CN108265035A - A kind of method of evolution bacteriophage host specificity - Google Patents

Abstract

The invention belongs to the field of biotechnology, and provides a method for using a springboard host to evolve phage host specificity, the obtained springboard host and phage. The present invention starts from known phages with clear genetic background, adopts the concepts and means of evolutionary biology and synthetic biology, transforms the lipopolysaccharide structure in the phage host to obtain a springboard host, and infects the springboard host with phage to obtain a phage with a mutated receptor binding protein , to change its host specificity so that it can recognize and infect new specific host bacteria. Experiments show that the obtained phages can resist Gram-negative bacteria and multi-drug resistant bacteria, produce plaques, and inhibit their growth, and have the potential to supplement and strengthen antibiotics in the treatment of bacterial infections.

Description

A method for evolving phage host specificity

technical field

The invention belongs to the field of biotechnology, and in particular relates to a method for evolving phage host specificity by using a springboard host.

Background technique

Phages, a class of viruses that infect bacteria, are the most ubiquitous and diverse species in the ecosystem. Phages are widely distributed in areas where bacteria are concentrated, such as soil or animal guts. Seawater is also one of the most dense natural sources of phages, with up to 70% of marine bacteria infectable by phages. The characteristic of phage is that it does not have a cell structure, and is mainly composed of a single nucleic acid DNA or RNA genome wrapped in a protein shell, which can encode as few as several or as many as several hundred genes. Phage must use resources such as energy and protein in the host cell to achieve its own growth and proliferation, and cannot grow or replicate independently. Phage recognizes the host by specifically binding to receptors on the surface of bacteria, and then injects its own genome into bacterial cells, and its infection has a high degree of host specificity. Phages can be lytic or lysogenic, or both. A lytic phage such as T7 replicates rapidly after entering a bacterial cell, lysing the cell to release progeny phages, which then infect new host cells. Lysogenic phages do not immediately lyse the host cell, they can integrate their genome into the host's DNA and replicate without damaging the cell, they are only activated when the host condition deteriorates and then lyse the cell.

Phages can recognize and infect bacteria to treat diseases. Certain rivers, including the Ganges in India, are believed to have magical healing powers in local traditions, and researchers later verified that they may be due to the presence of bacteriophages in the river water. For a century, in the former Soviet Union (now Georgia, Poland, Russia and other countries), phages were often used as an alternative to antibiotics to treat infectious diseases caused by bacteria. However, phages have long since ceased to be used to treat bacterial infections in developed Western countries. There are several reasons for this: 1) Although medical experiments have been carried out, the lack of basic understanding of phages makes these experiments unreliable; 2) the discovery of antibiotics It is widely marketed, and antibiotics are easier to produce, preserve and prescribe; 3) The research in the former Soviet Union continued, but most of the research results were published in Russian or Georgian and have not been circulated internationally for many years; 4) The method of evaluating the antibacterial potency of phage preparations Clinical trials lack sufficient controls, and incomplete protocols prevent important conclusions. In June 2009, the Journal of Wound Care reported the first standardized randomized double-blind clinical trial, which evaluated the safety and efficacy of a phage mixture preparation for the treatment of patients with venous lower extremity ulcers. The US FDA approved the study as Phase I clinical trial. In August of the same year, the Journal of Clinical Otolaryngology reported a clinical trial in Western Europe, concluding that the use of phage preparations to treat human chronic ear infections caused by Pseudomonas aeruginosa is safe and effective. In addition, there are many animal and other clinical trials evaluating the therapeutic effects of phages on burn infections, lung infections, etc. At the same time, researchers are also developing engineered viruses to overcome bacterial resistance, and engineering phage genes to carry enzymes that degrade biofilms and bacterial cell walls.

The diversity of phages in nature is extremely high, but people's understanding of them is poor. The effect of using naturally isolated phages to treat bacterial infections is somewhat unpredictable, and it is also a challenge to transform and mass-produce unknown phages. Furthermore, host-specific evolution of phages directly based on wild-type hosts and phages is nearly impossible due to the existence of strong interspecies evolutionary barriers. Therefore, it is of great significance to change the host specificity of phage so that it can recognize and infect new specific host bacteria.

