WO2001002555A1 - Method of making and identifying attenuated microorganisms, compositions utilizing the sequences responsible for attenuation, and preparations containing attenuated microorganisms - Google Patents

Abstract

A functional genomic approach for identification of mutants of microorganisms that are unable to grow under certain specific conditions is disclosed. In one aspect of the invention, a method is provided in which a library of signature tagged transposon mutants is constructed and screened for mutants attenuated in pathogenicity. The method is especially useful for identifying loci involved in pathogenicity. The method is well suited to identification of mutant actinomycetales, such as mycobacteria. The method is useful for, among other things, drug discovery and construction of vaccines.

Description

METHOD OF MAKING AND IDENTIFYING ATTENUATED

MICROORGANISMS, COMPOSITIONS UTILIZING THE SEQUENCES

RESPONSIBLE FOR ATTENUATION, AND PREPARATIONS CONTAINING

ATTENUATED MICROORGANISMS

BACKGROUND OF THE INVENTION

Field of the Invention

This invention relates to the field of genetic engineering and molecular biology. More particularly, this invention is directed to identification of genes or fragments of genes using the technique of signature tagged transposon mutagenesis.

Description of the Art

Bacteria of the Mycobacterium tuberculosis complex exhibit an extraordinary capacity to subvert and resist bactericidal responses of their hosts. This capacity has allowed mycobacteria bacilli to colonize one-third of the world population and kill nearly three million people annually (Dolin et al., 1994). During infection via the aerosol route, M. tuberculosis bacilli are inhaled into the alveoli of the lung where they reach and interact with mononuclear phagocytes, including macrophages. This interaction of M. tuberculosis with mononuclear phagocytes is part of the pathogenesis process. In vitro, M. tuberculosis has been shown to bind to many different phagocyte receptors, either directly or after opsonization, which allows internalization of the bacteria (Ernst, 1998).

It is unclear what ligands are involved in adherence to phagocytes in vivo. Indeed, many mycobacterial envelope molecules, as well as opsonins, can mediate the in vitro binding of M. tuberculosis to phagocyte receptors, but their availability at the infection site (for the host ligand) or their presence at the bacterial surface (for the mycobacterial molecules) is still unclear.

Once engulfed by phagocytosis, M. tuberculosis bacilli multiply within a specialized compartment called a phagosome, which does not acidify, possibly because of the exclusion of thei proton ATPase from the membrane of this organelle. The phagosome does not proceed through the standard maturation pathway and seems to be blocked at an intermediate stage of maturation (Clemens, 1996). This evidence indicates that M. tuberculosis has evolved virulence mechanisms allowing it to modulate the maturation of the phagosome and to resist the toxic molecules produced by the macrophages. However, the mycobacterial components responsible for these unusual features remain unknown.

The numerous studies concerning the interaction between M. tuberculosis and the host underscore the poor understanding of the mycobacterial factors and their role in the different steps of the infectious process. A genetic approach to identifying these mycobacterial factors would be to isolate mutants affected in one or several steps of the infectious process and to identify the compounds no longer produced, or produced in excess, by these strains.

Recently, major progress in investigation methods and genetic tools for mycobacteria have opened the way for such research. Hensel et al. (1995) described a method termed signature tagged transposon mutagenesis (STM) allowing the screening of large pools of mutants of Salmonella for those exhibiting an attenuated phenotype. Briefly, STM is a method in which mutants are generated by insertion of a transposon carrying a unique DNA sequence tag. A transposon is a translocatable genetic element that comprises large, discrete segments of deoxyribonucleic acid that are capable of moving from one chromosomal site to another in the same organism or in a different organism. A tag is a nucleotide sequence that can be used to identify and/or isolate nucleic acids to which it is linked. The tag can be labelled, for example with a radioactive isotope, although labelling is not required. Each clone can then be distinguished from others following detection of these tags by hybridization. These tagged mutants are pooled and inoculated within a host and allowed to multiply. After an appropriate time of infection, they are recovered, and those with an attenuated phenotype are identified by hybridization with the tags. This approach presents the advantage of allowing screening of a large collection of mutants in vivo with a limited number of animals. It was successfully applied to identification of virulence genes in four different pathogens: Salmonella typhimurium, Staphylococcus aureus, Vibrio cholerae, and Streptococcus pneumoniae (Hensel et al., 1995; Mei et al., 1997; Chiang and Mekalanos, 1998; Coulter et al., 1998; Polissi et al., 1998). This method requires an efficient insertional mutagenesis system for generating the tagged mutants and an animal model for the selection process. Two efficient transposon mutagenesis system were recently described that allow the construction of tuberculosis insertional mutant libraries (Pelicic et al., 1997; Bardarov et al., 1997). The difference between these two systems resides in the vector used to deliver the mobile element and to allow the selection of the transposition events. The first system is based on a plasmid carrying a thermosensitive replicon and the counter-selectable marker sacB (Pelicic et al., 1997). The second system uses thermosensitive mycobacteriophages unable to replicate at 39EC (Bardarov et al., 1997). Both vectors have been successfully used to deliver IS 1096 derivatives into the chromosome of bacteria of the M tuberculosis complex

SUMMARY OF THE INVENTION

A functional genomic approach for identification of mutants of microorganisms that are unable to grow in some specific conditions is disclosed. In the method of the invention, a library of signature tagged transposon mutants is constructed and screened for mutants attenuated in pathogenicity. As used herein, attenuated means weakened or lessened in degree, intensity, or detectability. The method is especially useful for identifying loci involved in pathogenicity. Knowledge of these loci is useful for drug discovery and for the construction of vaccines.

This invention provides a method of screening a library of mycobacterial insertional mutants for those with an attenuated phenotype. For this purpose, the signature tagged transposon mutagenesis (STM) method was adapted to mycobacteria using the mutagenesis system developed by Pelicic et al. (1997). Accordingly, the invention enables the identification of loci responsible for pathogenicity. The products encoded by these loci can be used as targets for antibiotics and subunit vaccines. In addition, the loci can be targets for the construction of attenuated strains. Thus, the products of these loci, and their sequences, can be used for the identification of host target sequences useful for therapeutic intervention. The sequences can be useful for the identification of host receptors and targets of the pathogenic effects. The knowledge of these host targets can lead to the discovery of new molecules and identification of host modulators.

Accordingly, the invention provides a method of making mycobacterial insertional mutants, a library comprising such mutants, and mutants containing plasmids, which allows signature tagged transposon mutagenesis. By insertional mutants, we mean mutants in which an insertion element, such as a transposon, has been incoφorated into the genome of the cell. Insertion of the element creates a mutation at the site of insertion, thereby generating a mutant. The insertion element can be delivered to the cell to be mutated through as part a plasmid. Thus, the invention provides plasmids that can serve as the basis for mycobacterial insertional mutants.

The invention also provides attenuated mutants of mycobacteria, such as M. tuberculosis. The attenuated mutants can be those unable to multiply within the lungs of mammals.

Accordingly, the invention also provides compositions comprising mycobacterium strains, mycobacterial nucleic acids, or mycobacterial polypeptides. The compositions can be used as part of immunogenic compositions, such as vaccines. As used herein, an immunogenic composition is a composition that elicits an immune response in an individual receiving it. A vaccinogenic composition, or a vaccine, is an immunogenic composition that elicits a protective immune response.

The invention is further directed to methods of treating individuals with the mycobacterial strains of the invention. The methods of treating can be methods of immunizing, methods of vaccinating, and/or methods of enhancing an already active immune response.

In addition, the present invention is directed to kits containing the mycobacteria strains and compositions of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 depicts the design of plasmid pCG113, which allows signature tagged transposon mutagenesis of M. tuberculosis. A unique Spel restriction site is present within IS 1096:: km, allowing the insertion of oligonucleotide tags.

