Advances in Mycobacterial Genetics: New Promises for Old Diseases

Immunobiol., vol. 184, pp. 147-156 (1992) Howard Hughes Medical Institute, Department ot Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, New York, USA

Advances in Mycobacterial Genetics: New Promises for Old Diseases WILLIAM R. JACOBS, Jr.

Introduction: Mycobacterial Infections and a Mycobacterial Vaccine

Historically, members of the genus Mycobacterium have been some of the most important players in the drama of infectious disease and immunology of man. Mycobacterium leprae causes leprosy, a disease that dates back to biblical times and continues to afflict millions around the world today. M. leprae was discovered by G. A. HANSEN in 1873 and found to be the first bacteria associated with human disease. Despite this early discovery, this bacillus has yet to be cultivated on artificial media and can only be propagated in the laboratory in nine-banded armadillos or footpads of mice. Twenty years ago, tuberculosis was thought to be a disease that was no longer to be a menace in the world with the presence of a vaccine and the development of effective chemotherapies to control this infection. However, in the last five years the world has seen alarming resurgence of tuberculosis. The World Health Organization estimates that there are 8 million new cases of tuberculosis and 3 million deaths resulting from this dreaded illness every year. An increasing number of reports of infections with drug-resistant Mycobacterium tuberculosis cells makes this resurgence so much more frightening. Since the introduction of isoniazid in 1954, the United States had thirty-two years of steady decline of the number of new cases of tuberculosis. This trend ended in 1986 and we have seen steady increases with a 5 % increase in 1989 alone. This increase is most likely a result of the epidemic of the Acquired Immunodeficiency Syndrome (AIDS), where M. tuberculosis infection appears to be one of the first signs of a loss of T cell function. AIDS patients also have a number of opportunistic infections, including infections by another mycobacterium, Mycobacterium avium. This bacterium rarely causes disease in immunocompetent individuals and yet is being found in greater than 40 % of AIDS patients in the US. Infections caused by M. avium are difficult to treat as the bacterium appears to be refractory to existing chemotherapies. It is quite clear that all of these mycobacteria continue to be significant cause of morbidity and mortality in man. The other Mycobacterium of notable significance is BCG (bacille Calmette et Guerin), an attenuated Mycobacterium bovis isolate that was developed by Drs. CALMETTE and GUERIN at Institut Pasteur earlier this

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century. BCG, unlike its parent, failed to cause tuberculosis when infected in animals, but instead provided protection against subsequent challenge with virulent M. tuberculosis. Early human trials showed a great degree of protection and the vaccine has been administered worldwide since 1948. A recent trial of BCG vaccination has shown no protection against M. tuberculosis, but it is unclear how this result correlates with earlier studies. Clearly, the components required for a protective immune response against tuberculosis must be more well-defined in molecular terms. In order to control mycobacterial diseases, a better understanding of the biology of the interactions of mycobacteria with their human hosts must be obtained. A major step towards reaching these goals has been recently crossed with the development of tools and methodologies to genetically manipulate the mycobacteria. This chapter describes some of these recent developments and how they may be used to increase our basic knowledge of mycobacteria. In addition, the use of BCG as a recombinant vaccine vector will be explored as a means of not only improving vaccines against mycobacteria, but also as a way to create new vaccines against a variety of diseases.