Contents of the invention

In view of this, the object of the present invention is to address the defects of the prior art and provide a method for evolving phage host specificity using a springboard host to change the host specificity of phage so that it can recognize and infect new specific host bacteria.

For realizing the purpose of the present invention, the present invention adopts following technical scheme:

A method for evolving phage host specificity by using a springboard host, transforming the lipopolysaccharide structure in the phage host to obtain a springboard host, thereby reducing the evolutionary barrier of the phage, and infecting the springboard host with a phage to obtain a phage with a mutated receptor binding protein.

Preferably, said modifying the lipopolysaccharide structure in the phage host is specifically knocking out genes related to lipopolysaccharide synthesis in the host.

Preferably, the phage is selected from at least one of T1, T2, T3, T4, T5, T6, T7, and P1 phages that recognize LPS on the surface of bacterial cells; the phage host is Escherichia coli; The lipopolysaccharide synthesis-related gene is selected from at least one of waaA, waaC, waaF, waaG, galU, waaO, waaR, waaU, gmhA, gmhB, and waaD.

Preferably, the infection is specifically mixing the phage with the springboard host and culturing on the culture medium.

The invention also provides the phage obtained by the method.

The present invention also provides a springboard host for bacteriophage, wherein genes related to lipopolysaccharide synthesis are deleted in the host cell.

Preferably, the host cell is Escherichia coli W3110; the lipopolysaccharide synthesis-related gene is selected from at least one of waaA, waaC, waaF, waaG, galU, waaO, waaR, waaU, gmhA, gmhB, and waaD.

The present invention also provides the application of the phage of the present invention in resisting Gram-negative bacteria.

Preferably, the Gram-negative bacteria are Salmonella Typhimurium and Acinetobacter baumannii.

The present invention also provides a method for inhibiting Gram-negative bacteria by mixing said phage with a substance containing Gram-negative bacteria.

The present invention also provides a pharmaceutical composition comprising the phage of the present invention.

It can be known from the above technical solutions that the present invention provides a method for utilizing springboard hosts to evolve phage host specificity, the obtained springboard hosts and phages. The present invention starts from known phages with clear genetic background, adopts the concepts and means of evolutionary biology and synthetic biology, transforms the lipopolysaccharide structure in the phage host to obtain a springboard host, and infects the springboard host with phage to obtain a phage with a mutated receptor binding protein , to change its host specificity so that it can recognize and infect new specific host bacteria. Experiments show that the obtained phages can resist Gram-negative bacteria and multi-drug-resistant bacteria, produce plaques, and inhibit their growth, which can supplement and strengthen antibiotics in the treatment of bacterial infections. Compared with the traditional means of directly transforming or evolving phages, the method of using a springboard host to evolve phage host-specificity described in the present invention greatly simplifies and promotes the process of transforming the host-specificity of phages, and has certain universality. Phage makes it important to recognize and infect new specific host bacteria

Description of drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings that are required in the description of the embodiments or the prior art.

Figure 1 shows the position of the knockout fragment of the waaC gene of the springboard host described in Example 1 of the present invention on the genome of Escherichia coli W3110;

Figure 2 shows the position (A) of the validation primers of the springboard host described in Example 1 of the present invention and the electrophoresis band (B) of the product of colony PCR with the primers, swimming lanes 1-5 are Kan resistance after homologous recombination PCR products of 5 single colonies randomly picked on the plate, lane 6 is the PCR product of colonies without homologous recombination (negative control), and lane 7 is DNAladder DL2000;

Fig. 3 shows the LPS structure comparison between the springboard host and the original host described in Example 1 of the present invention;

Fig. 4 shows the variation site of superphage described in Example 3 of the present invention compared with wild-type phage, wherein A is the partial sequencing result of gene12, and B is the partial sequencing result of gene17; the shaded part is the variation site;

Fig. 5 shows the phage plaques produced by superphages infecting target pathogenic bacteria-Salmonella and Acinetobacter baumannii in Example 4 of the present invention.

Detailed ways

The invention discloses a method for evolving phage host specificity by using a springboard host. Those skilled in the art can refer to the content of this article to appropriately improve the process parameters to achieve. In particular, it should be pointed out that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and products of the present invention have been described through preferred embodiments, and relevant personnel can obviously make changes or appropriate changes and combinations to the methods described herein without departing from the content, spirit and scope of the present invention to realize and apply the present invention. Invent technology.