Figure 2 shows the results of introduction of MT103 and the selected mutant in the lung of mice. BALB/c mice were inoculated intravenously with approximately 5xl0⁵ cfu of either wild type M. tuberculosis MT103, the attenuated control M. bovis BCG, or the selected mutants. The number of viable bacilli within the lung was measured three weeks post-infection. The value indicated represents the mean + standard error of the mean (SEM) obtained with at least three different mice. The value between the brackets indicates the number of mice used.

Figure 3 shows the genetic organization of one virulence gene cluster. The thin black line represents the M. tuberculosis chromosome, and the numbers below this line provide a frame of reference with respect to the sequence disclosed by Cole et al., 1998. The thick horizontal arrows represent the open reading frames (ORFs) of this region, with the corresponding gene designations indicated below. The short sequences presented in the figure show the overlap between the stop (TGA, TAA, TAG) and start (ATG, GTG) codons of some of the ORFs of this region. The different motifs of these arrows are associated with the putative functions of the proteins encoded by these ORFs. The short sequences presented show that there is an overlap between the stop and start codons of many of the ORFs in this region, suggesting that there is translational coupling of the ORFs. The vertical arrows below the chromosome line indicate the different insertions obtained in this region and the name of the different mutants.

Figure 4 shows the organization of the fadD30-pks6 gene cluster. The thin black line indicates the M. tuberculosis chromosome and the numbers below this line are the positions of the different ORFs (according to Cole et al., 1998). The thick horizontal arrows represent the ORFs of this region. The short nucleotide sequence presented shows that there is an overlap between the stop (TGA) and start (ATG) codons of the fadD30 and pks 6 ORFs, suggesting translational coupling. The vertical arrow below the chromosome line indicates an insertional mutant obtained in this region.

Figures 5 A and 5B to 20 A and 20B represent the genomic sequences (and their corresponding amino-acids sequence) of the ORFs or adjacent ORF thereof which have been disrupted by the insertion of the IS 1096 transposon in the attenuated mutants MYC2251 to MYC2266 respectively.

The Figures 5A to 20A disclose the disrupted ORF or adjacent ORF and the corresponding peptide with its putative function (if known).

The Figures 5B to 20B represent the localization of the transposon IS 1096 insertion site. The sequence depicted in bold indicates the insertion site of the transposon.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

In a first aspect of the invention, a method of screening a library of mycobacterial insertional mutants for those with an attenuated phenotype is provided. The method preferably utilizes a modified signature tagged transposon mutagenesis (STM) procedure based on the system developed by Pelicic et al. (1997). The method can be used to study genes and gene fragments that are important for the pathogenicity of microorganisms, including, but not limited to, actinomycetales such as Mycobacterium species, for example, M. tuberculosis. Adaptation of the STM method to organisms such as actinomycetales, and in particular, M. tuberculosis opens the way to a broader use of this procedure to target genes important in other steps of the infectious process. The present invention covers bacterial mutants that carry an insertion leading to a specific phenotype. It also covers genes or fragments of a gene encoding a function. Among these functions is binding to other molecules, such as DNA regulatory sites or molecules that interact with the fragment.

The present invention is directed to a method comprising: a) constructing and utilizing an insertion mutant library of an organism of interest, b) growing the organisms of the mutant library in different and defined conditions, and c) identifying the mutants/strains by ordering tags in order to recognize the presence or absence of tagged mutants by hybridization of the sequences corresponding to the tags under stringent conditions. The present invention further relates to a process for identification of the mutated loci selected according to the invention, characterized by: a) determining the nucleotide sequences flanking the insertion, and b) comparing the sequence with the sequences available in data banks, including the i genomic sequences of M. tuberculosis. The present invention further relates to the use of the signature tagged transposon mutagenesis (STM) method to isolate mutants of actinomycetales.

The present invention further relates to the use of mutants according to the invention, wherein said mutants are characterized by a specific phenotype, in particular, their growth in different environmental conditions or in attenuated phenotypes. The present invention further relates to a method according to the invention, wherein the insertion mutant library is in actinomycetales, in particular mycobacteria.

The present invention further relates to a process for assaying a library of mutants for growth of the mutants in different conditions.

The present invention further relates to purified nucleotide sequences identified by insertion according to the method of the invention. [The present invention further relates to purified nucleotide sequences according to the invention, which are chosen from among the following or complementary sequence thereof:

Rv2930 (fadD26), available in GenBank, Accession Number Z74697 (SEQ ID No. 37);

Rv0405 (pksό), available in GenBank, Accession Number Z84725 (SEQ ID No. 34);

Rv2452c, available in GenBank, Accession Number AL021246 (SEQ ID No. 31); Rvl395, available in GenBank, Accession Number Z80108 (SEQ ID No. 28); Rv3487c (HpF), available in GenBank, Accession Number Z95390 (SEQ ID

No. 26);

No. 14);

Rv0204c, available in GenBank, Accession Number AL021928 (SEQ ID No. 17); Rvl857 (modA), available in GenBank, Accession Number Z83859 (SEQ ID No. 11); Rv0450c (mmpL4), available in GenBank, Accession Number AL021932 (SEQ ID

No. 41); and

Rv3018c, available in GenBank, Accession Number AL021287 (SEQ ID No. 8). The present invention further relates to purified nucleotide sequences that are co- transcribed on the same mRNA with the identified sequences according to the invention. i The present invention further relates to purified nucleotide sequences of the DNA regions flanked by DNA sequences according to the invention and contain the sequences encoding products of the same metabolic pathway.

The present invention further relates to purified nucleotide sequences according to the invention, wherein the metabolic pathway is the synthesis of phtiocerol or phenolphtiocerols esterified by mycoserosic acids.

The present invention further relates to purified nucleotide sequences according to the invention, wherein the metabolic pathways include the products of ORF fadD26 (SEQ ID No. 36), ORF ppsA (SEQ ID No. 50), ORF ppsB (SEQ ID No. 52), ORF ppsC (SEQ ID No. 54), ORF ppsD (SEQ ID No. 56), ORF ppsE (SEQ ID No. 58), ORF drrA (SEQ LD No. 44), ORF drrB (SEQ ID No. 46), ORF drrC (SEQ ID No. 19), ORF papA₅ (SEQ ID No. 48), ORF mas (SEQ ID No. 62), ORF fadD28 (SEQ ID No. 60), and ORF mmpL7 (SEQ ID No. 22).

The nucleotide sequence of the following ORFs and their amino-acids sequence product thereof are available in GenBank under the Accession Number:

drrB, drrC and papA₅ ORFs; and

- Z83858 for mas, fadD28 and mmpL7 ORFs.

The present invention further relates to purified nucleotide sequences whose expression is regulated by the Rv2930 gene product (SEQ ED No. 36).

The present invention further relates to the use of the purified nucleotide sequences identified according to the method of the invention, for the selection of new drugs with an antibiotic activity.

The present invention further relates to the use of the purified nucleotide sequences identified according to the method of the invention for the selection of host factors or bacterial factors interacting with the products of said sequences. The present invention further relates to a kit for testing drugs having bacteriocidal or bacteriostatic effects, characterized by providing at least: selected gene products on a solid support or in a liquid; and substrates for measuring the activity of the gene products. The present invention further relates to a process for measuring the activity of the drugs characterized by the steps of: contacting the drugs with the gene expression products, revealing the activity by adding the appropriate substrate, and measuring the remaining activity.

The present invention further relates to a purified mutant bacteria constructed according to the invention, selected from among the following strains deposited at the Collection de Cultures de Microorganismes (CNCM) on June 15, 1999: Strain Accession Number

MYC2251 1-2227

MYC2253 1-2228

IMYC2254 1-2229

MYC2256 1-2230

MYC2257 1-2231

MYC2258 1-2232

MYC2260 1-2233

MYC2261 1-2234 MYC2262 1-2235

MYC2263 1-2236

MYCZ264 1-2237

MYC2265 1-2238

MYC2266 1-2239. The present invention further relates to purified gene products, wherein the genes encoding said products are identified according to the method of the invention.