Obtaining Basic Knowledge of the Mycobacteria: Fulfilling Koch's Molecular Postulate for the Mycobacteria A first step to gain understanding of an infectious disease is to identify the organism that causes the disease. In a similar fashion, a first step in understanding the characteristics of the infectious agent, i.e. a phenotype, is to identify the gene(s) that encodes that phenotype. How do we obtain such knowledge? In 1882, ROBERT KOCH wrote a milestone paper in which he demonstrated that tuberculosis is caused by the Mycobacterium tuberculosis (1). He reached this monumental conclusion and set the precedence for establishing whether a microorganism is the causative agent of a disease by experimentally fulfilling three conditions that have come to bear his name, i.e. KOCH'S postulate (Table 1). By analogy, «KOCH'S molecular postulate» (Table 1) contains the conditions that must be satisfied in order to establish whether a characteristic of a bacterium i.e. a phenotype, such as a virulence, results from the presence and expression of a particular gene. This postulate can be used to demonstrate that any phenotype, such as virulence, antibiotic-resistance, or the ability to grow on a particular carbon source results from the presence and expression of a specific gene, i:e. genotype. For example, to identify a gene required for virulence of M. tuberculosis, we would: 1) identify a mutant of M. tuberculosis that is avirulent because of a specific mutation within a specific gene, 2) clone the putative virulence gene from the virulent M. tuberculosis strain, and 3) introduce the cloned gene back into the avirulent mutant and demonstrate that this gene restores virulence. The conclusion from such an experiment would be that the «cloned gene» encodes a virulence determinant.

Advances in Mycobacterial Genetics . 149 Table 1. Comparison of KOCH'S postulate and KOCH'S molecular postulate KOCH'S postulate «To prove that tuberculosis is caused by the invasion of bacilli and the growth and multiplication of bacilli, it was necessary: 1) 2) 3)

isolate the bacilli from the body; to grow them in pure culture ... and by administering the isolated bacilli to animals, reproduce the same morbid material ... »

to

KOCH, ROBERT (1); a translation by BERNA PINNER and MAX PINNER with an introduction by ALLEN K. KRAUSE, Am. Rev. Tuberc. (1932) 25: 283. KOCH'S molecular postulate To prove that a phenotype, such as virulence, is caused by the presence and expression of a specific gene, it is necessary: 1) to isolate a mutant bacterium with a phenotype that differs from wild-type phenotype; 2) to clone the wild-type gene and 3) by introducing the wild-type gene back into the mutant bacterium, reproduce the wild-type phenotype.

In contrast to other bacteria, KOCH'S molecular postulate could not be fulfilled for M. tuberculosis or the bovine tubercle bacillus, M. bovis, until recently. The first condition requiring the isolation of mutants had first been demonstrated with the isolation of BCG (bacille Calmette Guerin), an avirulent mutant of M. bovis, by Drs. CALMETTE and GUERIN in 1909 (2). The isolation of M. tuberculosis H37Ra and M. tuberculosis H37Rv was another example of the early isolation of genetic variants of M. tuberculosis (3). Both of these efforts yielded avirulent mutants of M. bovis and M. tuberculosis, respectively. The second condition, requiring the cloning of genes, was achieved with the development of recombinant DNA technologies, that permitted efficient cloning of genes into cloning vectors in E. coli. This methodology is one of the most significant developments in the history of biology as it enables KOCH'S molecular postulate to be fulfilled for virtually any organism providing that its genetic material can be isolated. Numerous gene encoding protein antigens (4, 5) and enzymes (6) had been cloned in recent years from the slow-growing mycobacteria. This approach has already yielded considerable information about the nature of many important antigens found in mycobacterial infections. However, the testing of the function of genes required that the third condition of KOCH'S molecular postulate be fulfilled and necessitated that ways be found to introduce recombinant DNA into mycobacteria. Tremendous progress has been made in the last five years towards the development of both the methodologies and vectors that permit efficient transfer of recombinant DNA from E. coli to mycobacteria. The first such vector that allowed for efficient transfer was a hybrid molecule that replicated as a plasmid in E. coli and as a phage in mycobacteria. These shuttle phasmids (7) could be introduced in the M. smegmatis protoplasts as

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plasmid DNAs isolated from E. coli. The shuttle phasmid DNAs replicated in M. smegmatis as phages where they could be packaged in the mycobacteria phage heads. This permits the introduction of genes into other mycobacteria including the slow-growing mycobacteria, BCG and M. tuberculosis. This methodology was extended with the construction temperate shuttle phasmids, vectors that retained their ability to integrate site-specifically into the chromosomes of mycobacteria (8). These shuttle phasmids were instrumental in demonstrating that kanamycin and kanamycin-resistance genes functioned as selectable markers in mycobacteria. This knowledge combined with electroporation was subsequently used to develop plasmid transformation systems for M. smegmatis and BCG, and M. tuberculosis (8). Both plasmid and phasmid vectors provided vehicles by which cloned DNA fragments could be efficiently introduced into the mycobacteria. The ability to fulfill KOCH'S molecular postulates now provide a definitive means of identifying the genetic basis for virtually any phenotype expressed in the mycobacteria. The possession of such definitive knowledge provides the cornerstone upon which novel strategies to control M. tuberculosis infections can be devised.

