper tRNA codon recognition.
Because they fold over themselves, tRNA’s have a cloverleaf structure of which the stem part is the acceptor arm, where
the amino acids bind the loop that is the furthest away from this is the anticodon loop where codon recognition occurs.
Between the tRNA and the amino acid a high energy bond forms and when this bond breaks energy will be released that
is then used to form peptide bonds between the amino acids.
A amino acid attaches to a tRNA in two steps; coupling the amino acid to AMP and transfer of the amino acid to the
3’end of the tRNA. For both steps the enzyme aminoacyl-tRNA synthetase is needed and there is a different enzyme for
every different amino acid.
Because the ribosome blindly accepts every tRNA that has proper codon binding, the specify of translation has to come
from the aminoacyl-tRNA synthetases.

The ribosome in translation

A ribosome consists of three rRNA molecules and more than 50 proteins. It works highly efficient, adding 2-20 amino
acids per second.
In prokaryotes mRNA binds a ribosome immediately after transcription and as one ribosome moves along another can
bind right behind, polyribosomes.
The ribosome consist of is a large and a small subunit. The large subunit is 50S and causes formation of the peptide
bonds and the small subunit is 30S and contains a decoding center where tRNA bonds to the mRNA, where codon-
anticodon recognition occurs.
50S is made up of a 5S rRNA and a 23S rRNA which is coupled with many proteins. The 30S only contains a 16S rRNA and
proteins.

Formation of peptide bonds in translation

In the peptidyl transferase reaction amino acids are bonded. The ribosomes catalyze the formation of these bonds but
they are formed on the tRNAs. The tRNA contains energy to create the peptide bonds.
Ribosomes have three binding sites for tRNA molecules; the P-, A- and E-site. The A for a tRNA with an amino acid, P for
peptidyl-tRNA holding the chain and E for the tRNA that exits the ribosome.
The decoding center on the small subunit and the site for peptide bond formation on the large subunit are both located
deep in the interior of the ribosome. The mRNA goes in and out via tunnels, located in the small subunit. The tunnel fir
entry is small so only one mRNA strand fits in. The area between the tunnel of entry and the tunnel of exit is accessible
for tRNA, this is the P- and A-site. In the large subunit there is also an tunnel of exit but this is for the newly synthesized
chain and this is small so no premature folding can take place.

There are three stages of translation; initiation, elongation and termination.

Translation initiation
In the initiation stage there is formation of an 70S ribosome at the startcodon.
33

There are three things needed for the initiation of translation;

- Recruitment of the ribosome to the RNA to from the translational apparatus
- Placement of a methionine tRNA and its amino acid on the P site (the first tRNA does not go on the A-site)
- Precise positioning of the ribosome over the mRNA start codon
Of all three the last step is the most crucial because it makes an open reading frame and if it is off by even one base
position the protein being synthesized will not be correct.
Translation elongation
For other amino acids to be added to the methionine, three things need to happen;
- A tRNA carrying the next amino acid is loaded into the A-site.
- A peptide bond is formed between this amino acid and the amino acid in the P-site.
- tRNA moves, so the one on A moves to P and the one on P to E.
The ribosome has several mechanisms to prevent binding of the wrong amino acids;
- At the site where the mRNA codon and tRNA anticodon interact additional hydrogen bonds form to get the
orientation and to hold the two in place.
- Proteins that regulate elongation help to prevent incorrect binding of amino acids
- The process of accommodation ensures that the correct tRNA is used. There is tension on the tRNA anticodon
when binding the mRNA and only the correct tRNA can withstand this strain, the wrong one can’t and will
dissociate from the ribosome.

Translation termination
When A stop codon enters the A-site the translation stops. The stop codons ate recognized by proteins that cause the
translation complex (which consists of the ribosome and the new peptide chain) to break down. The peptide chain then
start to form a secondary and tertiary structure and the large and small subunit of the ribosome dissociate, searching for
a new mRNA strand.

Keep in mind
• The genetic code is based on combinations of three letters called codons.

• Each codon codes for a specific amino acid.

• There are three stop codons and one start codon in the code.
• Gene expression is the process of making a functional product based on the genetic information contained in
the DNA and consists of transcription and translation.
• Transcription proceeds through three steps: initiation, elongation, and termination.
• Translation uses messenger RNA, transfer RNA, and ribosomal RNA and occurs at the ribosome of the cell.
• The ribosome is made up of two subunits that contain RNA and protein.
• Amino acids are brought to the ribosome by transfer RNA molecules, which are specific for certain amino acids.
• While at the ribosome, peptide bonds form between adjacent amino acids.
• Translation stops when a stop codon enters the ribosome.

Regulation of gene expression

The regulation of gene expression is used to develop resistance to antibiotics. Bacteria are saving their energy so not all
their genes are expressed all the time.
Genes can be constitutive, so they are always being expressed. There is regulation of the expression of the other genes in
multiple stages, but mostly in transcription. At the level of transcription there are two types of regulation; positive
regulation (induction) with activators and negative regulation (repression) with repressors. The activators and repressor
recognize two sites at the DNA. At the promotor site RNA polymerase binds and this is adjacent to the operator site
where the regulatory proteins bind. These sites overlap so when a regulator protein binds the operator site, RNA
polymerase cannot bind the promotor and transcription is inhibited.

Induction
Induction turn genes on that are off.
An operon is a set of genes that is regulated, for example the lac operon. Lac genes code for enzymes that inhibit
bacteria to use the sugar lactose.
Glucose is the best molecule to get energy because it is easily converted and can provide 38 ATP. Because of this, genes
that break down glucose are constitutive. When there is no glucose present other molecules like lactose are used.
Because lactose normally is not used the genes for breaking it down are off so these are inducible. There are three of
these genes (for the breakdown of lactose) lac Z, Y and A, which lay adjacent to each other on the E. coli chromosome,
with the promotor on the 5’end of lac Z. The mRNA that is made of these genes is polycistronic but the three lac genes
have different functions. Lac Z codes for b-galactosidase which is an enzyme that cleaves lactose into galactose and
34

glucose. Lac Y codes for permease which is a membrane transport protein for lactose. Lac A codes for transacetylase
which removes toxic by-product of galactose from the cell.

So, when there is glucose present, the lac genes are off, they are repressed. The lac l gene codes for the lac repressor
protein and is constitutively expressed because having the lac genes active would have been a waste of energy. The lac
activator CAP and the lac repressor both bind the DNA at the operator site adjacent to the lac promotor region. The lac
repressor binds in such a way prevents the transcription of the lac genes until lactose is present and glucose is absent.
When no glucose is present, CAP recruits RNA polymerase and interacts with is what changes its configuration so that It
can bind and make mRNA. For the lac operon to be turned on the repressor must be inhibited through allosteric control.

Although in presence of glucose the lac genes are repressed, the expression is leaky so a few transcripts will be made
which results in a low level of b-galactosidase. Because of this small amounts of lactose can come into the cell and are
concerted to allolactose which binds the lac repressor (not on its active site), changing its shape so it can no longer bind
the operator site. when allolactose is no longer present the lac repressor returns to its original shape.
When glucose is present there are low levels of cyclic AMP (cAMP), what accumulates when energy levels decrease.
When there is no glucose anymore, the cAMP levels increase and cAMP binds CAP. This causes a conformational change
and the CAP-cAMP complex binds to the DNA what results in the recruitment of RNA polymerase to the promotor for
transcription of the lac operon. Because the binding of cAMP to CAP is allosteric, the conformation returns back to
normal when cAMP levels decrease and CAP falls off because RNA polymerase does not bind the DNA efficiently anymore
without CAP.

Repression
The turning off of gene is similar to feedback inhibition and occurs post-transcriptionally. When tryptophan accumulates
it becomes a co-repressor of its own synthesis. The repressor for tryptophan is always made but it is made in a
configuration that cannot bind the DNA. When there is excess tryptophan this will bind the repressor, changing its
configuration such that it is able to bind the DNA and represses the production of tryptophan. When there is no
tryptophan anymore to bind the repressor its configuration changes again and tryptophan is produced again.

Keep in mind
• The expression of a gene is carefully regulated.
• Genes can be constitutive (always “on”), inducible (“off” and can be turned “on”), or repressible (“on” but can
be turned “off”).
• Regulatory proteins control induction and repression through binding on the DNA at the site known as the
operator site.
• An operon is a set of structural genes that share a common promoter and operator and are regulated together
by a control gene.
• Gene expression is regulated at the level of mRNA production. When induced, message is made; when
repressed, message is not made.

Mutation and repair of DNA

Mutations often have adverse effects but they can also cause increased virulence and antibiotic resistance. Adaptation is
directly related to mutations so a bacterial cell depends on the balance between mutation and repair. Proofreading
handles most errors but some still manage to escape.

Replication errors
Missense mutations are point mutations that only affect one base. There are two kinds of point mutations;
- A purine is switched for a purine or a pyrimidine for a pyrimidine
- Transversion so a purine is switched for a pyrimidine or the other way around
A deletion or insertion is more serious which can be the result of a transposition of genes to another place on the
chromosome or from recombination. When the mutation involves more than one base the mutation is a frameshift.
The hot spots on the chromosome are more susceptible to mutation and have higher rates of spontaneous mutations.
Suppressor mutations can reverse primary function of a protein, ‘two wrongs make a right’. These can be intragenic, in
the same gene, or intergenic, in separate genes.

How DNA damage occurs.

DNA damage can be caused by;
- Hydrolysis
- Mutagens
35

- Deamination: water can cause deamination of cytosines, creating uracil that can bind to adenine and cause a
mutation.
- Alkylation: a C-G is changed into A-T after replication.
- Oxidation: for example, oxidation of guanine leads to creation of something that can bind to adenine and
cytosine so after replication G-C is replaced by A-T.
- Radiation: gamma and ionizing radiation cause double-strand breaks which are hard to repair and often lethal in
bacteria. Ultraviolet radiation causes the formation of thymine dimers, two thymine’s on the same strand bound
to each other. This causes DNA polymerase to stop at this site.
- Compounds that slip between two bases: for example acridine and ethidium cause shifts of the reading frame.
- Base analogs: are mistakenly placed in the growing DNA.