The present invention further relates to use of purified gene products according to the invention for the selection and screening of molecules interacting with or for their natural activity. In an embodiment, the method comprises the steps of: a) constructing an insertion mutant library of the organism of interest, wherein each mutant of the library contains at least one insertion consisting of a transposon associated with a tag, and wherein the transposon, the tag, or both are inserted in a gene or in its regulatory region, such as its promoter, within the genome of the mutated bacteria, b) growing the organisms of the mutant library in different and defined conditions, c) identifying mutated strains by ordering the tags on a solid support in order to recognize the presence or absence of the tagged mutants by hybridization of the sequence corresponding to the tags under stringent conditions, and, d) optionally, testing the ability of the mutants to multiply in vivo. As used herein, conditions are the milieu in which the organisms are maintained and/or grown, for example, the growth media and/or host cell used for maintaining or increasing the numbers of bacteria of the invention. The term "different conditions" signifies the type of conditions used to determine if a mutant has a detectable phenotype of interest. For example, different conditions can include the presence of a substance that is normally toxic to a wild-type organism.

In one embodiment, the method comprises: a) constructing an insertional mutant library of an organism or organisms of interest, wherein each mutant of the library contains at least one insertion in at least one gene and/or in a regulatory region of at least one gene of the organism or organisms of interest, the insertion comprising at least one tag and/or at least one transposon associated with a least one tag; b) growing organisms of the insertional mutant library in defined conditions; and c) identifying individual mutants in the insertional mutant library by hybridization, under stringent conditions, of the tags to known sequences.

In one embodiment, the method is used to identify and isolate mutants of actinonycetales. In this embodiment, the method can comprise performing signature tagged transposon mutagenesis (STM) on a collection of actinomycetales organisms.

Hybridization can be carried out at 65°C using the Rapid hybridization buffer of Amersham. Washing can be carried out under the following stringent conditions: a first washing with 2X SSC, 0.1% (w/v) SDS for 10 minutes; a second washing, performed twice successively, with IX SSC, 0.1% (w/v) SDS for 15 minutes; and a third washing in which the hybridized DNA is washed twice, successively, with 0. IX SSC, 0.1% (w/v) SDS for 15 minutes. More stringent conditions for hybridization and washing can be utilized, and the proper conditions for a given assay can be easily and rapidly determined without undue or excessive experimentation.

In an embodiment, the library is constructed using a delivery vector, for example, a plasmid, capable of transferring transposons with tags into a genome of a mycobacteria.

The method can be used to screen any sample or any bacterial library. In an embodiment, it is used to screen a mycobacterial library. In another embodiment, the method is used to screen anM tuberculosis library. In another embodiment, the method is used to screen anM bovis library. In another embodiment, the method is used to screen a library of the M avium/M. intracellulare group. In another embodiment, the method is used to screen an M. leprae library. Although the method can be applied to libraries derived from a single organism, it is not limited to screening a single bacterial library at a time. Rather, it is applicable to screening multiple libraries concurrently. That is, the method can be used to screen libraries of two or more different organisms, or two or more different strains of the same organism. Further, it can be used to screen samples containing genomic fragments of multiple organisms, including multiple species ϊMycobacterium. The sample could contain a library from at least two of the following organisms: M tuberculosis, M. leprae, the avium/M. intracellulare group, M. bovis, and M. paratuberculosis. The sample could contain a library from all of these organisms. For example, a screening method according to the invention can be applied to a sample containing a library of tuberculosis and a library of M leprae, or a sample containing a library of tuberculosis and a library ofM bovis and/or M intracellulare/M. avium.

In another aspect of the invention, a method of making mycobacterial insertional mutants is provided. The method utilizes a modified signature tagged transposon mutagenesis (STM) procedure, based on the system developed by Pelicic et al. (1997). In one embodiment, the insertional mutants are attenuated mutants. In one embodiment, the method comprises: a) constructing an insertion mutant library of the organism of interest, each mutant of the library containing at least one insertion consisting of a transposon associated with at least one tag, wherein the transposon, the tag, or both is inserted in at least one gene or in its regulatory region, such as its promoter, which is part of the genome of the mutated bacteria, b) growing the organisms of the mutant library in different and defined conditions, and c) identifying mutated strains by ordering the tags on a solid support in order to recognize the presence or absence of the tagged mutants by hybridization of the sequence corresponding to the tags under stringent conditions.

Accordingly, one aspect of the invention is a library comprising insertional mutants of an organism of interest. In one embodiment, the organism of interest is a mycobacteria species.

The present invention thus provides collections of bacteria comprising insertional mutants of an organism of interest. In one embodiment, the organism of interest is an infectious organism, such as an actinomycetales, in particular, a Mycobacterium species. In one embodiment, the mutants have specific phenotypes, in particular, mutants that show reduced virulence in eukaryotes, for example, in yeast or vertebrate hosts.

In one embodiment of the invention, attenuated mutants of actinomycetales, such as mycobacteria species are provided. Species for use in the invention include, but are not limited to, M tuberculosis, M avium/M. intracellulare, M. bovis, M. leprae, and M. paratuberculosis. The attenuated mutants are preferably those unable to multiply in some environmental conditions, in particular, in eukaryotic hosts, including the tissues of mammals in which they characteristically infect (i.e., their target tissues). Attenuated mutants can also show reduced growth rates in their target tissues. Attenuated mutants according to the invention include Mycobacterium strains: MYC2251, deposited under CNCM Accession No. 1-2227; MYC2253, deposited under CNCM Accession No. 1-2228; MYC2254, deposited under CNCM Accession No. 1-2229; MYC2256, deposited under CNCM Accession No. 1-2230; MYC2257, deposited under CNCM Accession No. 1-2231 ; MYC2258, deposited under CNCM Accession No. 1-2232; MYC2260, deposited under CNCM Accession No. 1-2233; MYC2261, deposited under CNCM Accession No. 1-2234; MYC2262, deposited under CNCM Accession No. 1-2235; MYC2263, deposited under CNCM Accession No. 1-2236; MYC2264, deposited under CNCM Accession No. 1-2237; MYC2265, deposited under CNCM Accession No. 1-2238; and MYC2266, deposited under CNCM Accession No. 1-2239, all of which were deposited with the Collection Nationale de Cultures de Microorganismes (CNCM: Paris, FRANCE) on June 15, 1999.

In an aspect of the invention, compositions comprising attenuated Mycobacterium strains are provided. The compositions can consist of a single Mycobacterium species or more than one species. The compositions can comprise at least one Mycobacterium species, and, in embodiments, can further comprise other immunogenic and/or infectious agents. Accordingly, the attenuated mutants and compositions of the invention can be used as part of immunogenic compositions, such as vaccines. When used as immunogenic compositions, additional physiologically acceptable components are preferably included, such as adjuvants, excipients, and the like. I In other embodiments, the present invention provides compositions for use as diagnostic tools and screening targets, as well as kits containing the compositions. The invention is further directed to methods of treating individuals with the mycobacterial strains of the invention. The methods of treating can be methods of immunizing, methods of vaccinating, and/or methods of enhancing an already active immune response. Methods of enhancing an already active immune response include immunizing an individual who has already been exposed to an immunogen. For example, enhancing an already active immune response can be administering a booster injection to an individual who has previously received an immunogenic composition, such as a vaccine. It can also include administering an immunogenic composition to a person who is known to be infected by an infectious organisms. For example, the immune response of a person who is known to be infected with a slow-growing Mycobacterium can be enhanced by administration of an immunogenic composition of the present invention.

The methods of treating can comprise administering an attenuated organism of the invention, or a composition comprising such an organism, to an individual in a quantity and quality sufficient to effect the desired response. Doses and administration schedules can be determined by those of skill in the art without undue or excessive experimentation.