Mycobacterium smegma tis, a Surrogate Host for the Analysis of Mycobacterial Genes Initial cloning of mycobacterial DNAs demonstrated that mycobacterial DNA was not expressed well in E. coli hosts, most likely resulting from inefficient transcription (9, 10). This observation is also common to other DNAs of a high guanine plus cytosine (G + C) content (mycobacterial DNAs range from 63 to 66 % G + C as compared to 50 % G + C in E. coil) such as Streptomyces, Caulobacter or Pseudomonas. The slow-growth of M. tuberculosis and BCG makes analysis of genes cloned in mycobacterial vectors difficult. M. smegmatis grows 10 times faster than M. tuberculosis and thus would be an ideal host for mycobacterial gene analysis, but it could not be transformed efficiently with plasmid DNA. Recently, however, we have isolated mutants of M. smegmatis that can be transformed with plasmids at five to six orders of magnitude more efficiently than the parent strain (11). These mutants, possessing the efficient plasmid transformation (Ept) phenotype, have a mutation that appears to uniquely affect plasmid replication. We have used these mutants to analyze the minimal DNA sequence necessary for replication of pAL5000 (14) and found that the knowledge gained from analysis in M. smegma tis directly applies to M. tuberculosis and BCG. In addition, these mutants have been useful in analyzing mycobacterial gene expression. By taking advantage of the fact the M. smegmatis has no residual ~-galactosidase activity, we have constructed expression probe plasmids that have a unique cloning site at the 5' end of a truncated E. coli lacZ gene. Random libraries of mycobacteriophage genomes have allowed us to identify sequences capable of

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expressing ~-galactosidase in M. smegmatis. Expression by these sequences are also expressed in BCG and M. tuberculosis (12). We have also found this to be the case for mycobacterial expression sequences identified using a promoter probe plasmid. These strains have permitted us to screen genomic libraries of the pathogenic mycobacteria such as M. tuberculosis, M. leprae and M. avium in the fast growing easily manipulable, M. smegma tis, strains. We have been able to demonstrate that genes from all three of these pathogens can express well in M. smegmatis, which serves as an excellent surrogate host to analyze the genes of slow growing pathogenic mycobacteria. Towards the Identification of Virulence Genes One of the most exciting areas of microbiology today is that of molecular pathogenesis. We are developing the means to identify the genes present within pathogenic mycobacteria that confer to those bacilli the ability to cause disease in mammalian hosts. Such approaches should help us understand the biology of the interaction mycobacteria and with the individual cells that they infect such as macrophages. In addition, these approaches should allow us to understand how the existing BCG vaccine works. Consistent with KOCH'S molecular postulate, we need ways to isolate and identify novel avirulent mutants of M. tuberculosis. Insertional mutagenesis using a selectable marker gene is essential for the mycobacteria as it provides a direct way to select for the growth of a pure culture of a mutant from a clump of mycobacterial cells. HUSSON and colleagues have demonstrated that gene replacement can be performed in the fast growing M. smegmatis (13). Also, recently a transposon has been identified and demonstrated to transpose in M. smegmatis as well (14). We wished to extend these strategies to be able to generate random mutations in mycobacteria, not only into M. sm egm atis, but also in the slow-growing mycobacteria such as M. tuberculosis, BCG and M. avium. KALPANA GANJAM, in our group, has recently extended the targeted mutagenesis strategy to a more random mutagenesis by constructing a library of M. smegmatis genes and then subjecting that library to transposon mutagenesis in E. coli. The resulting plasmids, devoid of a replicon that functions in mycobacteria, were transformed into M. smegmatis to yield insertional mutations via homologous recombination. She was successful in generating a number of auxotrophic mutations in M. smegmatis including those in biosynthetic pathways for pyridoxin and methionine (18). Upon extending this to BCG and M. tuberculosis, an observation unique for prokaryotes was made. Attempts to integrate a piece of homologous linear DNA into the BCG and M. tuberculosis chromosome resulted in the discovery that M. tuberculosis and BCG have the ability to incorporate linear DNA fragments randomly into their chromosomes (18). Further characterizations are underway, but this process may be random, and would then eliminate the need for transposon-generated mutations in these strains.