For realizing the purpose of the present invention, the present invention adopts following technical scheme:

A method for using a springboard host to evolve phage host specificity, characterized in that the lipopolysaccharide structure in the phage host is transformed to obtain the springboard host, thereby reducing the evolutionary barrier of the phage, and the phage infects the springboard host to obtain a phage with a mutated receptor binding protein .

Lipopolysaccharides (lipopolysaccharides, LPS) are usually composed of lipid A (lipid A), a phosphorylated core sugar, and a long-range O antigen inserted into the outer membrane. Core sugar is conceptually divided into two regions: the outer core and the inner core. The inner core is highly conserved, contains KDO (deoxy-D-manno-octulosonic acid) and heptose (L-glycero-D-manno-heptose), and is usually phosphorylated. The inner core sugar plays a crucial role in the basic barrier function of the outer membrane. The outer core contains a trihexose backbone that is differently modified with hexoses and acetylhexosamines in different strains. The outer core provides the binding site for the O antigen.

The method for evolving phage host specificity by using a springboard host in the present invention artificially transforms the original host of the phage to simplify the lipopolysaccharide structure of the original host to obtain a new phage host as an evolutionary springboard, that is, the springboard host. Then, phages are used to infect the springboard host, that is, a new phage host with a changed lipopolysaccharide structure, so that the gene of the progeny phage expressing the receptor binding protein will be mutated, and the specificity of the phage’s recognition of the host will be reduced, so that it can be widely recognized. Infect new host bacteria.

Wherein, in some embodiments, the modification of the lipopolysaccharide structure in the phage host is specifically knocking out genes related to lipopolysaccharide synthesis in the host.

Further, the lipopolysaccharide synthesis-related gene is selected from at least one of waaA, waaC, waaF, waaG, galU, waaO, waaR, waaU, gmhA, gmhB, and waaD.

The waaA gene encodes a Kdo transferase that integrates the innermost two Kdo residues into lipid IVA, the precursor of lipid A; the waaC gene encodes the enzyme responsible for the transfer of the first heptose to the Kdo in the LPS core; the w aaF gene encodes The enzyme responsible for transferring the second heptose to the Kdo component of the LPS inner core; the enzyme encoded by the waaG gene is responsible for adding the first glucose from the outer core from UDP-glucose to the second heptose residue in the inner core; the galU gene encodes UTP:glucose-1-phosphateuridylyltransferase; waaO gene encodes α-1,3glucosyltransferase, which is responsible for adding the second glucose to the first glucose; w aaR gene adds the third glucose to the second glucose; waaU is responsible for the heptane Attachment of sugar IV to glucose III residues in the outer core; gmhA gene encodes Sedoheptulose 7-phosphate isomerase, which catalyzes the first key step in the synthesis of LPS core components; gmhB gene encodes D,D-heptose 1,7-bisphosphatephosphatase; waaD gene encodes ADP-L-glycero-D-mannoheptose-6-e pimerase, is the last enzyme in the ADP-heptose precursor synthesis pathway of core LPS.

In some embodiments, the phage is T7 phage; the host of the phage is Escherichia coli W3110; and the lipopolysaccharide synthesis-related gene is the waaC gene. The enzyme encoded by the waaC gene is responsible for the transfer of the first heptose to the Kdo component of the LPS core. By knocking out the waaC gene in Escherichia coli W3110, the W3110 cell LPS does not contain heptose but only has Kdo, and a new phage host is obtained. Then, the T7 phage was used to infect the springboard host, that is, the new phage host that infects LPS and only retains Kdo, and the gene of the progeny phage expressing the receptor binding protein is mutated to obtain the mutated phage T7waaC. By mixing the mutated phage T7waaC with a new host bacterium, a new phage capable of recognizing and infecting a new host can be obtained. For example, the mutated phage T7waaC was mixed and plated with the target host Salmonella and Acinetobacter baumannii to obtain STT7waaC capable of infecting Salmonella and AbT7waaC capable of infecting Acinetobacter baumannii.

Further, preferably, the infection specifically includes mixing the phage with the springboard host and culturing on the culture medium.