Repair of DNA damage

There are two mechanisms of removing and repairing altered bases;
- Base excision repair: the damaged base is removed after which DNA polymerase fills the gap and DNA ligase
repairs the break. These enzymes all diffuse along the DNA for scanning for a particular type of damage. When a
base is removed it goes into a pocket of the enzyme. This all happens without distortion of the helix.
- Nucleotide excision repair: the enzymes do not recognize a specific type of damage but distortion in the helix
that trigger a chain of events, leading to the removal of short single strand sections surrounding it. After this the
gap is filled by DNA polymerase and the break repaired by DNA ligase.
- Photoreactivation: unlinks the thymine dimers (caused by ultraviolet radiation) , using the zyme photolyase that
binds the dimer in the dark, becomes activated in the light and then breaks the bond between the thymine’s.

Keep in mind
• Mutations have an important role in the infection process because pathogens can become resistant to
antibiotics through mutation.
• Bacteria depend on a balance between mutation and repair.
• Mutations can result from transposition of genes in the chromosome, point mutations, or frameshift mutations.
• Suppressor mutations can reverse the primary mutation.
• DNA can undergo spontaneous damage from hydrolysis or deamination and can also be damaged by chemicals
called mutagens.
• Repair of damaged DNA can be accomplished by excision repair or nucleotide excision systems in the cell.

Transfer of genetic information

Bacteria can transfer their genetic information by recombination. Virulence factors are genetically controlled and the
transfer of their genetic determinants can make harmless bacteria dangerous.
Recombination can occur in four ways of which three, transformation, transduction and conjugation, happen form
bacterium to bacterium, and one, transposition, shuffles the genes into a different order.

Transposition
In transposition transposons are randomly moved to other places on the chromosome. This can have negative and
positive consequences, but as negative ones usually cause cell death these are rarely seen.
In bacteria carry transposons genes for promoting resistance to antibiotics.

Transformation
When a cell dies its DNA can be released and taken up by bacteria to be combined with their DNA. The recipient cell must
be competent, the wall must allow uptake of large molecules. Some bacteria are naturally component and some after
chemical treatment. It is thought competence occurs via Ca2+ ions that shield the charge of the DNA to be able to move
through the membrane.
The process of transformation is very inefficient because only a small part of the competent cells take up DNA and when
they do it is only a small amount. In an experiment two strains of Streptococcus pneumoniae were used of which one
was encapsulated and causing pneumonia and one was nonencapsulated and harmless. Nonencapsulated bacteria did
not cause disease whereas encapsulated did. Killed encapsulated bacteria also didn’t cause disease but they did in
combination with live nonencapsulated bacteria. S. pneumoniae was then isolated and the cells where encapsulated.
This shows something from the death bacteria had been taken up by the live ones.

Transduction
In transduction genetic information is transferred using a bacteriophage. This can happen in both gram-negative and -
positive bacteria. The genetic information moves form one infected host to another, after which it undergoes
recombination into the host’s chromosome.
36

There are two kinds of transduction;

- Generalized transduction: this occurs random. A phage infects a bacterium, and the enzyme DNase (encoded by
the phage) gets activated. The DNase cleaves the DNA of the phage such it can be enclosed in viral particles, and
it cleaves the chromosomes of the bacterium (its host) in smaller pieces of which some also go into the viral
particles. When a viral particle infects a new cell the DNA from the vesicle recombines with the host DNA.
- Specialized transduction: this occurs specifically. The phage DNA gets incorporated in the host DNA after which
it is called a prophage. After a while the prophage excises itself from the host DNA to get enclosed in viral
particles. Usually the excision only contains prophage DNA but sometimes it also contains bacterial DNA. The
infected bacterium lyses, releasing the viral particles of which some contain only viral DNA and some both viral
and bacterial DNA. When a particle containing both types of DNA infects a new bacterium there is
recombination of the two.
Phage conversion is a development in pathogenicity or virulence which is related to transduction.

Conjugation
Conjugation requires direct contact between a donor and the recipient cell. It occurs in both gram-negative and -positive
bacteria but in gram-positive ones direct contact is the sticking together of the cell walls of two bacteria whereas in
gram-negative there has to be formation of a bacterial sex pilus on the donor cell. This pilus serves as a bridge and pulls
the cells closer to each other. The conjugation takes place in multiple steps;
- Recognition by the sex pilus on the donor cell, of the a receptor site on the wall of the recipient cell.
- An enzyme of the donor cell cause the plasmid DNA to unwind, starting a the origin of transfer.
- One of the single strands of the plasmid DNA stays in the donor cell and one goes to the recipient cell.
- Both strands are replicated after which both cells contain identical plasmids.
The recipient cell can now serve as a donor which can conjugate with another recipient cell.
Cells can also spontaneously lose their plasmid, then the cell is cured.
A replicated plasmid in the recipient cell can become incorporated into the chromosome, at the presence of
recombination sites on the chromosome, which are similar to the insertion sites in transposition. Such a cell is then called
a Hfr cell (high frequency of recombination). By being incorporated the DNA is not added but replacing an existing
segment which is then destroyed. Most cells cannot receive DNA from Hfr cells so they cannot pass the information they
receive to other cells.

Keep in mind
• Genetic recombination occurs in bacteria through transposition, transformation, transduction, or conjugation.

• Transposition is a specific form of recombination in which genetic elements called transposons move from one
place in the chromosome to another.
• Transformation involves the uptake of naked DNA from one cell to another.
• Transduction is caused by a virus transferring pieces of DNA from one cell to another.
• Conjugation occurs when DNA is moved from a donor cell (designated F+) to a recipient cell (designated F–).
• Each of the transfer mechanisms causes genetic recombination in the recipient cell and thus can be important in
making a pathogen more dangerous.

Genetics and pathogenicity

Genes for toxins and antibiotic resistance are found on plasmids so they are easily transported to other bacteria through
conjugation.
Dissimilation plasmids contain information to become more resistant to disinfectants and to be more able to adapt to
destructive environments

Summary
• DNA is the informational molecule of the cell.
• DNA is a double-stranded molecule made up of nucleotides (which consist of a phosphate, a deoxyribose sugar
and one of the four bases adenine, thymine, guanine, or cytosine).
• Nucleotides are bound together through complementary base pairing.
• RNA is a single-stranded molecule and contains uracil instead of thymine. It can be found in the form of
messenger RNA, transfer RNA, or ribosomal RNA.
• DNA is faithfully replicated by the enzyme DNA polymerase.
• DNA polymerase has a proofreading capability that prevents errors in replication.
• Replication occurs at the replication fork (a separation of the DNA strands) and is continuous on one strand and
discontinuous (made in pieces) on the other.
37

• DNA is transcribed into RNA by the enzyme RNA polymerase.

• The genetic code is based on codons, which are combinations of three nucleotides.
• Gene expression is the process of making a functional product by transcription and translation; it is highly
regulated.
• Mutations are changes in the DNA and are important in infectious disease because they can lead to antibiotic
resistance.
• Genetic recombination can occur in bacteria through transposition, transformation, transduction, or
conjugation.

Chapter 12
Virus structure
Viruses contain either DNA or RNA, which is enclosed by a protein coat called a capsid. Some viruses also have a cell
envelope made up of lipids from internal membranes or the plasma membrane from the host cell. There are many
different shapes and sized, both determined by the molecules of which they are constructed.

The virion
A virion is a mature infective viral particle. It has to withstand the environment before it infects a host cell and this
strength depends on the strength of its capsid. When entering the host cell, the virion has to shed the capsid.
So, the capsid gives protection against pH, temperature etc, together with the envelope. Interactions for making the
virion are reversible so it disassembles after entering the host.

The capsid
The capsid is made of subunits, capsomeres, and their binding gives structural symmetry. There three types of virus
shape; helical, icosahedral and complex.

Helical viruses
Helical viruses have a rod or filamentous shape. An example is the influenza virus which has a lipid envelope, a matrix
protein (a layer inside the envelope) and segmented RNA of which each segment is enclosed in its own helical capsid that
is held in place by nucleoproteins. The cell envelope consists of the glycoproteins hemagglutinin which serves in binding
and entering the host cell, and neuraminidase which serves in release from the host cell.

Icosahedral viruses
In icosahedral viruses, the capsid has 20 triangular faces. There are two types; simple and complex. In simple icosahedral
viruses, for example the Poliovirus, the capsid is made up of repetitive polypeptide capsomeres. Poliovirus infects via the
digestive tract and it can withstand harsh environments because of the stable protein-protein interactions in the capsid.
There is functional dichotomy in the capsid, it has to be strong to withstand harsh environments but it must also be able
to disassemble after contact with a receptor on the host cell.
In complex icosahedral viruses, for example the herpesvirus, proteins and lipids surround the envelope. In the
herpesvirus the virion contains three kind of proteins; those in the capsid, those forming the tegument (a protein layer
between the capsid and the envelope, and glycoproteins that form spikes on the envelope.

Complex viruses
Complex viruses are not helical or icosahedral.

Viral envelopes
Many viruses that infect humans and animals have an envelope which is formed when the viral glycoproteins and
oligosaccharides associate with membranes of the host cell. All envelopes contain a phospholipid bilayer, of which the
amount of lipids depends on the virus.

Envelope glycoproteins
Glycoproteins of the envelope are embedded in the phospholipid bilayer by domains of the proteins called spanners,
which associate with the bilayer with one part facing the capsid an one part facing away from the envelope. On the
exterior, the glycoprotein can form spikes for attachment to host cells.

Keep in mind
• Viruses are obligate intracellular parasites because they cannot live outside a host cell.
• Viruses come in a variety of sizes and shapes.
38

• Viruses contain either DNA or RNA but never both.

• The nucleic acid in a virion is surrounded by a protein coat called a capsid.
• The capsid is made up of repeating proteins subunits known as capsomeres.
• Some viruses are surrounded by an envelope composed of viral glycoproteins and oligosaccharides complexed
with host cell membranes.