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Even when mutants exist such as BCG and M. tuberculosis H37Ra, the complementation of a mutation to restore virulence is no simple matter. Currently, the only method that we have to distinguish virulent M. tuberculosis from avirulent M. tuberculosis is using a mouse or guinea pig animal model. Virulent M. tuberculosis will kill mice and guinea pigs, whereas avirulent M. tuberculosis will not. Screening for such a phenotype is complicated because we need to develop strategies for screening in vivo in animals. Towards that goal we have developed a number of strategies to overcome potential problems. One of the problems is the fact that plasmids, such as p YUB12, are lost in animals in vivo in BCG. To ensure that recombinant plasmids are not lost in vivo, M. H. LEE and G. HATFULL and my laboratory have developed novel E. coli-mycobacteria shuttle vectors that utilize the site-specific integration system of mycobacteriophage L5 (19). These vectors replicate in E. coli as plasmids, but integrate sitespecifically into the chromosomes of M. smegmatis, BCG, and M. tuberculosis. Once integrated, these recombinant molecules are stably maintained even when the BCG or M. tuberculosis recombinants are grown in animals. Other potential problems with screening in vivo include: 1) screening a large number of clones needed to represent an entire genome may be prohibitive; and 2) virulence determinants might be complex lipids or carbohydrates that require a large number of genes encoding the enzymes necessary to synthesize complex molecules. To overcome these problems, LISA PASCOPELLA has constructed episomal and integration-proficient shuttle cosmid vectors that enable large fragments of DNA to be cloned. She has constructed genomic libraries of M. tuberculosis, M.leprae and M. avium in these vectors and demonstrated that these recombinant molecules can be efficiently introduced into M. smegmatis, M. tuberculosis, and BCG. These vectors allow the entire genome of M. tuberculosis to be represented in as few as 100 clones and thus have reduced the number of clones necessary to be screened. Such in vivo experiments are underway. Moreover, in collaboration with JOHN BELISLE and PATRICK BRENNAN, we have identified cosmid clones of M. avium that confer to M. smegmatis the ability to express the unique glycopeptidolipid of serovariant 2. JOHN BELISLE has demonstrated that 23 kb of DNA is required to express this complex polysaccharide, a potential virulence determinant (21). Overall, we are hopeful that these approaches will allow us to identify virulence genes of the pathogenic mycobacteria. Applying Molecular Genetics Towards Chemotherapy Chemotherapy represents a potentially fruitful area for the application of molecular genetics. The possibility now exists to identify genes encoding the targets of anti-tuberculosis drugs, and the genes conferring resistance to those drugs. Previously, the lack of genetic systems have made it virtually

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impossible to identify, with certainty, the drug targets and mechanisms of resistance. For example it is likely that rifampicin acts at the RNA polymerase of M. tuberculosis. However, resistance to rifampicin in M. tuberculosis could result from a change in the RNA polymerase or from an altered membrane that blocks entry of the drug into mycobacterial cell. This type of information is essential in developing alternative strategies for treating drug resistant organisms. Additionally we should now have the tools available to clone the genes encoding the targets of action for isoniazid, ethambutol, and pyrazinamide. We should be able to identify the genes conferring resistance to these drugs in a variety of pathogenic mycobacteria. The ability to move genes in M. smegmatis permits its use as a surrogate host for the analysis of genes encoding drug targets for M. tuberculosis, M. avium and M. leprae. This should expedite the analysis of such genes, providing clues to drug mechanisms of action and perhaps novel therapeutic approaches. As information accumulates on drug resistances, we should develop sensitive assays for the early detection of drug resistant tuberculosis strains. It may be possible with PCR analysis to detect drug resistant tuberculosis within days instead of the current 6-12 weeks. Recombinant Mycobacterial Vaccines

