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Unveiling the Survival Strategies of the World's Most Effective Pathogen, Mycobacterium tuberculosis
Summary: William Jacobs has developed novel genetic approaches to make mutations and transfer genes in M. tuberculosis. With these tools, he has identified drug targets and novel virulence factors of M. tuberculosis, many of which are enzymes or products of complex lipid metabolism. These complex lipids are unique among bacterial pathogens and likely to contribute significantly to the pathogenic property of mycobacteria.
Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), has evolved to become the world's most successful pathogen. Because of its highly infectious nature and its ability to establish persistent infections in individuals with healthy immune systems, M. tuberculosis has infected more than one-third of the world's population, according to World Health Organization (WHO) estimates. Despite a highly effective chemotherapy and a vaccine of variable efficacy, TB continues to be one of the leading infectious causes of death in the world today. The vast majority of infected individuals are subclinically infected, with no symptoms. However, when these people suffer some compromise in their immune systems, they often develop TB, and this promotes the global increase in TB. Combined with these increases, strains of M. tuberculosis have emerged that are resistant to two or more antituberculosis drugs, typically called multidrug-resistant tuberculosis (MDR-TB). The combination of increasing incidence of TB due to increasing numbers of HIV infections and the emergence of MDR-TB prompted WHO to declare TB a global health emergency in 1993, a distinction never accorded to another disease. Clearly, novel interventions are needed.
Development of novel interventions requires new knowledge. For example, novel vaccines will require knowledge of protective antigens and of protective effector mechanisms. Understanding the mechanisms by which M. tuberculosis infects susceptible hosts, establishes and maintains persistent infections, and resists both innate and adaptive immune responses should aid in vaccine design and interventions. Knowing how M. tuberculosis is susceptible to existing drugs and how it develops resistance to these drugs, and identifying new targets, will lead to the development of improved and novel drugs and drug regimens.
Mycobacteriophages: Functional and Genomic Gold from Dirt
Viruses that infect mycobacteria (mycobacteriophages) were used by our lab to develop the first gene transfer systems for mycobacteria. We have also used mycobacteriophage vectors to generate (1) luciferase reporter phages for the rapid detection of M. tuberculosis and the assessment of their drug susceptibilities in developing world laboratories, (2) transposon mutagenesis systems for generating libraries of random mutants, and (3) specialized transducing phages for efficient generation of allelic exchanges in M. tuberculosis, a system that overcomes a highly efficient illegitimate recombination system of M. tuberculosis. The availability of the mycobacterial genome sequences and the ability to generate transposon mutants, targeted gene disruptions, and complementation analyses provide an unprecedented opportunity for the elucidation of the functions of mycobacterial genes. An understanding of gene function will provide knowledge needed to develop novel strategies to control TB.
Mycobacteriophages are a unique and novel set of unexplored evolutionary material. In collaboration with Graham Hatfull (HHMI Professor, University of Pittsburgh), we have isolated, characterized, and determined the DNA sequences of 14 independent mycobacteriophages. Most of these phages were discovered by our labs in soil samples from such diverse places as backyards, barnyards, or the Bronx Zoo. Several phages were isolated by high school students working in each lab. In collaboration with Reid Schwebach (Albert Einstein College of Medicine), we have developed Phagefinders, a summer research program for high school students. July 2004 marks our third year of this program. Surprisingly, only 50 percent of the genes in the mycobacteriophage database have homologs in the total genomic database, demonstrating a rich source of unexplored biological diversity. Astonishingly, the phage Bxz1, isolated from a zebra field at the Bronx Zoo, possesses a homolog of the human gene that encodes the protein Ro, to which autoantibodies are generated with the disease lupus. The discovery in Bxz1 may lead to understanding of the function of human Ro. Since it is estimated that 1031 phage particles exist on planet Earth, making them the most abundant biological entity, they merit continued study.
Growth and Multiplication in Mammalian Hosts: Overcoming Host Innate and Adaptive Immune Responses
M. tuberculosis has evolved the functions required to infect, replicate, and multiply in mammalian cells in the face of innate immune responses. Moreover, the tubercle bacillus has evolved strategies to survive an adaptive immune response. We have used a variety of mutagenesis strategies and screens to identify novel genes of M. tuberculosis required for these virulent functions. For example, we have found that M. tuberculosis must make most amino acids in order to replicate in mammalian cells, thus defining intracellular growth requirements. In addition, we have identified a number of new M. tuberculosis mutants that are defective in the ability to cause necrosis of lung epithelial cells. Studies aimed at elucidating the necrosis mechanisms should lead to an understanding of how M. tuberculosis spreads and causes pathology.