The infection cycle

During the infection cycle of animal viruses, the host is used for the production of virions and when the cell is completely
filled with virions they are released through exocytosis or by lysis.
When the virus is in a dormant state, latency, in which the genome of the virus and of the host cell combine to make a
provirus. The host cell still doesn’t make new virions but by replicating, the viral genome is also incorporated into new
cells.
Bacteriophages, that infect bacteria, also have two infection cycles. In the lytic infection cycle the bacteria are killed so
this can potentially be used in treating bacterial diseases, phage therapy.
The phages can also incorporate their genome into that of the host cell, producing a dormant state, lysogeny. Then a
prophage is formed.

The infection cycle of animal viruses contains multiple steps; attachment, penetration, uncoating, biosynthesis,
maturation and release.

Attachment
In attachment a virion binds specific receptors on the host cell. Some viruses only need interaction with one receptor, but
some also need a co-receptor, components on the host that interact with both the virus and the primar7y receptor.
Without these co-receptors the virus cannot complete the infection cycle.
The entry of a virus into the host cell needs the takeover of normal cell functions.
Sometimes causes the interaction between the virion and the receptor a conformational change in the capsomeres to
prepare for the uncoating. In other viruses the receptor only holds the virion in place until it has gained entry.

When virus meets host

Viruses can be highly specific or not at all when it comes to attachment to the host cell. Binding a receptor is not always
enough to cause infection. Besides having the right receptor, cell must also be permissive, containing all components
needed to produce new virions.
Usually, viral infections occur at the apical surface of epithelial cells and stay localized. When the virus is transported to
the basolateral surface it can go into the underlying tissues and spread to other sites in the body.
Many viruses only attach to lipid rafts, specific areas on the membrane that are rich in cholesterol, fatty acids and other
lipid so are more densely packed and contain less water. These areas are more reliable for stable attachment.

The rhinovirus causes infection of the upper respiratory tract. This rhinovirus binds to the glycoprotein receptor ICAM-1
on the host cell, which is an adhesion molecule that is normally involved in inflammatory responses. The inflammation
caused by rhinovirus increased the number of ICAM-1 receptors, allowing more binding of the virus to host cells. So, the
initial defensive response actually leads to more infection.

Receptor binding
For non-enveloped viruses, receptor binding is between a receptor on the host cell and structures on the viral capsid.
Enveloped viruses have an envelope around them, originating form host cells that were previously infected by the virus.
Proteins on this envelope interact with the receptor in the host cell. Influenza and HIV are both enveloped viruses. In
influenza hemagglutitin binds the host cell receptor (sialic acid). This hemagglutitin has a stalklike structure with a
globular protein, together forming a spike, that binds sialic acid. HIV also binds using spikes, these spikes bind the CD4
receptor on T-helper cells. In HIV binding of a co-receptor is needed.

Penetration and uncoating

After attachment to the host cell, the virion must gain access via penetration. First, the viral genetic material must lose its
capsid and envelope, uncoating.

Penetration and uncoating by non-enveloped viruses

When uncoating takes place at the plasma membrane, it is an easy process but when it occurs inside the host cell it is
harder because all viral particles are too large to move across the membrane without help.
Non-enveloped viruses can use receptor-mediated endocytosis where after binding of the virion to the receptor, a pit
forms in the membrane of the host. Then the membrane encloses the virion to form an endosome. When first formed
39

these are early endosomes and after traveling through the cytoplasm they fuse with the membrane or become late
endosomes (endosomes that have been in the cell for a while). The environment in late endosomes is more acidic than in
early endosomes which causes the start of uncoating of the virion, which is completed after fusion of the late endosome
with a lysosome.
Some non-enveloped viruses form a pore in the host plasma membrane. Binding of the virus to the receptor causes
conformational changes of the capsomeres such that they become hydrophobic and are attracted to the plasma
membrane. Then the hydrophobic part of the virus is inserted in the membrane forming a pore through which genetic
material can enter the cell.

Penetration and uncoating by enveloped viruses

With enveloped viruses, the envelope can fuse with the host cell membrane. This fusion is mediated by fusion proteins
and results in the formation of a fusion pore which permits the virion to move across the membrane into the host cell.
Sometimes the binding of the virion to the receptor causes a conformational change in which fusion proteins are
expressed and in other cases the presence of co-receptors is needed for fusion to take place. In for example HIV a co-
receptor is needed, the CD4 receptor on T-helper cells. All co-receptors for HIV are normal surface receptors for
chemotactic cytokines.

Cytoplasmic transport of viral components

With viral infections there is compartmentalization in which the synthesis of RNA/DNA, viral proteins and capsid proteins
occurs in specific locations in the host cell. After synthesis, all compounds move to other sites for assembly of the virions.
There are two ways in which viral components can move through the host cell;
- Using membrane-enclosed vesicles in conjugation with host cell cytoskeletal structures.
- Direct association of viral compounds with cellular transport mechanisms
Sometimes chaperones can be used to facilitate the movement through the cell.

Transport of the viral genome into the nucleus

Some viruses need to be in the host’s nucleus for their replication so they have to penetrate the phospholipid bilayer
that surrounds it. This involves pores in the nuclear membrane that are normally usd by host cell proteins.
DNA viruses are too large to get into the nucleus intact so while moving through the cytoplasm the capsid is removed
and only the viral genome enters the nucleus.
Retroviruses are RNA viruses that carry the enzyme reverse transcriptase. This enzyme converts the viral RNA into a DNA
copy that enters the nucleus to integrate in the host’s chromosome. This occurs with breakdown of the nuclear
membrane during mitosis.

Keep in mind
• Viruses can go through a lytic cycle in which the host cell fills with virions and bursts.
• In some cases, viruses cause a lysogenic infection in which the viral genome becomes integrated with the host
cell’s chromosome.
• Host cell receptors used by viruses represent a small fraction of the cell membrane proteins.
• Many different viruses can share the same receptor.
• Virus–receptor interactions facilitate the infection process by enhancing virus entry into host cells.
• For enveloped viruses, penetration occurs through a fusion event between the viral envelope and the host cell’s
plasma membrane.
• This fusion is catalyzed by a specialized viral glycoprotein called a fusion protein, whose function is highly
regulated.
• Once fusion has occurred, the virus is released into the cytoplasm of the host cell, where various mechanisms
allow the uncoating of the virus.
• Penetration mechanisms of non-enveloped viruses are thought to be through host cell endocytotic pathways
that are routinely used by host cells for the normal importation of molecules.

Biosynthesis
DNA viruses – replication
Viruses that have DNA genomes can use mechanisms of the host cell for the replication of their genome. Replication of a
viral genome required synthesis of at least one viral protein and expression of several viral genes, so the replication start
immediately after infection but has to wait until a sufficient amount of these components has been made.
The virus inhibits the DNA replication of the host cell to get all proteins involved in replication contributing to the viral
DNA synthesis.
40

In the nucleus of the host cell, compartments are formed that contain both the templates and the replication machinery.
These compartments are required for the viral DNA synthesis to take place. It allows exponential replication, increases
the local concentration of proteins involved in replication and allows new DNA to immediately serve as a template.
This localized high concentration of templates and proteins is also useful for gene expression because these are also
needed for transcription.
The areas do not form randomly.

DNA viruses- transcription

Transcription of viral DNA is done by the host’s RNA polymerase. Some viruses can bring in their own RNA polymerase.
Single- and double stranded viruses undergo a different process.

Double-stranded DNA viruses

When the genome of the viruses is double stranded, it is in the same conformation as the host’s DNA so transcription can
start immediately after entering the nucleus and one strand is transcribed.

Single-stranded DNA viruses

When a virus genome is single-stranded, it must first be converted to double strands, using the host cells DNA
polymerase. After this the new strand is used for transcription.
The transcription of the virus DNA is coordinated with synthesis of the viral DNA, because proteins needed for the
replication are made first and those needed to assembly the virion are made next so only after the viral DNA synthesis
has began. This means there is only production of proteins to build virions when there us newly synthesized viral DNA to
put in them. This is different from RNA viruses because in these all genes are expressed continuously so all at the same
time.
When a cell is infected with a DNA virus, the host cell transcription stops and part of the transcriptional machinery is
converted to be used by the virus. In this way the virus can use the resources the host would normally use for this, and
there is no competition for translation.

Keep in mind
• Replication of DNA viral genomes uses the same mechanisms as the host cell but also requires several viral
proteins.
• Viral DNA replication occurs at specialized sites in the host cell, which contributes to the efficiency and
productivity of the viral infection.
• Transcription of dsDNA genomes can begin immediately that viral DNA enters the nucleus; however, for ssDNA
genomes, the single strands must be converted to double strand before transcription can begin.
• Transcription of viral DNA is performed by the host cell’s RNA polymerase (except for poxviruses, which bring
their own RNA polymerase).
• Viral genomes are transcribed at a very high rate, to make as many new virions as possible.

RNA viruses – replication and transcription

RNA strands can be (+) or (-), depending on if they can be directly used as mRNA. The original strand is used as a
template to make a new strand. When this new strand is used as a template, the sequence of this new one will be the
same as the original strand. This means the first new strand can be used as a template for synthesizing copies of the
original strand.

Double-stranded RNA viruses

The genomes of double-stranded RNA viruses have both a (+) and a (–) strand. Viral RNA polymerase copies the (-) strand
into mRNA that is used for the production of viral proteins and it can be used as a template make a double stranded
genome for the new viral particles.

(+) Single stranded RNA viruses

This (+) can be immediately used as mRNA and can be directly translated. The replication consists of two steps; copying
of the strand into a (-) strand and using this (-) strand as a template to produce more (+) strands for the new virions.

(-) Single-stranded RNA viruses

This (-) strand cannot directly be used as mRNA, first a (+) strand should be made using viral RNA polymerase. This new
(+) strand is mRNA and can be used as a template for making more (-) strands for new virions.