The high incidence of tuberculosis infections around the world and the inability of BCG to protect certain populations clearly mandates that we need a better tuberculosis vaccine. While it is unclear how to generate a protective response against M. tuberculosis infections, the possibilities of

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Figure 1. The Ideal Vaccine: Recombinant BeG. BeG can act a vaccine vector for the elicitation of immune responses to a wide range of diseases. Genes encoding antigens from many different pathogens (such as Measles, Human Immunodeficiency Virus, Leishmania and M. Jeprae) would be cloned into expression vectors in BeG and this recombinant BeG would hopefully provide protective immune responses to a large number of diseases with a single immunization.

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Figure 2. Macrophage presentation of Antigens Expressed in BeG. BeG has the ability to engender a wide array of immune responses since it lives within a key antigen-presenting cell. Although, the mechanisms of how antigens expressed by BeG are processed are not well understood (Hence a black box!) recent data suggests that BeG can elicit a full range of cellular and humoral immune responses to the cloned foreign antigens.

introducing recombinant DNA into BCG has made possible the ability to test a dream that BARRY BLOOM has advocated for sometime, i.e. the development of recombinant BCG vaccines to generate the «Ideal Vaccine» (Fig. 1). By cloning and expressing foreign antigen genes into BCG it may be possible to make a multi-valent vaccine that would protect not only against tuberculosis but also against the pathogen from which the cloned gene originates. There are a number of good reasons that make BCG an extremely attractive vehicle. First it is a very safe vaccine; over two billion doses have been administered worldwide and the mortality statistics are very low. Second, mycobacteria are excellent adjuvants, being the active component of complete Freund's adjuvant. Third, BCG is one of the only live bacterial vaccines that is recommended to be given at birth thereby ensuring that more children receive the vaccine. In addition, BCG has the ability like M. tuberculosis to persist for a long time within a human host and elicit a long lasting T cell immunity. The ability to dissect BCG genetically and add new antigens to BCG provides new tools with which we can study the immune response elicited when live BCG is introduced into the mammalian host. Considerable effort has been devoted in recent years towards developing expression vectors capable of expressing foreign antigen genes in BCG. Preliminary analysis and work done in collaboration with scientists at Medimmune Inc., Gatherburg, MD, the University of Pittsburgh, and BARRY BLOOM, and my laboratory here at Einstein have recently demonstrated that a full range immune responses (antibodies, T helper and cytotoxic T cell responses) can be produced to cloned foreign antigen genes (20). Further work is underway how to optimize immune responses and

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how to alter the immune responses in vaccine preparation (Fig. 2), but this provides a powerful new approach to make a variety of vaccines against viral, parasitic, and bacterial infections. In addition these vectors provide a novel set of tools for us to dissect the immune responses against mycobacterial infections and generate a better tuberculosis vaccine.