It will be appreciated that this invention additionally encompasses immunogenic compositions comprising recombinant mycobacterium strains described above. The invention also encompasses a vaccine composition containing a recombinant mycobacterium according to this invention in combination with a pharmaceutically compatible excipient. The present invention also pertains to a vaccine composition for immunizing humans and mammals against a pathogenic strain of mycobacteria, comprising an immunogenic composition as described above in combination with a pharmaceutically compatible excipient (such as, for example, saline buffer), and optionally in combination with at least one adjuvant such as aluminum hydroxide or a compound belonging to the muramyl peptide family.

Various methods for achieving adjuvant effect for the vaccine include the use of agents (such as aluminum hydroxide or phosphate (alum), commonly used asθ.05 to 0.1 percent solution in phosphate buffered saline, admixture with synthetic polymers of sugars (Carbopol) used as 0.25% solution. Another suitable adjuvant compounds consist in DDA (dimethyldioctadecyl-ammonium bromide), as well as immune modulating substances, such as lymphokines (e.g., IFN-gamma, LL-1, IL-2 and IL-12) or IFN-gamma inducers compounds, such as poly I:C. The vaccine composition according to the present invention is advantageously prepared as an injectable form (either as liquid solution or suspension). However, solid forms suitable for solution in or suspension in, liquid prior injection may also be prepared.

In addition, if desired, the vaccine composition may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, or adjuvants which enhance the effectiveness of the vaccines.

The vaccine compositions of the invention are administered in a manner compatible with the dosage formulation, and in such amount as will be therapeutically effective and immunogenic. The quantity to be administered depends on the subject to be treated including, e.g., the capacity of the individual's immune system to induce an immune response.

Suitable dosage ranges are of the order of 10 to 10 cfu (colony forming units) at an attenuated recombinant mycobacteria concentration of about 10 cfu/mg. Most preferably, the effective dose is about 10 cfu. The dosage of the vaccine will depend on the route of administration and will vary according to the age of the patient to be vaccinated and, to a lesser degree, the size of the person to be vacciated. Most preferably, the vaccine composition according to the present invention is administered via an intradermal route and in a single boost.

In the case of patients affected with immunological disorders such as, for example, immunodepressed patients, each injected dose preferably contains half the weight quantity of the attenuated mycobacteria contained in a dose for a healthy patient.

In the case of neonates, the dose will be approximately four times less than for an adult, and in the case of young children (4-6 years old), the dose will be approximately half the dose used for an adult healthy patient. In some instances, it will be necessary to proceed with multiple administrations of the vaccine composition according to the present invention, usually not exceeding six administrations, more usually not exceeding four vaccinations, and preferably one or more, usually at least about three administrations. The administrations will normally be at from two to twelve week intervals, more usually from three to five week intervals. Periodic boosters at intervals of 1-5 years, usually three years, will be desirable to maintain the desired levels of protective immunity. In an aspect of the invention, kits containing the attenuated organisms of the invention are provided. In an embodiment of this aspect of the invention, kits containing attenuated mycobacteria strains are provided. The kits can be used, for example, to package and contain the reagents and strains necessary for administration of a composition according to the invention to an individual. For example, the kits can contain an attenuated Mycobacterium species formulated in a vaccine composition. In an embodiment, the kits contain some or all of the equipment, reagents, and organisms necessary for vaccinating an individual. In embodiments where the contents of the kit are to be used in vivo, the materials to be introduced in vivo may be sterilized before administration. In one embodiment, the kit comprises at least one of the additional reagents and or equipment necessary for vaccinating a patient. In one embodiment, the kit comprises all of the additional reagents and/or components necessary for vaccinating a patient. In one embodiment, the kit contains multiple organisms.

In another embodiment, the invention provides a kit for testing for drugs having antibacterial effects. The kit contains at least the following components: selected gene products on a solid support or in a liquid; and substrates for measuring the activity of the gene products.

The invention also provides for use of the mutants and the purified nucleotide sequences identified according to the method of the invention for the identification and/or selection of molecules, such as new drugs, with an antibiotic activity. Thus, in one embodiment, the invention provides a method of identifying molecules or compounds that have antibiotic activity. The method comprises contacting a mutant of the invention with at least one molecule or compound and determining whether the molecule or compound affects the growth or viability of the mutant, wherein an effect on the growth or viability indicates that the molecule or compound has antibiotic activity.

The invention also covers targeted genes. These genes can be inserted in a specific vector for expression. For example, a gene identified by the methods of the invention can be isolated and cloned into a vector. The gene can then be expressed in a host organisms, such as a vaccine strain. The vaccine strain can then be administered to an individual such that the individual mounts an immune response against the gene identified by the methods f the present invention. In this way, the individual can develop an immune response to a virulent organism without being exposed to the entire organism. Thus, bacterial sequences involved in pathogenesis that are identified by the methods of the present invention can be used for the construction of live vaccines. Hosts and targeted bacterial genes involved in, or suspected of involvement in, pathogenicity are also included in the invention.

EXAMPLES

The following examples serve to illustrate representative embodiments of this invention. The examples are not to be construed as limiting the scope of the invention, but are presented to further clarify specific embodiments of the invention.

Example 1: Bacterial strains and growth conditions.

Escherichia coli XL 1 -Blue was used for cloning experiments. It was grown on Luria-Bertani medium (LB) supplemented with the appropriate antibiotics. M. tuberculosis strain MT103 and M bovis BCG Pasteur were grown on Middlebrook 7H9, 7H10, or 7H11 (Difco) supplemented with ADC or OADC (Difco) and 0.2% glycerol or 0.05% Tween 80. When required, kanamycin (Km) 20μg/ml and sucrose 2% were added to the growth medium.

The growth rates of the different M tuberculosis mutant strains in vitro were compared with that of the wild type MT103 by first growing all strains to an OD₆oo nm of approximately 0.5 in 7H9 supplemented with ADC and Tween 80. These cultures were diluted in fresh medium to reach an ODβoo nm of 0.01 and then incubated at 37°C with 5% CO₂. At different time points, the OD₆oo nm was measured using a culture previously inactivated for 1 hour at 80°C. The growth indicated in Table 1 is the ratio between the doubling times of the mutant and the wild type strains. This experiment was repeated twice independently with similar results. Table 1 : Phenotypic characterization of the J 6 attenuated mutants.

Disrupted ORF In vitro Intracellular

Mutant Putative function or adjacent ORF growth index ' growth index &

1A20 Rv2930 (fadD26) Lipid metabolism 1.28 ND

1A22 Rv2930 (fadD26) Lipid metabolism 1.24 ND

1A29 Rv2930 (fadD26) Lipid metabolism 0.92 0.55 +/- 0.08

1B8 Rv0405 (pksδ) Polyketide synthase 1.20 ND

1B22 Rv2930 (fadD26) Lipid metabolism 1.25 ND

2B3 Rv2452c Unknown 0.93 ND

2B26 Rvl395 Transcriptional activator 0.86 ND

4BK Rv3487c (IφF) Lipid degradation 1.06 0.17 +/- 0.04

6A22 Rv2930 (fadD26) Lipid metabolism 1.10 ND

8B152 Rv2942 (mrnpLT) Proton dependent 1.02 0.19 +/- 0.03 transporter

9A29 Rv2938 (drrQ ABC transporter 1.13 0.16 +/- 0.01

15A19 Rv0507 (mmpL2) Proton dependant 1.02 ND transporter

19A126 Rv0204c Unknown 0.96 0.53

22A17 Rvl857 (modA) Molybdenum fixation 0.82 ND

23A26 Rv0450c (mmpL4) Proton dependant 1.03 ND transporter

23A44 Rv3018c PPE family 0.93 ND

BCG - - - 0.11

' The in vitro growth index is the ratio between the doubling times of the mutant and the wild type strains in 7H9/ADC incubated at 37°C with 5% CO₂. This experiment was repeated twice independently with similar results. & The intracellular growth index is the ratio eight days post-infection of mutant to wild type cfu number divided by the ratio one day post-infection of mutant to wild type cfu number. In each experiment, the number of cfu was evaluated in four independent wells for each strain and each time point. The experiment was performed at least twice for each strain. The indicated value is the mean of two independent experiments +/- the standard error of the mean. ND, experiment not done. Example 2: DNA manipulations and hybridizations.