Retroviral transcription and integration

41

Retroviruses have an RNA genome and contain the enzyme reverse transcriptase, and RNA-dependent DNA polymerase.
Each virion contains 50-100 copies of this enzyme. Once a virus has uncoated and nucleotides become available, reverse
transcriptase starts, in the cytoplasm, and new DNA copies go to the nucleus to integrate in the host’s chromosome. The
viral RNA is then degraded because the template is not needed anymore.

Viral control of translation

During infection, viruses use the host’s translational machinery and often this machinery is then modified such that it
prefers viral RNA. This modification can cause a signal to the host’s defenses. The host has two genes coding for enzymes
that prevent RNA (both host and viral RNA) from binding with ribosomes and stop protein synthesis. This will cause the
cell to die but sometimes that is better than keeping the viral infection. Many viruses have proteins that neutralize this
host defense.

Keep in mind
• RNA virus replication is more complicated than DNA virus replication.
• RNA viruses have either single-stranded or double-stranded RNA.
• Single strands can be (–) or (+).
• In all cases, RNA viruses use a template strand of RNA to make new viral genomes.
• Retroviruses are RNA viruses that contain the enzyme reverse transcriptase.
• Reverse transcriptase can convert RNA into DNA, which can than be integrated into the host cell’s
chromosomes.

Maturation
During maturation, the new viral components move to specific sites in the host cell, intracellular trafficking, where they
will assemble.

Intracellular trafficking
Because some components are made in the nucleus and some in the cytoplasm they have to be brought together. Some
components have to travel a great distance and then simple diffusion is not useful and microtubules have to be used,
which costs a lot of energy. For the virus this is not a problem because it uses energy form the host cell.
The sites where the components go to, to be assembled, are determined by some factors;
- Whether the virions will have an envelope: viral envelopes are derived from the plasma membrane so
enveloped viruses will assemble adjacent to the membrane. Sometimes a envelope is not derived from the
plasma membrane but from a specific organelle. Then the site is near this organelle.
- The type of genome, RNA or DNA
- The mechanism of genome replication
Sometimes the assembly of non-enveloped viruses is in the cytoplasm and sometimes in the nucleus. When it takes place
in the nucleus all needed proteins have to be transported to the nucleus.

From their site of assembly, the viral proteins go to the cell surface through vesicles. First, the proteins go from the ER to
the Golgi apparatus, where they.. p.286

Keep in mind
• Virus replication is a complicated process in which the host cell manufactures new viral components from the
template provided by viral DNA and RNA.
• Viruses are completely reliant on host cell machinery for translation.
• Intracellular trafficking is crucial for viral reproduction.
• Intracellular trafficking requirements can be quite complex, with transport of viral macromolecules over long
distances in the cell.
• The assembly of different viral components occurs at different sites and requires that all viral proteins be sorted
in the Golgi.

Assembly
To be able to do reproduction, all steps of assembly should be completed. These steps are different for enveloped and
non-enveloped viruses.
Non-enveloped;
- Formation of structural subunits for the capsid
- Assembly of the capsid
- Association of the viral genome within the capsid
Enveloped;
42

- Formation of structural subunits for the capsid

- Assembly of the capsid
- Association of the viral genome within the capsid
- Assembly of the viral envelope glycoproteins

Assembly of capsid subunits

The capsid is made up of capsomeres. The assembly of these capsomeres differs between RNA and DNA viruses, and it
there are several mechanisms. There are always way more capsomeres produced than needed because they have to find
each other in the cytoplasm. This, in combination with locating the production of the subunits at distinct assembly sites,
increases the chance on random interactions and so on successful virion assembly.
Without assembly of the capsid there will be no infection.

Assembly of the viral genome

The virion is assembled during synthesis of the viral genome or the genome is inserted into a already formed capsid.
Empty capsid can play an important role in the defense of the virus against the host’s immune response.

Release
Release of the new virions can take place in two ways; lysis of budding off. In lysis the host cell dies immediately and in
budding, the host cell remains alive while releasing the virions.

Budding from the plasma membrane

The process in which enveloped virions bud off form the plasma membrane consists of four steps; bud formation, bud
growth, fusion of the bud membrane and separation from the host. During bud formation and growth, the bud forms and
pushes against the membrane. To become separated from the host, the bud uses exocytosis.

In some viral infection, there is assembly of virions but these are non-infectious and immature. To be converted to
mature and infectious, viral enzymes are needed. This happens late during assembly or after release.

Keep in mind
• All virions complete a set of assembly reactions.
• Capsomere proteins are assembled first.
• The number of capsomeres produced is always far in excess of the number required for the number of virions to
be assembled.
• Some viruses use host cell proteins called chaperones to assemble viral capsids.
• New virions are released through lysis, which kills the host cell, or by budding from the host cell, which allows
the host cell to survive for a period.
• Some viruses are released in an immature non-infectious state and must be activated enzymatically before they
can infect host cells.

Spread of viruses
To infect a next host cell, which can be both near and far away, the virion has to leave its host cell and come into the
extracellular environment where it is susceptible for the host’s defense mechanisms (not when the next cell is very
close). The spread can also take place via syncytia, many infected cells fused into one gigantic cell. Using a syncytium, the
virion can move far without having to leave this cell and get exposed to the host’s defenses.
Besides this, some viruses can produce decoy virions, empty capsids or non-infectious virions which confuse/distract the
host’s defenses. Also, can some viruses keep proteins of the host in their membrane as camouflage.

Summary
• Viral structures include nucleic acids (either DNA or RNA), and a capsid protein coat made of capsomere
subunits.
• Some viruses are surrounded by envelopes composed of viral glycoproteins, oligosaccharides, and host cell
membrane lipids.
• Viruses may lyse the host cells by using a lytic infection cycle or they may be dormant in a latent infection.
• A lytic infection cycle involves the steps of attachment, penetration, uncoating, biosynthesis, maturation, and
release.
• Host cell receptors are used to facilitate virus attachment and entry into host cells.
• Synthesis of new viral components involves complex replication mechanisms of the viral DNA or RNA.
• Intracellular trafficking and assembly of viral components in the host cell occur during the maturation stage of
the viral infection cycle.
43

• Newly assembled viruses may be released through lysis of the host cell or by budding from the surface of the
host cell.

Chapter 13
Patterns of viral infection
Viral infections can be acute or persistent and there are several types of persistent infection.
Different viruses have different incubation periods, time between exposure to the pathogen and the onset of symptoms,
in which the virus is replication, and the host is starting a response.

Acute infections
Acute infections, for example influenza, polio and rhinovirus, have a rapid production of virions and rapid elimination of
the virus by the hosts defense.
When the innate immune response limits the infection, it is asymptomatic.
A problem with acute infection is the incubation period because by the time people get symptoms, they have already
transmitted the disease.

Antigenic variation
Usually, when a patients survives a acute infection he is immune to re-infection.
The adaptive immune response causes development of a memory but some viruses can escape because of structural
changes in their virions, antigenic variation.
There are two forms of antigenic variation; antigenic drift and antigenic shift. In antigenic drift there is a small change in
the structure, caused by mutations, occurring after the infection has begun. In antigenic shift there are large changes in
the structure, caused by getting new genes.

Persistent infections
When defenses are not working properly and cannot clear the virus from the host persistent infection can be caused.
Because of this these infections are often seen in patients after organ transplantation because of the immunosuppressive
therapy.
There are three types; chronic with continues and long-term production of virions, latent with no virion production and
slow infection with a prolonged incubation period followed by progressive disease and virion production.

Because the immune system only responds to certain peptides in the infected host cell, the cell becomes easily invisible.
Cytotoxic T-lymphocyte (CTL) escape mutants are viruses that mutate their peptides, which develop because of error-
prone replication and selective pressure.
When CTL escape mutants occur early, a persistent infection develops, which doesn’t happen when they don’t occur
early.
Some viruses only infect tissues with reduced immunosurveillance to avoid a CTL response. For example the human
papillomavirus infects a part of the skin that is terminally differentiated so where there is no local immune response.

Chronic infections
When a virus remains in the body indefinitely you get a long-term infection which is chronic. For example, Hepatitis C,
which causes liver problems but few symptoms.

Latent infections
Three characteristics of latent infections;
- Absence of a productive infection so no large-scaled production of virions
- Reduced or absent host immune response
- Persistence of an intact viral genome so that productive infections can occur later
Latent viruses can be re-activated, for example because of stress or trauma.

Slow infections
In slow infections are usually fatal brain infection, causing ataxia (loss of motor control) and dementia. There may be no
symptoms for a long period but when the first signs appear death usually follows quickly.

Keep in mind
• Viral infections can be acute or persistent.
44

• Viruses differ in their incubation periods.

• Most acute viral infections result in lifelong immunity.
• The process by which virions change structure is called antigenic variation.
• Persistent infections last for long periods and can be chronic or latent.

Dissemination and transmission of viral infection

A virus can only cause infection when having access to a susceptible and permissive host, so a host with the right
receptor (susceptible) and the gene products needed for infection (permissive).
In theory one virion is enough to start an infection but in practice we see many virions are needed, depending on the
virus, age, site of infection and health of the host.

Portals of entry
Respiratory tract
Viruses can come into the respiratory tract through breathing and can infect every part of the tract but in the lower tract
is more dangerous because of the risk of causing pneumonia. From the respiratory tract the virus can go to other parts of
the body.

Gastrointestinal tract
Because of stomach acid, bile, digestive enzymes, immunoglobulins and phagocytotic cells, the gastrointestinal tract is a
hard route of infection and viruses must be resistant, for example picornaviruses. For some viruses this harsh
environment is required for infection, for example reoviruses which use the digestive enzymes to be converted from
non-infectious to infectious particles.
M cells in the intestinal epithelium transport the viruses to lymphocytes and macrophages for destruction. Some viruses
evade this by entering the lymphatics or the blood stream and become systemic, other viruses stay in the M cells,
replicate and eventually destroy the M cells causing inflammation and diarrhea.

Genitourinary tract
The genitourinary tract is the entry point for sexually transmitted viruses. It has defenses in the lining mucous, but these
can be compromised. This tract can be used in both women and main to gain entry to underlying tissue, for example HIV
(infecting lymphocytes) and herpes (infecting sensory neurons) and disseminate to the rest of the body to cause
persistent infections.