In some embodiments, the infection is that T7 phage is shaken and mixed with the springboard host at MOI=1, and after standing for 20 minutes, LB medium containing 0.6% agar at a temperature of 50° C. is added, shaken and mixed, and poured into The pre-prepared lower layer LB solid medium (agar content 1.5%) was left at room temperature for 1 hour, and after the upper layer agar was solidified, it was placed in a constant temperature incubator at 37° C. and incubated overnight.

The invention also provides the phage obtained by the method. The host specificity of this phage changed, and it could recognize and infect new host bacteria, and it was named superphage 1.0.

The present invention also provides a springboard host in which the genes related to lipopolysaccharide synthesis are deleted.

Wherein, the host is Escherichia coli.

The lipopolysaccharide synthesis-related gene is selected from at least one of waaA, waaC, waaF, waaG, galU, waaO, waaR, waaU, gmhA, gmhB, and waaD.

In some embodiments, the springboard host is Escherichia coli W3110 with waaC gene deletion

In a certain embodiment, the present invention mixes superphage 1.0 (such as mutated phage T7waaC) with Gram-negative bacteria such as Salmonella and Acinetobacter baumannii as target hosts, respectively, and plate them, and obvious phage plaques can be seen after culture. It shows that the super phage of the present invention can resist Gram-negative bacteria, produce phage plaques, and inhibit their growth. Therefore, the present invention also provides the application of the super phage in resisting Gram-negative bacteria.

Wherein, the Gram-negative bacteria may be common Gram-negative pathogenic bacteria, including but not limited to Salmonella and Acinetobacter baumannii.

Further, the present invention also provides a method for inhibiting Gram-negative bacteria, specifically mixing the superphage of the present invention with a substance containing Gram-negative bacteria.

The present invention also provides a pharmaceutical composition comprising the above-mentioned phage. Wherein the phage is the phage obtained by the method of utilizing springboard hosts to evolve phage host specificity, that is, super phage 1.0.

In order to further understand the present invention, the present invention will be described in detail below in conjunction with specific examples. Unless otherwise specified, the reagents involved in the examples of the present invention are all commercially available products and can be purchased through commercial channels.

The construction of embodiment 1 springboard host

The Kan resistance gene fragment was amplified by PCR using a pair of primers containing homology arms, and then the corresponding fragment of the waaC gene on the Escherichia coli W3110 genome was replaced by homologous recombination to obtain the correct waaC knockout strain. The primers containing the homology arms are

F: AGTTTAAAGGATGTTAGCATGTTTTACCTTTATAATGATGATAACTTTTC (SEQ ID NO. 1);

R: TACTGGAAGAACTCAACGCGCTATTGTTACAAGAGGAAGCCTGACGGATG (SEQ ID NO. 2).

The PCR reaction system is:

The PCR reaction procedure is:

95°C for 5 minutes;

28 cycles of 95°C for 30sec, 54°C for 30sec, and 72°C for 1.5min;

72°C for 10 minutes.

The method of homologous recombination is specifically as follows: first, Dpn I is used to treat the PCR product containing the homology arm and the Kan resistance gene fragment, and then the PCR product is purified by gel recovery; about 100 ng of the purified fragment is added to 100 μl of freshly prepared expression λ-red Escherichia coli electrotransformation competent cells with recombinant enzymes, mixed gently, transferred to the electroporation cup, and then subjected to electric shock (1.8kV); immediately after electric shock, 1ml LB medium was added, and the resuspended bacteria solution was recovered (37°C, 220rpm) After 2 hours, 20% was spread on the LB plate containing Kan; after culturing overnight at 37°C, the single colony grown on the resistant plate was verified by PCR.

The present invention designs verification primers for the upstream and downstream sequences of waaC to perform PCR amplification, and verifies that the obtained waaC knockout strain is a springboard host. Wherein said verification primers are:

F: ATGAGCCATATTCAACGGGA (SEQ ID NO. 3);

R: TTATCGACGAATGCAATTATCA (SEQ ID NO. 4).

The amplification results are shown in Figure 2B.

Figure 2B shows the electrophoresis bands of 5 single colonies and 1 wild-type colony (control) selected for PCR verification after successful homologous recombination. Amplified bands were seen in 2 of the 5 single colonies, and the amplified product was about 900bp (lane 1 and lane 3), and no amplified band was seen in 3 colonies (lane 2, lane 4, and lane 5). However, no amplified band was seen in the wild-type colony (lane 6).