Chromosomal DNA from M tuberculosis and M bovis BCG was extracted as described previously (Torrea et al., 1995). All nucleic acid manipulations were performed according to standard molecular biology techniques (e.g., Sambrook et al., 1989) or recommendations of the enzyme manufacturer. For amplifying the tags from the recovered pools of mycobacteria, the culture was inactivated for 1 hour at 80°C. The killed bacteria were boiled and then frozen in a dry ice- alcohol bath 4 or 5 times to break the cells. Ten μl of this extract was used for the PCR amplification in lOOμl volume with pTag3 and pTag4 as primers using the same conditions as described for tag production. The PCR products were gel purified using the QiaexII kit (Qiagen) and labelled with the megaprime labelling kit (Amersham Pharmacia Biotech) and [³²P] dCTP (ICN). In the labelling reaction, the primers from the kit were replaced by pTag3 and pTag4 (5 pM each). The radioactive probe was digested with Hindlll and purified on a Nick-Column (Pharmacia) to remove the non-specific arms of the tags. Pre- hybridization, hybridization, and washes were carried out at 65°C using the rapid hybridization buffer (Amersham) as suggested by the manufacturer. BioMax MS X-ray films (Kodak) were exposed for 4h to 24h at - 80°C depending on the signal strength. Example 3 : Construction of plasmid pCG 113.

The transposon Tn5367, derived from IS1096, was extracted from pYUB285 on a HindϊH fragment (McAdam et al., 1995). It was cloned into the E. coli vector pBluescript KS-linearized with HindRl. The resulting plasmids were named pCG106 and pCG107 according to the orientation of the insert. The kanamycin gene from plasmid pUC4K (Amersham Pharmacia Biotech) was extracted on a Pstl fragment, blunt-ended using the T4 DNA polymerase, and ligated into pBluescript KS- linearized with Smal. An EcόRl-NotI fragment containing the kanamycin gene was extracted from this new plasmid and blunt- ended. This fragment was used to replace the kanamycin gene present on an MZwI fragment in pCG107. Both orientations were obtained and the plasmids were named pCGl 10 and pCGl 11. Finally, the plasmid pCGl 13 was produced by inserting the blunt-ended EcoRV- Kpnl fragment of pCGl 10 containing the transposon into the pPR23 vector cut with No/I two

Double-stranded DΝA tags were produced by PCR amplification using a variable oligonucleotide pool RTCG as template DΝA. RTCG (5'-AAA CTA ACT AGT TAC AAC

CTC AAG CTT-[Ν]₂₀-AAG CTT GGT TAG ATG ACT AGT ATT AAA-3'; SEQ ID

NO:l) is highly similar to RT1 designed by Hensel et al. (1995), where [N]₂₀ is a section of

20 nucleotides, which can be the same or different. The only difference resides in 12 bases of both extremities that introduce a Spel restriction site. The primers used to amplify the tags were pTagl (5'-AAA CTA ACT AGT TAC AAC CTC-3'; SEQ ID NO:2), pTag2 (5'- TTT AAT ACT AGT CAT TCT AAC C-3'; SEQ ID NO:3), pTag3 (5'-TAG TTA CAA CCT CAA GCT T-3'; SEQ ID NO:4), and pTag4 (5'-TAG TCA TTC TAA CCA AGC TT- 3'; SEQ ID NO: 5). For the cloning, the tags were produced by PCR amplification using RTCG as the template and pTagl and pTag2 as primers. The PCR was performed using the Amplitaq kit (Perkin-Elmer) according to the manufacturer recommendations in a lOOμl volume¹ containing 200 pg RTCG, pTagl, and pTag2 (lOOpM), 1.5 mM MgCl₂, and 250 μM each of dATP, dGTP, dCTP, dTTP, IX Taq buffer, and 5U of Amplitaq. The cycles used were the same as described by Hensel et al. (1995). PCR products were purified using the QiaexII kit (Qiagen), digested with Spel, and ligated into the vector pCG113 linearized with Spel and dephosphorylated. The ligation mixture was electroporated into E. coli and transformants selected on LB containing Km. The plasmids harbored by the E. coli transformants were analyzed by restriction enzyme digestion to verify that the tags were incoφorated into pCGl 13. Example 5: Construction of tagged M. tuberculosis mutant libraries.

To perform a signature tagged mutagenesis inM tuberculosis, the plasmid pCGl 13 was constructed (Figure 1). It consists of a ts-sacB vector carrying an IS1096 derivative with a unique restriction site permitting the insertion of the DNA signature tags. It allows efficient counter-selection of the plasmid at 39°C on sucrose and isolation of large number ofM tuberculosis transposition mutants.

Eighty base-pair (80 bp) tags (comprising 20 bp invariable arms flanking a highly variable 40 bp region) were amplified by PCR and cloned into pCGl 13 (see below). The resulting plasmids were transformed into E. coli for amplification and verification. A pool of 5000 E. coli transformants was used to prepare plasmid DNA. These plasmids were transferred into M tuberculosis MT103 by electroporation and transformants selected on 7H10 medium containing Km. Fifty five transformants were retained for producing 55 independent mutant banks (Pelicic et al., 1997). A library of 1927 mutants picked randomly from 51 banks was organized and grown in microtiter plates containing 7H9 medium supplemented with Km. The library was organized such that the same wells from different microtiter plates contained different mutants with the same tag, but mutants from different wells of the same microtiter plate harbored different tags. To verify that the colonies formed under these conditions were the result of random transposition events, chromosomal DNA was extracted from 112 clones randomly picked among 16 of the 55 libraries. DNA was digested with BamΗl and analyzed by Southern blotting using an internal IS 1096 DNA fragment as a probe. It appeared that the majority of the tested mutants exhibited hybridizing patterns, suggesting that the tagged IS 1096 derivatives transposed at different locations within the M tuberculosis chromosome.

Into each microtiter plate was added a bovis BCG mutant tagged following the same protocol as used for M tuberculosis, resulting in each microtiter plate containing 47 M tuberculosis mutants plus the tagged BCG. They were stored at -20°C in 15% glycerol. Ten μl of the 48 strains from each plate were pooled and inoculated in 5 ml of 7H9. These pools were grown to an ODβoo of approximately 0.4 and stored at -20°C in 15% glycerol. A series of membranes containing the 51 different tags used was produced by amplifying the different tags by PCR with pTag3 and pTag4 and transferring them onto Hybond N+ (Amersham Pharmacia Biotech) using a Minifold I apparatus (Schleicher et Schull). The 51 different tags present in this collection showed no strong cross-hybridization, indicating that they were suitable for the screening procedure.

Example 6: Screening of the M. tuberculosis insertional mutant bank.

The initial multiplication of M tuberculosis within the lung is one step in M tuberculosis pathogenesis. Therefore, the mutant bank made in Example 5 was assayed for attenuated organisms in an in vivo lung multiplication assay. Infection of BALB/c mice 6 to 8 weeks old (purchased from CERJ, Le Genest St Isle, France) was performed as described previously (Jackson et al., 1999). Briefly, 5xl0⁵ cfu in 0.5 ml of PBS of either input pools or individual strains were injected intravenously. A total of 1968 clones (41 pools each containing 47 M tuberculosis mutants plus one tagged BCG) were used for this screening. Approximately 5x10⁵ cfu of each pool was inoculated intravenously in at least two mice per pool.

One day post-infection, two animals were sacrificed and the bacterial load in the spleen, liver, and lung were evaluated. More than 5x10³ cfu were recovered from the lung, resulting in a probability of greater than 99% for each strain among the 48 to be present in the lung (if each strain was equally represented in the pool).