References 1. KOCH, ROBERT. 1882. Die Aetiologie der Tuberculose. Bed. klin. Wschr. XIX: 221. 2. CALMETTE, A., and C. GUERIN. 1909. C. R. Acad. Sci., Paris 149: 716. 3. STEENKEN, W., W. H. OATWAY, and S. A. PETROFF. 1934. Biological studies of the tubercle bacillus. III. Dissociation and pathogenicity of the Rand S variants of the human tubercle bacillus (H37). J. Exp. Med. 60: 515. 4. YOUNG, R. A., B. R. BLOOM, C. M. GROSSKINSKY, J. IVANYI, D. THOMAS, and R. W. DAVIS. 1985. Dissection of Mycobacterium tuberculosis antigens using recombinant DNA. Proc. Nat!' Acad. Sci. USA 82: 2583. 5. YOUNG, R. A., V. MEHRA, D. SWEETZER, T. BUCHANAN, J. E. CLARK-CURTISS, R. W. DAVIS, and B. R. BLOOM. 1985. Genes for the major protein antigens of the leprosy parasite, Mycobacterium leprae. Nature 316: 450. 6. JACOBS, W. R., M. A. DOCHERTY, R. CURTISS III, and J. E. CLARK-CURTISS. 1986. Expression of Mycobacterium leprae genes from a Streptococcus mutans promoter in Escherichia coli K -12. Proc. Nat!' Acad. Sci. USA 83: 1926. 7. JACOBS, JR., W. R., M. TUCKMAN, and B. R. BLOOM. 1987. Introduction of foreign DNA into mycobacteria using a shuttle phasmid. Nature 327: 532. 8. SNAPPER, S. B., L. LUGOSI, A. JEKKEL, R. MELTON, T. KIESER, B. R. BLOOM, and W. R. JACOBS jr. Jl988. Lysogeny and transformation of mycobacteria: Stable expression of foreign genes. Proc. Nat!' Acad. Sci. USA 85: 6987. 9. CLARK-CURTISS, J. E., W. R. JACOBS, M. A. DOCHERTY, L. R. RITCHIE, and R. CURTISS III. 1985. Molecular analysis of DNA and construction of genomic libraries of Mycobacterium leprae. J. Bact. 181: 1093. 10. JACOBS, W. R., M. A. DOCHERTY, R. CURTISS III, and J. E. CLARK-CURTISS. 1986. Expression of Mycobacterium leprae genes from a Streptococcus mutans promoter in Escherichia coli K-12. Proc. Nat!. Acad. Sci. USA 83: 1926. 11. SNAPPER, S. B., R. E. MELTON, S. MUSTAFA, T. KIESER, and W. R. JACOBS JR. 1990. Isolation and characterization of efficient plasmid transformation mutants of Mycobacterium smegmatis. Molec. Microbio!. 4: 1911. 12. BARLETTA, R. G., S. B. SNAPPER, J. D. CIRILLO, N. D. CONNELL, D. D. KIM, W. R. JACOBS, JR., and B. R. BLOOM. 1990. Recombinant BCG as a candidate oral vaccine vector. Res. Microbio!. 141: 931. 13. HUSSON, R. N., B. E.JAMES, and R. A. YOUNG. 1990. Gene replacement and expression of foreign DNA in mycobacteria. J. Bact. 172: 519. 14. MARTIN, c., J. TIMM, J. RAUZIER, R. GOMEZ-Luz, J. DAVIES, and B. GICQUEL. 1990. Transposition of an antibiotic resistance element in"mycobacteria. Nature 345: 739. 18. GANJAM K., B. R. BLOOM, and W. R. JACOBS JR. 1991. Insertional mutagenesis and illegitimate recombination in mycobacteria. Proc. Nat!' Acad. Sci. USA, 88: 5433. 19. LEE, M. H., L. PASCOPELLA, W. R. JACOBS JR., and G. F. HATFULL. 1991. Site-specific integration of mycobacteriophage L5: Integration-proficient vectors for Mycobacterium smegmatis, BCG, and M. tuberculosis. Proc. Nat!' Acad. Sci. USA 88: 3111. 20. STOVER, C. K., V. F. DE LA CRUZ, T. R. FUERST, J. E. BURLEIN, L. A. BENSON, L. T. BENNETT, G. P. BANSAL, J. F. YOUNG, M. H. LEE, G. F. HATFULL, S. B. SNAPPER, R. G. BARLETTA, W. R. JACOBS JR., and B. R. BLOOM. 1991. New use of BCG as a vector for vaccines. Nature 351: 456.

156 . WILLIAM R. JACOBS, Jr. 21. BELISLE, J. T., L. PASCOPELLA, J. IMAMINE, P. J. BRENNAN, and W. R. JACOBS, JR. 1991. Cloning of biosynthetic genes of complex surface glycolipids of mycobacterium. J. Bact. 173: 6991. Dr. WILLIAM R. JACOBS, JR., Howard Hughes Medical Institute, Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, New York 10461, USA