Eyes
The inner surface of the eyelids and the sclera can be portals of entry for viruses. However, because of the constant
flushing of tears the chance for getting an infection is small, among others because tears contain IgA. If the cornea is
damaged, Herpes viruses can cause an eye infection that totally destroys the cornea and can cause latent infection of the
optic neurons.

Skin
The skin is the most effective barrier but can be a portal of entry when being broken, for example because of a bite of an
insect that also serves as a vector to transmits viruses. When using a vector, the virus avoids exposure to the
environment. When remaining in the epidermis the infection is local bt when moving to the dermis the infection can
become systemic because then it gets access to the blood and lymph.

Dissemination pathways
When a virion is released from the apical surface of the epithelial cell, a localized infection is causes but when released
from the basal surface into the underlying tissue it can spread systemically.

Bloodstream
Hematogenous dissemination happens via the blood stream and is the best route for dissemination. Viremia is when
there are virions present in the blood and active viremia when these are also replicating. Most viruses spend only
between 1-60 minutes in the blood but some travel in it for years, for example Hepatitis B and C. This makes sense
because they target hepatocytes and the liver is the major blood-filtering organ.

Nervous system
Neurons are present in the whole body, so they are often used by viruses to disseminate, for some viruses as primary
target of for some not.
The virions move through the neurons via microtubules. When the virions disseminate from neurons that are connected
to the spinal cord and brain, there will be severe effects like viral meningitis and encephalitis.
45

From the central nervous system, viruses can come into the periphery where they cause local infections. Beneficial for
humans is that this direction used most often and not the other way around.

Internal organs
Systemic infection mostly occurs via the liver, spleen or bone marrow. Viruses in the liver via the blood, infect the Kupffer
cells and then the hepatocytes via translocation. The liver tissue gets destroyed. In some places in the brain, the
basement membrane of the capillary epithelium is very thin and can be passed by viruses. They come into the choroid
plexus, where there is production of cerebrospinal fluid, from where they can disseminate through the central nervous
system.
Besides this, viruses can attach to blood vessel walls so they can invade organs easily or move out the vessels to other
tissues.

Viral transmission
Viral transmission can be in only one species or form animal to humans, zoonotic diseases.
In acute infection the transmission must be efficient so with enough virus particles. This is not needed in persistent
infection because the virions are produced continuously.

Transmission via the respiratory tract

Sneezing, coughing and contact with saliva can spread infection. In the nose there are large droplets and in the lungs
smaller ones. Large droplets fall on the ground more quickly. Smaller drops can stay in the air longer and because they
can more easily come into the alveoli they can cause more severe infections.
Sneezing is the best way of transmission but occurs less. Sneezing spreads way more droplets but the exposed individuals
have to be close by.
Geography and season influence transmission because respiratory infections are seen more often in winter because the
droplets can be kept in the air for longer and there are more cracks in the mucosa through which the viruses can enter.

Transmission via the epidermis

When systemic infections leave the blood vessels and result in the destruction of host cells, there will be skin lesions or
rashes. When these spots appear, the infection is already going, and the person had already transmitted it to others.
Transmission can also occur via the skin through direct contact.

Transmission via bodily fluids

Some viruses are present in semen and vaginal secretions, and sexually transmitted. Some of these viruses can also be
transmitted via blood.

Transmission via the fecal-oral route

Besides used by many bacterial infections, the fecal-oral route is also common for viral infections. Viruses that replicate
in the kidney can come into urine that can contaminate food and water supplies. This is not a good environment to be in
so the viruses should withstand this.

Fetal infection
When pregnant women have viremia, the fetus can be exposed to the virions which can cause developmental defects.
Besides this, babies can get infected during natural birth and by breastfeeding when the mother is infected with HIV.

Keep in mind
• Viruses are disseminated between tissues, prgans, and organ systems within a host.
• Viruses can be transmitted between hosts
• Portals of entry, including the respiratory, gastrointestinal and genitourinary tracts, and the yes and skin, allow
viruses access to the host’s body.
• For an infection to be established, there must be adequate number of virions, permissive host cells, and an
ineffective host defense response.
• Dissemination occurs through the blood stream, nervous system and internal organs.
• Viral transmission occurs via respiratory tracts, epidermis, bodily fluids, the fecal-oral route and fecal infection.

Virulence
Virulence is how fit a pathogen is to fight the host, so how harmful.
This can be measured in three ways;
- Lethal dose 50% (LD50): how much viruses is needed to cause the death of 50% of infected individuals
- Infectious dose 50% (ID50): how much virus is needed to infect 50% of a population
46

- Paralytic dose 50% (PD50): how much virus is needed to paralyze 50% of infected individuals.
Virulence can be influenced by age, gender, health and route of infection. Besides this, the virulence can be affected by;
- Changes in replication ability of the virus this can also affect the virulence
- Mutation or turning off of cytopathology genes, limiting destructiveness
- Change in host cell function required for the virulence

Virulence and host susceptibility

Some infections are less severe in the elderly because, for example because smaller alveoli so less are for infection and
weaker muscles that are less efficient in propelling virus particles over large distances by sneezing and coughing.
Besides age, also gender can play a role because men are more susceptible for viral infections and pregnant women
more to hepatitis A/B/E.

Vaccine development
Because there is no cure for viral infections, it is the best to prevent them with vaccination. With most viral infections,
the memory is lifelong and herd immunity is caused.
The first vaccine was for smallpox. Milkmaids did get all the severe symptoms other people got because they got cowpox
instead of smallpox. When stuff from their lesions were given to uninfected non-cow persons, they did get smallpox.
There are three kind of vaccines;
- Live attenuated: made up of intact viral particles that were mutated or showed poor growth. The vaccine
contains infectious virions so there is a chance of causing symptoms.
- Inactivated (killed): made up of a virus that is death or non-infectious which is safer because there are no
infectious virions.
- Subunit vaccine: made up of immunogenic parts if the virus. This type is the safest because there are no intact
virions present. However, because if this is can be less effective.
Vaccination can be active or passive. In active immunization, the antigen is given what causes an immune response
whereas in passive immunization an already-formed antiviral product like antibodies are given. Long-lasting immunity
only arises with active immunization.
Because the development of a vaccine costs hundreds of millions of dolors, there are little companies willing to take the
risk.

Recombinant DNA vaccines

In recombinant DNA (rDNA) technology genetic material from multiple sources is combined and novel sequences are
inserted in viruses. This method is way quicker then original methides and can be used when a new vaccine is needed
with an outbreak.
Sometimes a dial immunological response can be caused in which both antibodies and T-cells are produced in the
vaccinated animals.

Viral culture
To make vaccines, viruses have to be grown in cells so it is needed to grow cells outside of organisms, using cell cultures.
Some cell types are easy to grow while others are very hard. In all cells enzymes are used for the isolation of cells from a
tissue after which they are cultured in medium with nutrients and grow factors.
There are different kind of cell lines;
- Primary: have to be grown from an organ from scratch every time (monkey kidney)
- Semi-continuous: can be grown for some time until the cells are too old or differentiated (embryo kidney)
- Continuous: can be grown for a very long time (HeLa)

Viruses and cancer

Viruses are involved in 20% of cancer and there is a relationship between viruses and liver- and cervical cancer. This
malignancy is a side effect of infection.

Oncogenic viruses
Retroviruses are able to inactivate genes that suppress formation of tumors.
There are five oncogenic viruses; Epstein-Barr, hepatitis B/C, human lymphotropic virus and human papillomavirus (HPV).
Viral proteins of these viruses can override mechanisms for ensuring cell division only takes place when necessary,
resulting on uncontrolled cell growth. 3

Keep in mind
• Virulence refers to the capacity of a virus to cause disease.
47

• Virulence varies from one virus to another and can be affected by the route of infection, by the age and health
of the host, and in some cases by the sex of the host.
• Susceptible hosts can be infected and transmit the disease, but immune hosts cannot be infected.
• Vaccines have been very effective in limiting viral diseases.
• Vaccines can be composed of live attenuated virus, inactivated virus, or subunits of the virion (parts of the virion
that can elicit an immune response).
• There are strict requirements for vaccines, including minimal side effects coupled with maximum protection
from infection.
• Viral vaccine development and production rely on embryonated chicken eggs, on primary, semi-continuous, or
continuous cell lines; on adult or embryonic stem cells; or on recombinant DNA technology.
• Oncogenic viruses (oncoviruses) can induce cancer in humans.

Summary
• Viral infections can be acute or persistent.
• Persistent infections last longer and can be chronic or latent.
• Latent viral infections do not produce large numbers of virions, but these infections can be reactivated later and
release virions.
• Viruses can be disseminated (move to other parts of the infected host’s body) through the respiratory, digestive,
and genitourinary tracts as well as the nervous system and internal organs.
• Transmission of virus from one host to another can be through the fecal– oral route of infection, fomite
transmission, and iatrogenic mechanisms (by health care workers).
• Virulence varies from one virus to another and can be affected by the age of the host, the health status of the
host, and the route of infection.
• Vaccination has been very effective in limiting viral diseases.
• Vaccines can contain live attenuated virus, inactivated virus, or viral sub-units.
• Some oncogenic viruses (oncoviruses) have been implicated in the development of cancer.

Chapter 14
Parasites and their infections
There are two main groups of parasites; protozoans (single-celled eukaryotes) and helminths (multi-cellular worms).
Most are not able to infect humans and play a role in ecology, but some live in the host.

Significance of parasitic infections

Many people are infected by parasites, for example by Plasmodium/malaria or Ascaris.

Protozoan morphology and pathogenesis

Protozoans have a membrane-bound nucleus, cytoplasm and are often facultative anaerobe and heterotroph. There
often is a trophozoite (active) stage in which the protozoan is growing and reproducing, and a cyst (dormant) stage in
which the protozoan moves between hosts and is protected against environmental challenges. With most protozoa,
reproduction happens by binary fission but some undergo simple fission followed by sexual reproduction, gametogony.