The construction of embodiment 2 superphage

Shake and mix T7 phage with a plaque-forming unit (PFU) of 10 ⁷ /ml with the springboard host at MOI=1, let it stand for 20 minutes, add LB medium containing 0.6% agar at a temperature of 50°C, shake and mix Evenly, pour the pre-prepared lower layer LB solid medium (agar content 1.5%), let it stand at room temperature for 1 hour, and put it into a 37°C constant temperature incubator after the upper layer of agar solidifies. After overnight cultivation, usually 1-2 Phage plaques are called "Super Phage 1.0".

Example 3 Sequencing of Superphage 1.0

The products of genes11, 12, and 17 of T7 phage are the main receptor-binding proteins responsible for recognizing and binding LPS, wherein gene11 encodes tail tube protein A, gene12 codes tail tube protein B, and gene17 codes tail fiber protein. Pick the superphage 1.0 obtained in Example 2, use the springboard host to further amplify the culture, then extract the phage DNA, use PCR to amplify T7 phage and gene11, gene12, gene17 fragments of the superphage, perform Sanger sequencing after purification, and then compare The mutation sites of superphages were analyzed, and the results are shown in Figure 3.

The results in Figure 4 show that gene12 carries a base mutation (A is replaced by G), and gene17 carries a base mutation (G is replaced by T), both of which are sense mutations.

Example 4 Superphage Infects Pathogenic Bacteria

Shake and mix the T7 phage or superphage 1.0 with a PFU of 10 ⁹ /ml and the target pathogen at a ratio of MOI=10. After standing for 20 minutes, add 3 mL of 50°C LB medium containing 0.6% agar, shake and mix, Pour into the pre-prepared lower layer LB solid medium (agar content 1.5%), let stand at room temperature for 1 hour until the upper layer of agar is solidified, put it into a 37°C constant temperature incubator, and cultivate it overnight. The observation results are shown in Figure 5. The results in Figure 5 show that no phage plaques appeared in the control without adding phage and the control adding T7 phage, only the plate with superphage 1.0 could be observed, usually 1-2 per ten plates, the phage The phages in the plaques are the phages produced by superphage 1.0 after further evolution, which can recognize the surface receptors of the target pathogenic bacteria.

Claims (11)

1. A kind of 1. method using springboard host evolution bacteriophage host specificity, which is characterized in that in transformation bacteriophage host Lipopolysaccharides structure obtain springboard host, Phage Infection springboard host obtains the bacteriophage that receptor binding protein morphs.
2. 2. according to the method described in claim 1, it is characterized in that, the lipopolysaccharides structure in the transformation bacteriophage host is specific To knock out lipopolysaccharides synthesis related gene in host.
3. 3. according to the method described in claim 2, it is characterized in that, the bacteriophage is selected from identification bacterial cell surface lipopolysaccharides At least one of T1, T2, T3, T4, T5, T6, T7, P1 bacteriophage；The bacteriophage host is escherichia coli；The fat Polysaccharide synthesis related gene is in waaA, waaC, waaF, waaG, galU, waaO, waaR, waaU, gmhA, gmhB, waaD It is at least one.
4. 4. according to the method described in claim 1-3 any one, which is characterized in that described to infect specially bacteriophage and springboard Host's mixing, is cultivated on culture medium.
5. 5. the bacteriophage that claim 1-4 any one the methods obtain.
6. 6. the springboard host of a kind of bacteriophage, which is characterized in that lipopolysaccharides synthesis related gene lacks in host cell.
7. 7. springboard host according to claim 6, which is characterized in that the host is escherichia coli；The fat is more Sugared synthesis related gene in waaA, waaC, waaF, waaG, galU, waaO, waaR, waaU, gmhA, gmhB, waaD extremely Few one kind.
8. 8. application of the bacteriophage described in claim 5 in anti-Gram-negative bacteria.
9. 9. application according to claim 8, which is characterized in that the Gram-negative bacteria is motionless for salmonella and Bao Man Bacillus.
10. A kind of 10. method for inhibiting Gram-negative bacteria, which is characterized in that by bacteriophage described in claim 5 with containing leather orchid The material mixing of family name's negative bacterium.
11. 11. include the pharmaceutical composition of bacteriophage described in claim 5.