Additional studies suggest that M. tuberculosis can secrete both offensive and defensive weapons that protect it from innate immune responses. Genes required for the synthesis and export of unique lipids of M. tuberculosis, such as phthiocerol dimycocerosate (PDIM), are essential for lung growth. We have also discovered a novel accessory secretion system that utilizes a homolog of the well-characterized SecA protein and demonstrated that the secA2 gene is required for virulence. Two of the proteins that are not secreted include catalase peroxidase and superoxide dismutase, proteins that cells use to protect themselves from oxidative killing mechanisms. This secretion system appears to be common to all gram-positive pathogens. Thus, secreted lipids and secreted proteins appear to play roles for survival in the face of innate immune responses. We also hypothesize that M. tuberculosis has mechanisms to protect it from adaptive immune responses. Our first such candidate gene, pcaA, encodes an enzyme required to decorate mycolic acids with specific cyclopropanes. Mutants of M. tuberculosis that lack this enzyme grow normally in the acute phase of M. tuberculosis growth but fail to cause a persistent long-term infection. These studies suggest that these cyclopropanated lipids hide M. tuberculosis from a sterilizing immune response. We are devoting considerable efforts to identifying other mechanisms that M. tuberculosis uses to evade host immune responses.
Evading Killing Effects of the Antituberculosis Drug Isoniazid
Isoniazid (INH) remains one of the frontline drugs in treating TB with potent bacteriocidal activity. Our lab has spent numerous years elucidating the mechanism by which INH kills M. tuberculosis cells and the mechanisms by which M. tuberculosis becomes resistant to INH. We had identified a candidate target gene, inhA, and demonstrated that resistance could be mediated by overexpression of inhA or with structural mutations within inhA. Biochemical analyses allowed us to determine that inhA encodes an enoyl reductase, an enzyme involved in mycolic acid biosynthesis. INH is a prodrug that is activated by a catalase peroxidase enzyme. In collaboration with James Sacchettini (Texas A&M; University), we have determined the three-dimensional structure of the InhA protein and have shown that the activated INH binds to NAD, which forms an adduct that inhibits the InhA enzyme. Knowledge of the target inhibition is leading to numerous structural studies to identify novel inhibitors and new TB drugs. An understanding of INH action will require knowledge of the entire set of mutations that confer INH resistance, which is a complex phenotype. Resistance to or susceptibility to INH has only been shown to be transferable with alleles of katG or inhA in M. tuberculosis strains, and yet there exist many INH-resistant clinical isolates for which no mutated alleles of katG or inhA have been described. We are attempting to identify the unknown INH-resistance alleles and to elucidate novel mechanisms of INH resistance. Such knowledge should lead to an understanding of the mechanisms by which INH leads to the lysis of M. tuberculosis cells and to strategies to make novel antituberculosis agents.
Development of Tuberculosis Vaccine Candidates: Remaking BCG from M. tuberculosis
We want to test the hypothesis that an attenuated M. tuberculosis strain when used as a vaccine might elicit a more robust immune response to challenge with M. tuberculosis than vaccines derived from an M. bovis BCG (bacillus Calmette–Guérin) strain. Moreover, by deleting genes encoding immunosuppressive functions, we hypothesize it will be possible to make the vaccine candidate more immunogenic. To test these hypotheses, we have constructed deletion mutants of M. tuberculosis. By deleting the primary attenuating mutation of BCG, namely ΔRD1, and the individual genes in this region, we have demonstrated that this region encodes secreted proteins and secretion systems to secrete the proteins that mediate necrosis and egress of M. tuberculosis outside of lung epithelial cells and macrophages. To make the resulting strain safer and more immunogenic, we have deleted genes required for the synthesis of the vitamin pantothenate. The resulting strain mc26030 contains both the RD1 deletion and the deletion of panCD genes and is significantly safer than BCG in immunocompromised mice. We are evaluating the efficacy of these strains to protect animals against aerosol challenges with virulent M. tuberculosis. To enhance immunogenicity, we are also incorporating a number of newly discovered mutations that inactivate functions that alter immunomodulating functions of M. tuberculosis.
This work is also supported by a grant from the National Institutes of Health and the Ellison Medical Foundation.
Last updated: August 1, 2007
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