There are four types of protozoa that can infect humans, classified by means of locomotion;
- Ameboids: move by pseudopods, bulges of cytoplasm
- Ciliates: move using cilia
- Flagellates: move via flagella
- Sporozoans: only exhibit motile structures in the gamete stage

The way in which protozoa are pathogenic varies.

Helminth morphology and pathogenesis

Helminth worms have elongated, cylindrical or flat bodies and can be 1mm-10m long. The body is covered by a cuticle
which can be smooth or has ridges, spines or tubercles, and at the and there can be suckers, hooks or plates.
A parasite has organs, like nervous and excretory systems.

There are three types of helminths that can infect humans;

48

- Nematodes (roundworms): cylindrical body with a dietary canal from mouth to anus. There are two types, the
ones stays in the gastrointestinal tract and use only host to complete their life cycle, and the ones that infect
blood and tissues and use multiple hosts.
- Cestodes (tapeworms): flat ribbon-shaped body with suckers and hooks. The neck region proglottids,
reproductive segments with both male and female gonads. There is no digestive tract, so nutrients are absorbed
across the cuticle. Some use one and some use two hosts to complete their life cycle.
- Trematodes (flukes): leaf-shaped body with a blind branched dietary canal (so with one opening) and two
suckers of which one is an oral sucker via which nutrients comes in and waste goes out, and one is a distal
sucker for attachment.

Most worms cannot increase in number while they are in the host. The severity of a helminth infections is dependent on
the number of worms, so on the number of repeated infections.
The worms are nourished by bodily fluids, lysed tissues and intestinal contents. They can cause loss of iron, compromised
organ function and cancer.
There is damaged caused by both the infection and the immunological defense. The cuticle and enzymes protect the
helminths against this defense responses. For example, Schistosoma incorporates host’s antigens in its cuticle for
protection.

Life cycles and transmission pathways of protozoans

There is variation in life cycles and ways of transmission, among others in the number of hosts that have to be infected
for the life cycle to be completed.

Parasites that use a single host

Some parasites need only one host to complete their life cycle and the transmission to another host depends on their
ability to survive in the external environment. For example, Ascaris lumbricoides produces eggs that are passed through
via the stool. They are not infectious and have to mature in the soil so it cannot be directly transmitted from host to host.

Parasites that use multiple hosts

Some parasites need multiple hosts to complete their life cycle, including a definitive host in which sexual reproduction
occurs, and an intermediate host in which asexual development occurs. Plasmodium, which causes malaria uses a
mosquito to infect humans. Sexual reproduction takes place in the mosquito, making this the definitive host and the
human the intermediate host.

Keep in mind
• Parasites can be protozoans or helminths (although not all protozoans and not all helminths are parasites).
• Parasitic infections affect hundreds of millions of people throughout the world and cause millions of deaths each
year.
• There are three classes of helminth that infect humans: nematodes (roundworms), cestodes (tapeworms), and
trematodes (flukes).
• Some parasites have a life cycle that involves a single host, whereas others use more than one host.
• Pathogenic mechanisms for both protozoan and helminthic infections vary and depend on the specific parasite.

Examples of protozoan infections

Malaria (Plasmodium)
Malaria is a parasitic infection of human red blood cells caused by Plasmodium, a sporozoan protozoan. There are four
Plasmodium species that malaria in different degrees, of which P. falciparum is the worst. The transmission;
- Depends on density and feeding habits
- Mortality mostly in infants and immunocompromised people
- Sometimes transmission is seasonal, then infection is seen in all ages

Life cycle of Plasmodium

A female mosquito (definitive host) ingests male and female Plasmodium gametocytes via the blood of an infected
person (intermediate host). Fertilization takes place and the zygote penetrates the gut wall to form an oocyst in whih
many sporozoites are formed that are released in the mosquito when the cyst bursts. Some sporozoites penetrate saliva
glands. When biting takes place, the sporozoites in the saliva come in the blood stream of the human, go to the liver and
attach to hepatocytes. In these cells they produce daughter cells, merozoites, which are released when the hepatocytes
rupture. Then they attach to red blood cells and undergo phagocytosis to come inside the cells, ring stage. When the red
blood cells rupture, the merozoites are released after which some go in destroying red blood cells and others transform
into the gametocyte form that circulates in the blood until ingested by a mosquito.
49

Pathogenesis of malaria
Symptoms; fever, anemia and circulatory changes (low blood pressure), caused by the destruction of red blood cells.
In P. falcipiarum, red blood cells stick to walls of capillaries in the brain, cerebral malaria, causing delusions, paralysis,
coma, and acute jaundice (yellow skin).
At the start of the life cycle there is a cold stage with shaking and chills, after which the temperature increases and the
patients reaches the hot stage with fever and sweating.

Treatment of malaria
The treatment consists of two factors; the Plasmodium species causing the infection and the immune status of the
patient. With P. falciparum, treatment should start as soon as possible. All three forms of the parasite should be
destructed, there is not yet a drug that does that.

Other examples of protozoan infections are;

- Taxoplasmosis (Toxoplasma gondii)
- Amebiasis (Entamoeba histolytica)
- Trichomonias (Trichomonias vaginalis)
- Trypanosomiasis (Trypanosoma species)

Keep in mind
• Protozoan parasitic diseases include malaria, toxoplasmosis, amebiasis, trichomoniasis, and trypanosomiasis.
• Protozoan parasites may use humans as intermediate host and another animal as their definitive host.
• Malaria (caused by Plasmodium species) is spread by the bite of Anopheles mosquitos.
• Toxoplasmosis (caused by Toxoplasma gondii) is spread by housecat feces.
• Amebiasis (caused by Entamoeba histolytica) is acquired by the ingestion of fecally contaminated water.
• Trichomoniasis (caused by Trichomonas vaginalis) is a sexually transmitted disease.
• Trypanomiasis (caused by Trypanosoma species) is spread by the bite if tsetse flies and kissing bugs.

Examples of helminthic infections

- Intestinal nematodes: Enterobiasis and Ascariasis
- Tissue nematodes: Trichinosis
- Cestodes
- Trematodes: Paragonimiasis, Clonorchiasis and Schistosomiasis

Keep in mind
• Nematodes cause tissue, blood, and lymph infections, and can be caused by intestinal nematodes, such as
Enterobius and Ascaris, and by tissue nematodes, such as Trichinella spiralis.
• Cestodes (tapeworms) are the largest intestinal parasites and have a scolex, which incorporates both muscular
sucking disks and in some cases attachment hooks called a rostellum.
• Trematodes (flukes) can infect the blood, liver, and lungs.

Fungal infections
Fungi play a role in the environment and in the production of food and other products.
Acute fungal infection are rare, they are mostly subacute or chronic.

Fungal structure and growth

Fungal cells contain organelles, a nuclei with linear chromosomes etc. The fungal plasma membrane differs from the
bacterial membrane because it contains ergosterol that makes it stronger. Also the cell wall is different, being composed
of the polysaccharides mannam (on the surface, antigenic), glucan (glucose polymers) and chitin (major component,
forms aerial hyphae).
Fungi are heterotroph and usually obligately aerobic, sometimes facultative anaerobic but never obligately. S
Some to sexual and some asexual reproduction. Asexual reproduction, canidia, happens by mitotic division and budding,
and sexual reproduction by spore formation, in which donor nuclei and recipient cells fuse and meisosis takes place.

Yeats and molds

There are two kinds of fungi; unicellular yeasts and multicellular molds. Yeasts grow by budding, by making blastoconidia
(buds). Molds form hyphae, extensions, that can intertwine to form a mycelium. Within hyphae septa are formed and
they contain multiple nuclei.
Part of the mycelium serves as an anchor and part as aerial hyphae, which contain the reproductive structures.
50

Dimorphism
Some fungi species can grow both as a yeast and as a mold, dependent on the environment, dimorphism. Yeast form
with in vivo circumstances so a temperature of 35-37 degrees and an enriched source of nutrients. Molds form in
ambient temperatures and minimal nutrients.

Keep in mind
• Fungi are mostly harmless commensal organisms that cause no problems for humans.
• Fungi are eukaryotes that have the sterol ergosterol incorporated in their plasma membrane and the
polysaccharides mannan, glucan, and chitin in their cell walls.
• Fungi are heterotrophic, metabolically diverse, and either aerobic or facultatively anaerobic.
• Fungi reproduce either sexually or asexually.
• Fungal growth can be in a mold or yeast form, but some fungi are dimorphic and can grow in either form
depending on the environmental conditions.
• Medically important fungi are classified and distinguished by ribosomal RNA typing.

Fungal infections (mycoses)

There are different types of fungal infections;
- Superficial mycoses: occur in non-living skin, hair and nails and are often caused by Trichophyton
- Cutaneous and mucocutaneous: occur in the skin, eyes, sinuses, oropharynx, external ears and vagina. An
example is oral thrush, caused by candida albicans.
- Subcutaneous: development of cysts and granulomas
- Deep mycoses: seen in immunocompromised patients and caused by inhalation or digestion of fungi or fungal
spores or via injection. The infection can move through the blood and lymph or moves to the skin

Pathogenesis of fungal infections

The pathogenesis of a fungal infection can be divided in three classes;
- Adherence: some species can adhere to mucosal surfaces by a surface adhesion molecule on the fungus and a
receptor on the target cell.
- Invasion: some fungi come in the tissue by a break in the skin and some by inhalation. For inhalation, spores
need to be small enough to go past the respiratory defenses. These small ones can stay in the air for a long time.
Some dimorphic fungi can change their morphology to become more invasive, for example Candida albicans can
grow hyphae to spread.
- Tissue injury: fungi don’t produce exotoxins inside but outside the host. The primary tissue injury is caused by
the host’s defense.

Keep in mind
• Medically important fungi can be divided into four categories of mycoses (diseases caused by fungi).
• Superficial mycoses do not involve tissue responses and include infection of the hair shafts and superficial skin.
• Mucocutaneous mycoses are associated with the skin, eyes, sinuses, oropharynx, external ears, or vagina.
• Subcutaneous mycoses are localized infections of the subcutaneous tissues.
• Deep mycoses can be localized or systemic, and are usually restricted to patients who are
immunocompromised.
• The pathogenesis of fungal infections involves adherence, invasion, and tissue injury.