Three weeks later, mice were euthanized with CO₂ and the organs were removed aseptically and homogenized (Jackson et al., 1999). Serial dilutions of organ homogenates were plated on solid medium supplemented with kanamycin. For the recovered pools used for the hybridization studies, between 5xl0³ and lxlO⁴ colonies were collected and subjected to tag amplifications. These tags were labelled and used to probe, by Southern blotting, a membrane onto which the 51 different tags had been transferred. Tags giving a strong hybridization signal with the probe from the input pool and weak signals with the probes from the two recovered pools identified candidate attenuated mutants.

As mentioned above, a tagged BCG mutant was included as an internal control in each pool. Indeed, following intravenous inoculation in mice, this strain was shown to multiply in the lung far less than virulent M tuberculosis (only one log of increase at the peak) (Jackson et al., 1999). So the prediction was that BCG should be weakly represented amongst the 5xl0³ to lxlO⁴ colonies recovered from the lung. As expected, the spot corresponding to the BCG tag gave very weak hybridization signals with the majority of the probes obtained from the recovered pools of bacteria.

With the 1927 M tuberculosis mutants, the initial screen identified 79 candidates potentially attenuated for multiplication within the lung of mice. These mutants were analyzed further by testing independently their multiplication within the lungs of mice. They were inoculated intravenously in at least three mice and, three weeks post infection, cfu in the lung were determined. Of the 79 strains, 3 exhibited a severe attenuation (a difference of more than two logs compared to the wild type strain), 9 had a mild attenuation (between one and two logs of difference) and 4 had a weak attenuation (between 0.6 log and one log of difference) (Figure 2). The others showed no major difference compared to MT103. The results demonstrate that the signature tagged transposon mutagenesis method is a powerful strategy to identify genes important for multiplication of M tuberculosis within the lungs of mice. To perform this study, the transposon mutagenesis system developed by Pelicic et al. (1997) was adapted, and the original STM procedure (Hensel et al., 1995) was modified in a way similar to the one used in S. aureus (Mei et al., 1997). Pools of 48 mutants were used instead of the 96 as described in the original methods (Hensel et al., 1995; Mei et al., 1997). In spite of this reduction in the pool complexity, the hybridization patterns obtained with the recovered pools derived from two mice infected with the same mixture are not always superimposable. Some mutants seemed lost in one mouse but still present in the other. This probably generates a number of false negative mutants that appeared to be non-attenuated when tested individually. First, this may represent a technical artifact of the PCR due to the competition of the different amplicon. However, this is unlikely since the false attenuated clones do not correspond to the same tag in the different experiments. Second, some of the bacteria of the input pools might have not reached the lung. Indeed, it has been shown that after intravenous inoculation, the distribution of the inoculum is approximately 90% in the liver, 9% in the spleen and 1% in the lung (Lefford, 1984). With an inoculum size of 5xl0⁵ cfu corresponding to 48 strains, the chance of having at least one bacillus of each strain in the lung is higher than 99% even after the inoculum reduction due to the organ distribution. However, if some strains were very under-represented in the input pools, it is possible that they did not seed in the lung. This phenomenon might be amplified by a difference in aggregation between the strains. A third explanation is that a significant portion of the bacilli that seed in the lung do not replicate (or not to the same extent). A comparable phenomenon was observed by Chiang et al. (1998), who estimated that for V. cholerae, a minimum of 1000 cfu of a wild type strain was required to establish a successful colonization of the infant mouse.

The selection scheme was designed to investigate the initial multiplication within the lung of mice. During this step of the infectious process, M tuberculosis is believed to multiply within mononuclear phagocytes in the absence of a specific immune response, which appears in the lung approximately 3 weeks post-infection and is marked by an inflection in the growth curve of M tuberculosis. Screening 1927 mutants of M tuberculosis allowed the identification of at least 16 strains with an attenuated phenotype in the lung, both in competition assays and in individual assays. None of the selected mutants were impaired for growth in standard liquid medium. This result showed that none of the mutations affect genes involved in central metabolism. This is consistent with the fact that none of the mutated genes exhibits similarity with housekeeping genes from other bacteria. Thus, the mutated loci seemed to be important only in specific environments, such as those encountered in mice or in the macrophage phagosome.

Experiments with mouse bone marrow macrophages showed that all the tested strains, including M bovis BCG, were able to multiply. However, four strains, 4B18, 9A29, 8B152, and M bovis BCG exhibited a weak defect in their intracellular multiplication in comparison with the virulent M tuberculosis strain. It is possible that this defect accounts for the attenuation observed in vivo, although it is important to note that the route of entry in phagocyte and the subsequent response triggered might be different in vivo and in vitro. After intravenous inoculation, mycobacteria are likely to be coated by opsonins when they encounter phagocytes, whereas in in vitro multiplication assays, they are free of such compounds. Moreover, the activation state of the phagocytes in which M tuberculosis multiplies are also likely to be different in vivo and in vitro. Example 7: Mapping of transposon insertion sites and sequence analysis. To identify the transposon insertion site, chromosomal DNA from the strain of interest was prepared. The insertion site was amplified using a ligation-mediated PCR (LMPCR) as described previously (Prod'hom et al., 1998). The product was purified and directly sequenced using an ABI 373 DNA sequencer with the Taq Dyedeoxy terminator cycle sequencing kit (Perkin-Elmer Cetus). The sequences were mapped on the H37Rv genome using the Blastn program (Altschul et al., 1997; Cole et al., 1998) and were compared with Genbank and Prodom database entries using Blast 2.0 and PSI-Blast programs (Altschul et al., 1997). Transmembrane structure predictions were performed using TMHMM 1.0 program (Sonnhammer et al., 1998). Example 8: Characterization of the attenuated mutants.

The characteristics of the attenuated mutants were studied with the goal of determining whether the strains showing a multiplication defect in vivo behaved like the wild type in axenic culture. The growth curves of these mutants were compared to that of the wild type (Table 1). None of these strains appeared to have a growth defect in 7H9, suggesting that the mutations did not affect genes involved in the basic metabolism ofM tuberculosis.

The mutations leading to the attenuation phenotype were mapped precisely using amplification of the adjacent regions of the IS 1096 derivatives by LMPCR, sequencing, and comparison with the published M tuberculosis genome sequence (Cole et al., 1998) (Table 1). Five of the 16 mutants (1A22, 1A20, 1B22, 1A29 and 6A22) showed insertions of the mobile element within, or just upstream of, the fadD26 open reading frame (ORF) ofM tuberculosis. Mutants 1A20, 1 A22, 1B22, and 6A22 contain the same mutation, suggesting that these clones might be siblings or that this site might be a hot spot of insertion. By hot spot, it is meant a region where insertion occurs at a greater frequency than seen in the genome as a whole. They are located 112 bp upstream of the putative start codon of fadD26, while the 1A29 insertion was mapped 583 bp after the putative start of the gene, which is 1881 bp long in total. The fadD26 gene product shows strong similarity to acyl-CoA synthases, which are enzymes involved in lipid metabolism. The fadD26 gene is located just upstream of the ppsA, B, C, D, E genes, which encode enzymes required for the biosynthesis of phthiocerol and phenolphthiocerol. Interestingly, the genetic organization revealed that the expression of the fadD26 and pps genes might be coupled.

Indeed, the stop and start codons of all these genes (with the exception of ppsD and ppsE) overlap, suggesting a translational coupling (Figure 3). Thus, the identified mutants might be affected in their ability to metabolize lipids.

Two other mutants might also be affected in their lipid metabolism. Mutant 4B18 exhibits a mutation located 136 bp upstream of the lipF ORF, which encodes a protein showing similarity to lipases, while 1B8 harbors an insertion within the pksό ORF, an ORF having sequence similarity with the polyketide synthase.