Summary
• Parasitic infections in humans can be caused by protozoans and helminths.
• These infections affect hundreds of millions of people around the world, and millions die from these infections
each year.
• Parasitic protozoans are classified into four groups; ameboids, ciliates, flagellates, and sporozoans.
• Parasitic helminths are found in three classes: nematodes, cestodes, and trematodes.
• Medically important fungi can be divided on the basis of the types of infection they cause: superficial mycoses,
subcutaneous mycoses, mucocutaneous mycoses, or deep mycoses.
• Deep mycoses are the most serious fungal infections and can be either localized or systemic.

Chapter 19
51

Source of antibiotics
Antibiotics are part of bacterial self-protein
Many microorganisms produce antimicrobial compounds to keep other microorganisms away. With this production they
use control mechanisms to prevent self-destruction. There are several ways to ensure their own safety against these
compounds;
- Only produce these toxic compounds when they are not dividing.
- Keep the levels of the antibiotics they produce low in the cell and they are exported out of the cell in inactive
form to become activated by extracellular enzymes.
- Modify their own cell wall. Genes for this modification are clustered with the antibiotic producing genes so
when these are on, the others will be made automatically.

Keep in mind
• Penicillin was discovered accidentally
• Many microorganisms produce toxic substances as part of their survival mechanisms and have developed
methods to protect themselves from the antibiotics they produce.

Antibiotic spectra
The first antibiotics were natural compounds.
Broad-spectrum: effect on both gram-positive and -negative bacteria
Narrow spectrum: effect on just one group
They can have different effects at different doses and can be bacteriostatic or -cidal.

Antibiotic structure
Penicillin is a four-sided ring structure, a beta-lactam. Derivatives with this same structure are also called beta-lactams.
These contain an extra side chain which determines the activity, resistance to stomach acid and half-life time.

Penicillin can be bound in two natural forms; penicillin G and V.

Synthetic: Penicillin
Semi-synthetic: ampicillin etc.

Natural penicillin has a very narrow spectrum and is only effective against a small group of gram-positive bacteria.
Changing the side chain to ampicillin broadened the spectrum by including gram-negative bacteria. Also these semi-
synthetics can be modified even further to increase efficiency.

Keep in mind
• Antibiotics are classified by their spectra of reactivity, either broad or narrow, and their effect, cidal or static.
• Penicillin is composed of a core ring structure known as the beta-lactam ring.
• Natural penicillin is found in two forms, G an V, both of which have a narrow spectrum of activity.
• Modification or addition of side chains to the core structure of naturally occurring antibiotics can increase their
effect and their spectrum of activity.

Antibiotic targets
One criteria for antibiotics is that they should have selective toxicity, so being toxic for the pathogen but not for the host.
To minimize toxicity, antibiotics were created against specific bacterial targets, divided into five groups;
- Bacterial cell wall
- Bacteria plasma membrane
- Synthesis of bacterial proteins
- Bacterial nucleic acids
- Bacterial metabolism

The bacterial cell wall

Because the cell wall is found in both gram-negative as -positive bacteria, and not in human this is the best target.
Antibiotics that target the bacterial cell wall are; penicillin, cephalosporins, carbapenems and monobactams (all beta-
lactams) and glycopeptides.
- Penicillin: the cell wall is composed of peptidoglycan, which consists of NAG and NAM. These are crosslinked by
penicillin-binding proteins (PBPs). Penicillin can bind these proteins and prevent the crosslinking, resulting in a
weak cell wall.
52

Penicillin is the most active during the growth phase so the more rapidly the bacteria are dividing, the more
severe the effect.
Because gram-negative bacteria only have a thin layer of peptidoglycan, these are less sensitive.
Beta-lactamase breaks can break open the beta-lactam ring by hydrolysis, causing resistance.
- Cephalosporins: have a greater effect on gram-negative bacteria, they are naturally broad spectrum. They are
less susceptible for beta-lactamase. Cephalosporins can go through porins in the outer layer of gram-negative
bacteria. The side chains are continuing to be modified, increasing the spectrum.
- Carbapenems: are even less susceptible for beta-lactamases, they reduce their activity by binding them. One
form that is currently used is imipenem.
- Monobactams: have a different structure and are not susceptible for beta-lactamases. They are only effective
against gram-negative bacteria.
- Glycopeptide antibiotics: for example Vacomycin (derived from Streptomyes), inhibit synthesis of the cell wall,
not in the same way as beta-lactams but by binding the amino acids making up the peptidoglycan molecules.
They are only effective against gram-negative bacteria.
- Peptide antibiotics effective against mycobacteria: mycobacteria (causing tuberculosis and leprosy) have
modified cell walls by incorporation of mycolic acids, waxy components that are protective and cause resistance.
- Polypeptide antibiotics: used for superficial infections of gram-positive and -negative bacteria and inhibit the
transport of building blocks. For example, Polymyxin B modifies cell wall permeability.

The bacterial plasma membrane

The bacterial plasma membrane is involved in many functions, making it a good target, but is quote similar to the human
plasma membrane so the antibiotics against the plasma membrane need restrictive use.

Synthesis of bacterial proteins

Both bacteria and eukaryotes have ribosomes, but these are very different, solving the problem of selective toxicity.
Ribosomes in the mitochondria of eukaryotes are similar to prokaryotic ribosomes so there can be some antibiotic
interference.
Macrolides act by blocking elongation of the peptide chain on the 50S subunit, inhibiting translocation of the ribosome.
They have a narrow-spectrum and are effective against gram-positive bacteria and used against respiratory infections.
Tetracyclines are bacteriostatic and block arrival of tRNA at the A-site. Unfortunately, many bacteria have become
resistant against them and there are side effects.
Aminoglycosides target the 16S part of the 30S ribosomal subunit and work in combinations with other antibiotics. They
are effective against gram-negative and not against -positive. They cause side effects can only be given by injection.

Bacterial nucleic acids

Because any disruption in the function nucleic acids results in, these would be perfect targets but a big problem is the
selective toxicities, because they are the same in bacteria and humans. However, quinolones and rifamycins have been
found, which are harmful to bacterial nucleic acids but not to the same in humans.
Quinolones block DNA replication and repair. In replication, the enzymes topoisomerase and gyrase make cuts in the
DNA, allowing it to uncoil and separate. Topoisomerase has a different structure in bacteria so using this as target gives
selective toxicity.
Rifamycins bind to RNA polymerase in an allosteric way (not at its active site) what changes its shape, making it
dysfunctional. No protein synthesis is possible, which is lethal. All rifamycins have selective toxicity because human RNA
polymerase is 100 times less sensitive.

Bacterial metabolism
Several metabolic pathways are specific for bacteria so interrupting these would inhibit bacterial growth without
affecting the host. Folic acid is needed for the synthesis of nucleic acids and an intermediate in this pathway is PABA.
Some drugs (sulfa) inhibit enzymes that incorporate PABA in the pathway and this is competitive inhibition because this
drug has a similar structure so its incorporated instead of PABA, what is lethal because folic acid is needed to survive.
There is selective toxicity because this is not the case in humans, they get folic acid from their diet.

Keep in mind
• Antibiotics must satisfy the criterion of selective toxicity, so they must react against the invading organism but
not against the host.
• Most antibiotics have some side effects.
• There are five targets for antibiotics: the bacterial cell wall, the bacterial plasma membrane, protein synthesis,
metabolic inhibition, and nucleic acids.
• The bacterial cell wall is the most easily selectively toxic because humans cells don’t have a cell wall.
53

• Nucleic acids and the plasma membrane are the least selectively toxic targets because they are very similar in
bacteria and in host cells.

Antiviral drugs
With viruses, are obligate intracellular parasites and there are some problems for therapy;
- many drugs would be dangerous or lethal for the non-infected cells as well
- viruses cannot be grown in the lab, making it hard to test potential antiviral drugs
- successful drugs must eliminate all virions because escape of even one into the blood can restart the whole
cycle

Different kind of antiviral drugs;

 Nucleoside analogs: are similar to nucleosides for DNA synthesis and are converted to nucleotide analogs in the
eukaryotic cell to be incorporated in the new chain, resulting in termination. However, the DNA of viruses and
oof the host cell is the same so there is no selective toxicity. This problem is not so bad, because viral DNA
polymerase is more likely to use to analogs than host cell DNA polymerase, so they are useful for viruses that
carry their own DNA polymerase like retroviruses and herpesviruses. Also, they are not effective in a latent
state, only when there is synthesis of the viral DNA.
Acyclovir is a nucleoside analog of guanine and is produced as a prodrug. It is activated by enzymes only present
in an infected cell, reducing selective toxicity.
AZT is activated by enzymes in a thymine analog and can be incorporated by reverse transcriptase to terminate
DNA synthesis. Because reverse transcriptase has no proofreading capability this is selectively toxic.
 Nucleotide analogs: do the same as nucleoside analogs but don’t need initial activation. An example is cidofovir,
a cytosine analog.
 Non-nucleoside analogs: also inhibit viral DNA polymerase but by allosteric inhibition so by binding the enzyme
away from the active site, inhibiting its function. An example is foscarnet but its toxic so only used as a last-
resort.
 Inhibiting viral assembly and release
- Protease inhibitors: are anti-retroviral drug that affect the viral protease enzyme. They cause
production of non-infectious viral particles. These inhibitors are very specific so there is the risk of
developing resistance.
- Neuraminidase inhibitors: target neuraminidase of the influenza virus and prevents budding off of new
virions.
 Inhibiting viral uncoating: Amantadine is a drug against influenza A, that binds viral proteins, inhibiting the
uncoating of the virion. However, the protein target often mutates so resistance develops.
 Inhibiting viral entry: before having gained entry, the virus is more accessible to drugs, these drugs inhibit
proteins needed for viruses to fuse with the plasma membrane.
 DRACO: (double-stranded RNA activated caspase oligomerizer) is selective for infected cells and causes
apoptosis based on the length of dsRNA. Uninfected cells don’t have long dsRNA so are unharmed.

Keep in mind
• Viruses present problems for therapy because they are obligate intracellular microbes; once inside the host cell,
selective toxicity is hard to achieve.
• Potential targets for antiviral drugs are specific viral proteins, which are selected on the basis of knowledge of
viral life cycles.
• Many currently available antiviral drugs target viral DNA polymerases, including reverse transcriptase, and viral
proteases.
• Viral entry and uncoating are also potential targets.

Antifungal drugs
Because of the increase in immunocompromised patients there has been an increase in secondary fungal infections.
Selective toxicity is often a problem in finding drugs because fungi are eukaryotes so many things are the same as in the
host. However, two differences are the composition of the plasma membrane and the presence of a cell wall.
 Antifungals that affect the plasma membrane: the fungal membrane contains ergosterol, which is not present in
the human membrane. This is directly affected by polyenes and indirectly by azoles ana allyamines.
- Polyenes: are produced by Streptomyces and interact with ergosterol, increasing the permeability.
There are lethal side effects like kidney failure, and it used intravenously so only in health care facilities
possible.
- Azoles: there are two classes: imidazoles and triazoles, both inhibiting ergosterol production.
54

 Antifungals that affect the cell wall: echinocandins inhibit synthesis of glucan of the cell wall.
 Antifungals that affect nucleic acid synthesis: flucytosine and pentamidine

Keep in mind
• Fungal infections have become more prevalent since the appearance of immunocompromised individuals,
especially in those infected with HIV.
• Because fungi and host cells are eukaryotes selective toxicity is hard to achieve and side effects of antifungal
drugs can be serious.
• Targets for antifungals are ergosterol in the fungal cell membrane, and the fungal cell wall, neither of which is
present in animal cells,

Drugs for parasitic infections

Protozoa are intracellular parasites, because of this many drugs are not selectively toxic and cause severe side effects.
 Anti-protozoan drugs: protozoa can produce cytokines to down-regulate the host’s immune responses. Two
targets of these anti-protozoan drugs are asexual reproduction and folate metabolism.
- Antimalarials: act against the asexual stage of Plasmodium while in red blood cells, destroying the
parasites. For example, chloroquine affects the pH what cause there to be more toxic metabolites that
interfere with the synthesis of nucleic acid.
- Folate antagonists: disrupt the folic acid metabolism, disrupting nucleic acid synthesis and repair
- Drugs for anaerobic protozoa: metronidazole
- Drugs for Trypanosoma and Leishmania
 Anti-helminthic drugs

Keep in mind
• The development of drugs that are useful against parasitic protozoans and helminths has been slow because
diseases caused by these organisms do not often occur in developed countries.
• Selective toxicity is hard to achieve because parasites are eukaryotes, like humans; many antiparasitic drugs
have serious side effects.
• There are several antimalarial drugs based on quinine, but they have similar resistance issues to antibiotics.
• Metronidazole is one of the most widely used anti-protozoan drugs and is also used as an antibiotic; it has good
selective toxicity.
• Ivermectin is used to treat several types of intestinal worm infection.

Summary
• Microorganisms produce toxic chemicals as part of their natural defense to protect themselves from other
microorganisms.
• The term antibiotic is generally used for chemicals that act against bacteria.
• Antibiotics can be broad or narrow spectrum, and have a cidal or static activity.
• Chemical modification of natural antibiotics can broaden their spectrum.
• Antibiotics and all anti-microbial compounds must be selectively toxic to be used therapeutically - they must
harm the pathogen but not the host.
• The five targets for antibiotics are the cell wall, the plasma membrane, the ribosome, nucleic acids, and
metabolic synthesis pathways.
• Viruses present problems for antibiotic treatment because they are obligate intracellular parasites, so selective
toxicity is hard to achieve once they are inside the host cell.
• Targets for antiviral drugs are viral DNA polymerases such as reverse transcriptase and viral entry into the host
cell.
• Selective toxicity is hard to achieve for antifungal drugs because fungi are eukaryotes.
• Drugs against parasitic protozoans and helminths have been slow to be developed because these infections
occur mostly in underdeveloped countries.
• Protozoans and helminths are also eukaryotes and present selective toxicity issues.
• Many antifungal and antiparasitic drugs have significant side effects
• There are many more antibiotics than there are antiviral, antifungal or anti parasitic drugs
• The choice of drug with which to treat an infection depends on several factors including; the route of
administration, side effects, and costs.

Chapter 20
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Development of antibiotic resistance

When increasing the amount of antibiotic present, there will be an evolutionary pressure for bacteria to become
resistant to this antibiotic. This relies on mutations. Many factors are involved in the development of antibiotic
resistance;
 Bacterial growth and mutation rates: because of the short generation time, there is an high potential for
mutations which can cause the ability to survival after exposure to antibiotics and when this antibiotic is present
there will be selected for these mutations. Because the resistant cells die, there will be a population that is
resistant because of vertical gene transfer.
 Plasmids and conjugation: via conjugation there can be transferring of plasmids between bacterial cells,
horizontal gene transfer. When the plasmid has a gene for resistance, the recipient bacterium will also gain
resistance. The plasmids with those genes integrate in specific sites in the chromosome, in resistance islands.
 Inappropriate clinical use of antibiotics: often virus infections cause cold-like symptoms, so they get a
prescription for an antibiotic, which won’t be affective against the virus. This, and not finishing a treatment,
contributes to development of resistance. Besides this, some people discard their unused drugs into water
sources, introducing them to the environment, creating pressure to develop resistance. Also, incorrect use of
antibiotics can destroy the normal flora in the body.
 Use of antibiotics in the food chain: antibiotics are used as feed additives to promote growth of food animals.
This can cause contamination of the environment and ground water, resulting in pressure for resistance. Also
can antibiotics in food affect the normal microbiota.
 Immunocompromised population: immunocompromised persons have increased needs for antibiotics.
 Health care facilities: in hospitals there is everything needed for the development of resistance like many
bacteria, immunocompromised people, use of antibiotics.
 Lifestyle: in large cuties there are more people is small aries, making the passing over of infections and of
resistant pathogens easy. This is made worse by less health care and poor sanitation. Also traveling plays a role.

Keep in mind
• Like the production of antibiotics antibiotic resistance is a natural phenomenon
• Mutations that confer resistance to antibiotics are selected for if there is an evolutionary pressure that makes
processing them an advantage by substantially increasing the chance of survival the more an antibiotic is used
the greater is the chance of resistance developing
• The genes for anti-bacterial resistance can be bought for one generation of bacteria to the next (vertically) and
between bacteria of the same generation and bacteria of different species horizontally
• Inappropriate use of antibiotics in clinical and non-clinical settings, is the major cause of rapid increase in
resistant populations of bacteria.
• An increase in a number of immunocompromised individuals and modern lifestyle are also contributory factors.
• Hospitals are ideal settings for the development and spread of antibiotic resistance.

Timeline of antibiotic resistance

Guidelines to decrease the chance on resistance;
- Optimal use of all antibacterial drugs
- Control of certain antibiotic classes and selective use of broad-spectrum antibiotics
- Use of antibiotics in rotation or cyclic patterns
- Use of combination therapy
- Evaluation of routes of resistance
- Implementation of global changes

Susceptible testing
In susceptibility testing involves;
- Culturing the infective agent
- Isolating and identifying each organism
- Creating a list of likely drugs, based on the identity of the pathogen
- Testing this list using the Kirby-Bauer method to determine the extent to which a drug affects the plated
pathogen. Any disk that inhibits pathogen growth is identifiable by the zone of inhibition around it.

The disk method is used to compare effectiveness of different compounds and various concentration of one. A larger
inhibition zone does not mean the compound is more powerful, there are differences in diffusion rate.
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The E-test can be used to determine the minimal inhibitory concentration (MIC), the lowest concentration of
antibiotic that prevents growth.

To know whether the pathogen is bacteriostatic or -cidal, the broth dilution test can be used, which determines the
minimum bactericidal concentration (MBC). This involves incubating an organism in wells containing decreasing
amounts of the antibiotic. Microbes that do not grow are recultured in nutrient broth without the antibiotic. When
there is growth this means the antibiotic inhibited the growth of the organism but did not kill it, so it is
bacteriostatic. When there is no growth the organisms was killed and the antibiotic is bactericidal.

Keep in mind
- Resistance can develop very quickly in less than a year in some cases.
- The time it takes organisms to develop resistance to antibiotic varies, but there is no doubt that the more an
antibiotic is used the more quickly resistance occurs.
- The therapeutic lifespan of an antibiotic is based on how quickly resistance develops.
- Steps can be taken to lengthen the therapeutic lifetime of antibiotics.

Mechanisms of resistance
Mechanisms or protection;
- Inactivation of the antibiotic
- Efflux pumping of the antibiotic
- Modification of the antibiotic target
- Alteration of the pathway
These methods counteract with methods used to find potential antibiotics because we look for targets that are lethal to
the bacteria while the bacteria have ways to change these targets.

Inactivation of antibiotic
When bacteria can activate an antibiotic, usually by enzymatic breakdown, it will be resistant against it. Beta-lactamase is
produced by bacteria to attack beta-lactam antibiotics. We want antibiotics that are not affected by beta-lactamases;
- Antibiotics with another structure
- Antibiotics augmented with lactamase inhibitors
Many bacteria have the AmpC gene which codes for beta-lactamase and is turned on by the presence of antibiotics
containing a beta-lactam ring. AmpC is not inhibited by lactamase inhibitors and its expression is controlled by other Amp
genes. AmpG proteins binds peptidoglycan fragments that were created when the antibiotic destroyed the cell wall, and
transports the fragments to induce transcription of AmpC. In this way, after the cell wall has been destroyed, beta-
lactamase is turned on to stop this destruction.
Mutations in Amp genes result in an overexpression of AmpC, causing resistance.

Efflux pumping of antibiotic

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