Five mutants, 8B152, 9A29, 19A126, 15A19, and 23A26 have insertion within or close to ORFs encoding proteins with predicted transmembrane domains. The ORFs are, respectively, mmpL7, drrC, Rv0204c, mmpL2, and mmpL4. The mmpL2, mmpL4, and mmpL7 putative genes belong to a family of large M tuberculosis genes sharing sequence similarities as well as common structural features (Cole et al., 1998). The mmpL encoded polypeptides exhibit sequence similarities with the product of genes tpl ofM leprae and actII-3 of Streptomyces coelicolor, the latter being involved in antibiotic transport (Fernandez-Moreno et al., 1991). The predicted structure of these proteins, with more than 10 predicted transmembrane domains and two large periplasmic loops, is also similar to those of proton-dependant transport proteins involved in the translocation of various molecules, including drugs, lipooligosaccharides, and sugars (Saier et al., 1998). The clone 9A29 shows an insertion within the dπC gene. The putative product of this ORF exhibits similarities with the DrrB proteins ofM tuberculosis and Streptomyces peucitius. The genetic organization of the drrA, B, and C genes in tuberculosis suggests that their expression may be coupled since their start and stop codons overlap. DπA is a 331 amino acid long polypeptide, which exhibits all the features of the cytoplasmic components of ABC transporter; i.e., an ATP-binding cassette followed by an ABC transporter family signature. DrrB and DπC are, respectively, 283 and 268 amino acids long and they are both predicted to have 6 transmembrane domains. They also exhibit strong similarities with each other and membrane subunits of ABC transporters. All these features of DπA, DrrB, and DπC suggest strongly that these three polypeptides form an ABC transporter.

The clone 19A126 also contains a mutation within an ORF, Rv0204c, encoding a predicted integral membrane protein. This polypeptide shows no sequence similarity with entries of protein database. A last insertion (clone 22 A 17) occurs in the mod A ORF encoding a protein associated with the membrane. This putative gene encodes a polypeptide exhibiting the sequence signature of lipoproteins. Therefore, it is likely that this protein is covalently modified with a fatty acid after translocation, and remains anchored to the membrane. The product of modA shows strong similarities with periplasmic proteins involved in the fixation of molybdate (47% identity over 249 amino acids and 28% identity over 276 amino acids with ModA of Arthrobacter nicotinovorans and E. coli, respectively). This gene is located upstream the modB, modC and modD ORFs that encode proteins exhibiting similarities with molybdate ABC transporters.

In the strain 2B26, the transposon insertion occuπed within the Rvl395 ORF. The polypeptide encoded by this ORF exhibited the features of transcriptional activators of the AraC/XylS family, i.e., a conserved carboxy-terminal region with a helix-turn-helix motif and the Prosite profile PS01124 typical for this family of proteins.

The mutant 23 A44 exhibited an insertion within a gene, Rv3018c, of the PPE family. The last two mutants, 24 A7 and 2B3, contain mutations in the Rv0726c (GenBank Accession Number Z84395) and Rv2452c ORFs, encoding proteins with no sequence similarities with protein database entries. j The attenuated mutants allowed the identification of 13 loci important for multiplication in vivo. Most of the mutated ORFs are likely to be essential for the pathogenicity; however, it is possible that the transposon exerts an action on neighboring gene expression through polar effects. Four of the 13 loci are grouped on a 50 kb region of the M tuberculosis genome

(Figure 3). This region comprises 13 ORFs, of which the following are involved in biosynthesis of compounds of the cell envelope: phthiocerol, phenolphthiocerol, and mycoside B (Azad et al., 1997; Azad et al., 1996; Fitzmaurice and Kolattukudy, 1998).

Interestingly, these molecules are restricted to eight species of mycobacteria (M tuberculosis, M. bovis, M. africanum, M. marinum, M. ulcerans, M. leprae, M. gastri, and

M kansasiϊ), seven of them being pathogenic (Daffe and Laneelle, 1988). j The first mutated ORF of this region is fadD26, is an ORF having similarities with i an acyl-CoA synthase gene. As previously mentioned, the expression of this ORF might be coupled to those of downstream genes ppsA to ppsE, which were shown to encode subunits of a polyketide synthase producing phthiocerol and phenolphthiocerol (Azad et al., 1997). Because of the expression coupling, it is likely that FadD26 is linked either to the synthesis or transfer of these long chain diols. Recently, Fitzmaurice and Kolattukudy (1997, 1998) demonstrated that an enzyme, FadD28, exhibiting more than 55% identity to FadD26, is specifically involved in the transfer of mycoserosic acids onto phthiocerol and phenolphthiocerol. These diols are not only esterified by mycoserosic acid but also other lipids, and FadD28 does not seem to be required for this process (Fitzmaurice and Kolattukudy, 1998). Therefore, FadD26 might be involved in the transfer of these other substrates onto phthiocerol and phenolphthiocerol. This hypothesis is now under investigation. Interestingly, both fadD28 and mas, the gene encoding the enzyme responsible for the synthesis of mycoserosic acid, are located on the 50 kb region (Figure 3). The two other mutated ORF of this region, drrC and mmpL7, both putatively encode proteins exhibiting similarities with transporter molecules. So far, their substrates remain unknown. Moreover, the enzyme MAS has been suggested to be associated with the cell wall but lacks a typical signal sequence suggesting that a secretion system other than the general secretory pathway is involved. All these data suggest that this 50 kb region might be involved in the biosynthesis and translocation of lipid molecules that are important for the pathogenicity ofM tuberculosis. Another insertion, in the mutant 1B8, was found in another region that exhibits common features with the 50 kb region described above. The mutant 1B8 is described in Figure 4. This figure represents the genetic organization of the fadD30-pks6 gene cluster. The mutant 1B8 contains ORFs having similarities with acyl-CoA synthase and polyketide synthase genes, and proton-dependent transporter genes. 1B8 harbors a mutation in pksό, the putative gene of a polyketide synthase showing 42% identity over 1018 amino acids to PpsA. Upstream of pksό is located the fadD30 ORF encoding a protein sharing 45% identity over 583 amino acids to fadD28. Upstream of fadD30 is found mmpLl, another gene of the mπipL family. This similar genetic organization suggests that all the genes identified in the 50kb region might be involved in the biosynthesis and translocation of lipid molecules. This finding is interesting since it has been believed for many years that compounds of the cell envelope, including many different lipids, are key players in the interaction of M tuberculosis with its host and in its pathogenicity.

Three other mutants, 19A126, 15A19, and 23A26, contain insertions in ORFs having similarities with integral membrane protein-encoding genes. Two of these genes belong to the mmpL family and might encode proton dependant transporters. However, the vicinity of these two ORFs does not present the same organization as that of the mmpLl and mmpL7 genes mentioned above.

Another attenuated mutant, 4B18, showed an insertion just upstream of the lipF ORF, which encodes a polypeptide with similarity to lipid esterases. The association of lipase production with pathogenicity has been suggested by several works concerning non- mycobacterial pathogens. For example, Camilli and Mekalanos (1995) showed that the production of a lipase by V. cholerae was induced following infection in an infant mouse model. However, a null mutant for the gene encoding this lipase did not appear to be attenuated in a competition assay. Other pathogens, such as Pseudomonas cepacia and Pseudomonas aeruginosa were shown to secrete lipases. Enzymes purified from these organisms were shown to have several effects in vitro on eukaryotic cells. They inhibit the phagocytic functions of alveolar macrophages and modulate the release of inflammatory mediators by different cells of the immune system (Konig et al., 1996; Straus et al., 1992). Another possible function for these lipases might be to degrade host lipids to provide fatty acids for the pathogen. The strain 2B26 harbors a mutation within a gene encoding a transcriptional regulator of the AraC/XylS family. Members of this family are widely distributed in bacteria and are mainly involved in carbon metabolism, stress response, and pathogenesis (Gallegos et al.,^! 1997). Therefore, this regulator might be involved in the adaptation of M tuberculosis to the particular environments encountered within the host.

The mutant 23 A44 contains an insertion within a gene of the PPE family. This family consists of 68 members encoding polypeptides with common primary sequence features (Cole et al., 1998). It has been suggested that this PPE family of genes might be involved in a kind of antigenic variation. However, the fact that the mutation in Rv3018c led to a phenotype shows that the 68 genes of this family are not completely redundant. Example 9: Multiplication of selected mutants within mouse bone-marrow macrophages.

One of the features ofM tuberculosis is its ability to survive and multiply within professional phagocytes (e.g., macrophages). This ability was tested with attenuated mutants of the invention. Mutants 1A29, 8B152, 4B18, 9A29 (which represented 4 of the most attenuated strains), and 19A126 (a weakly attenuated mutant) were used to infect

5xl0⁴ mouse bone-marrow macrophages from 7 to 8 weeks-old BALB/c mice infected at

I a multiplicity of infection close to 0.1 bacilli per macrophage. The course of multiplication was followed over a period of 10 days and compared to those of control strains MT103 and M. bovis BCG (Table 1). Briefly, the procedure is summarized as follows. To prepare bacteria for macrophage infection, each strain was grown to an ODβoo of approximately 0.4, aliquotted, and stored at -20°C in 15% glycerol. Before macrophage infection, the bacterial aliquot was thawed, vortexed, and centrifuged for 15 min at 4000 φm. The pellet was resuspended in phosphate saline buffer (PBS) containing 0.01% Tween 80. Large aggregates were pelleted by centrifiigation for 10 min at 20 x g. The upper 500 μl of a 1 ml suspension was used for the infection. The number of viable cfu in this stock was evaluated by plating serial dilutions on solid medium following the same treatment used prior to infection. In each experiment, the number of cfii was evaluated in four independent wells for each strain and the experiment performed. The intracellular growth index provided in Table 1 is the ratio eight days post-infection of mutant to wild type cfu number divided by the ratio one day post-infection of mutant to wild type cfii number. [All the tested strains, including the control strain M bovis BCG, were able to multiply within macrophages. However, several mutants (8B152, 4B18, 9A29), as well as BCG, exhibited a reduced growth rate compared to the wild type strain. Eight days post- infection, the multiplications of the cfu number for 8B152, 4B18, 9A29, and BCG strains were five to ten times less than the one observed with the virulent strain MT103 (Table 1). By comparison, mutants 1 A29 and 19A126 appeared unaffected by their insertion regarding the multiplication within macrophages.

When an ORF having similarities with a gene is identified using the genetic method of insertion mutagenesis, this allows the identification of other genes that are co-transcribed on the same messenger RNA. When the gene identified by this method has the characteristics of a regulatory sequence, all genes regulated by this regulator are of interest. The genes could be identified by the fixation of the regulatory genetic regions in close vicinity to the promoter.

Some genes could be used as a read out for the search of new antibiotics, particularly when genes code for enzymes, the enzymatic activity could be used as a read out.

As an application of the invention, a kit can contain several products such as enzymes (expressed from the genes identified by insertion mutagenesis and then purified) and specific substrates for revealing the activity or the absence of activity in the presence of drugs to be tested.

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Claims

1. A method of screening a library of mutants comprising: a) constructing an insertional mutant library of an organism or organisms of interest, wherein each mutant of the library contains at least one insertion in at least one gene and/or in a regulatory region of at least one gene of the organism or organisms of interest, the insertion comprising at least one tag and/or at least one transposon associated with a least one tag; b) growing organisms of the insertional mutant library in defined conditions; and c) identifying individual mutants in the insertional mutant library by hybridization, under stringent conditions, of the tags to known sequences.

2. The method of claim 1, wherein identifying mutants in the mutant library comprises hybridizing the tags to sequences that are attached to a solid support.

3. The method of claim 1, wherein the method identifies mutants having an attenuated phenotype.

4. The method of claim 3, wherein the phenotype is virulence.

5. The method of claim 4, further comprising testing the ability of the mutants to multiply in vivo.

6. The method of claim 1, wherein the organism of interest is an actinomycetales.

7. The method of claim 6, wherein the actinomycetales is a mycobacterium.

8. The method of claim 7, wherein the mycobacterium is chosen from M tuberculosis, M. bovis, M. leprae, M. avium, M. intracellulaire, and M paratuberculosis.

9. A method of making a libraiy of mycobacterial insertional mutants, comprising constructing an insertional mutant library of a mycobacterial orgamsm of interest, wherein each mutant of the library contains at least one insertion in at least one gene and/or in a regulatory region of at least one gene of the organism of interest, the insertion consisting of at least one tag or a transposon associated with at least one tag.

10. The method of claim 9, wherein the method further comprises growing organisms of the insertional mutant library in different and defined conditions.

11. The method of claim 10, wherein the method further comprises identifying individual mutants in the insertional mutant library by hybridization, under stringent conditions, of the tag or tags to known sequences.

12. A mycobacterial insertional mutant library, wherein the library comprises mutant organisms having at least one insertion in at least one gene and/or in a regulatory region of at least one gene, the insertion consisting of at least one tag or at least one transposon associated with at least one tag.

13. The library of claim 12, wherein the library contains mutant organisms that show an attenuated phenotype.

14. The library of claim 13, wherein the attenuated phenotype is reduced virulence in eukaryotes.

15. An attenuated mycobacterial mutant made by the method of claim 9.

16. The mutant of claim 15, wherein the mutant is chosen from the following Mycobacterium strains, all of which were deposited at the Collection de Cultures de Microorganismes (CNCM) on June 15, 1999:

Strain CNCM Accession Number

MYC2251 1-2227;

MYC2253 1-2228

MYC2254 1-2229

MYC2256 1-2230

MYC2257 1-2231

MYC2258 1-2232

MYC2260 1-2233

MYC2261 1-2234

MYC2262 1-2235

MYC2263 1-2236

MYC2264 1-2237_.

MYC2265 1-2238; and

MYC2266 1-2239.

17. A composition comprising an attenuated Mycobacterial mutant made by the method of claim 9.

18. The composition of claim 17, which is an immunogenic composition.

19. A vaccine comprising the immunogenic composition of claim 18. _!

20. A kit containing the mutant of claim 15.

21. The kit of claim 20, wherein the kit further comprises at least one of the additional reagents and/or equipment necessary for vaccinating a patient.

22. The kit of claim 21, wherein the kit further comprises all of the additional reagents and/or components necessary for vaccinating a patient.

23. The kit of claim 21 , wherein the kit contains multiple organisms.

24. A method of treating an individual suffering from a mycobacterial infection, suspected of being infected with a mycobacterium, or having been exposed to an infectious mycobacterium, comprising administering the attenuated mycobacterium of claim 15 to the individual.

25. The method of claim 24, wherein the attenuated mycobacterium is present in an immunogenic composition.

26. A vector for making an insertional mutant library of an organism of choice, wherein said vector comprises: a) a temperature sensitive origin of replication that is functional in the organism of choice; b) a sacB gene; and c) an insertion sequence, wherein the insertion sequence comprises at least one nucleotide sequence that is a tag.

27. The vector of claim 26, wherein the insertion sequence is a transposon.

28. The vector of claim 26, which is pCGl 13.

29. The method of claim 1, wherein the method is used to identify mutated loci within genomes of organisms of the library by determining nucleotide sequences flanking the insertion and comparing the sequences with sequences in data banks.

30. A method of identifying and isolating mutants of actinomycetales, said method comprising performing signature tagged transposon mutagenesis (STM) on a collection of actinomycetales organisms.

31. The method of claim 30, wherein said mutants display an attenuated phenotype.

32. The method of claim 30, wherein the attenuated phenotype is growth characteristics in different environmental conditions.

33. A method of identifying compounds that have antibiotic activity, said method comprising contacting the mutant of claim 15 with at least one compound and determining whether the compound affects the growth and/or viability of the mutant, wherein an effect indicates that the compound has antibiotic activity.

34. A kit for testing drugs having bacteriocidal or bacteriostatic effects, comprising selected gene products and substrates for measuring the activity of the gene products.

35. The kit of claim 34, wherein the selected gene products are on a solid support or in a liquid.