John D. McKinney, Ph.D.
The Rockefeller University

Genetic Analysis of TB Persistence: Identification of New Drug Targets.

“Following infection, the incubation period of tuberculosis ranges from a few weeks to a lifetime.” This remark from a leading epidemiologist encapsulates the chief mystery and challenge of tuberculosis (TB): the ability of the pathogen to persist indefinitely in the tissues in the face of the acquired immune response. Although in most cases infection is effectively contained by host immunity, failure to eliminate the “enemy within” means that TB can flare up again if the immune system is weakened. Nearly 2 billion individuals worldwide, including 10-15 million in the US alone, are asymptomatically infected with Mycobacterium tuberculosis. Over the course of a lifetime, 100-200 million of these latent infections will reactivate and develop into full-blown TB – an enormous burden of future disease arising from infections that are already established. At present, virtually nothing is being done to reduce this vast and pervasive reservoir of contagion because effective and practicable tools are lacking. In recognition of this unmet need, the National Academy’s Institute of Medicine in a recent report stressed that “The first priority for research is development of an understanding of latent infection.”

What are the immune mechanisms that maintain TB latency and block reactivation? Why is infection contained but not eradicated? What is the physiologic state of persistent mycobacteria? What are the mechanisms that defend the pathogen against the onslaught of host immunity? We are taking a molecular-genetic approach to address these “persisting problems” in TB. Our studies exploit recent technological advances that permit the direct analysis of M. tuberculosis in its natural environment, the mammalian lung. A long-term goal of our research is the development of new and more effective strategies for TB control. With 10 million new cases and 2-3 million deaths each year attributed to TB, the need could hardly be greater.

In vivo drug tolerance of M. tuberculosis. One of the key obstacles to TB control is the inadequacy of current drug therapies. Effective treatment of TB requires administration of multiple drugs for at least 6 months, a regimen that many patients are unwilling or unable to complete without close supervision. Prophylactic therapy of latent TB, where the patient has no clinical signs or symptoms, is especially problematic. Why is TB so difficult to cure? Our recent studies indicate that during later stages of infection mycobacteria may be in the stationary phase of growth. This could explain the recalcitrance of in vivo mycobacteria to conventional antimicrobials, which target cellular growth processes such as DNA replication and cell wall biogenesis. If correct, then development of “better, faster, cheaper” therapies for TB will hinge on the elucidation of bacterial pathways that are essential for non-dividing persistence in the lungs.

In vivo metabolism of persistent mycobacteria. The term “parasite” is derived from the Greek parasitos, meaning “One who eats at another’s table.” This definition underscores a central but poorly understood feature of the parasitic lifestyle, the exploitation of the host as a substrate to fuel the pathogen’s metabolism and growth. We have undertaken a systematic analysis of the in vivo metabolism of M. tuberculosis. Our studies to date indicate that in vivo mycobacteria switch to a diet of fatty acids at late stages of infection. This switch is triggered by the host immune response and mutant bacteria that cannot make the switch fail to persist. One of the pathways involved, the glyoxylate cycle, is an attractive target for drug development because it is absent in human cells. Current efforts are focused on elucidation of the host mechanism that forces the switch in bacterial metabolism, and development of glyoxylate cycle inhibitors as potential anti-TB drugs.

Mycobacterial persistence factors. TB infection is biphasic: an early acute phase of exponential bacterial growth in the lungs leads into a prolonged chronic phase in which bacterial numbers are stabilized by the emergent host immune response. The ability to persist indefinitely in the lungs is the key feature of TB pathogenesis, yet little is known about the mechanisms involved. Our studies on the glyoxylate cycle indicate that genetically distinct pathways are required for early-stage growth and late-stage persistence. Building on this conceptual foundation, we have initiated genetic screens to identify other pathways that are specifically required for late-stage persistence of M. tuberculosis in the lungs. The “persistence factors” identified in these screens may also be attractive targets for development of novel anti-persistence drugs.

Immune evasion. The ability to persist indefinitely in the lungs of healthy individuals indicates that M. tuberculosis has evolved effective mechanisms of defense against the onslaught of the immune response. We have initiated genetic screens to identify mycobacterial “defense factors” by comparing the growth and persistence of transposon-induced mutants in normal mice v. mice with specific immune deficiencies. These studies will elucidate the biology of the host-pathogen interface, and could point the way to new strategies to enhance the immune system’s ability to kill persistent mycobacteria.

In vivo gene statement of M. tuberculosis. We are analyzing M. tuberculosis gene statement in mouse and human tissues via quantitative real-time RT-PCR with fluorescent probes (“molecular beacons”). Our studies indicate that adaptation of M. tuberculosis to life in the lung involves induction of pathways for alternative carbon metabolism (including the glyoxylate cycle, fatty acid b-oxidation cycle, and gluconeogenesis), iron scavenging, and microaerobic stress response. We have uncovered significant differences in M. tuberculosis gene statement in mouse v. human lungs, which underscores the importance of studying the pathogenesis of infectious disease in the natural host. Our current efforts are focused on the comparative analysis of M. tuberculosis gene statement in the lungs of humans with latent infection v. active disease. Our aim is to identify mycobacterial correlates of protection and pathogenesis, respectively, as a powerful new metric for evaluation of candidate TB vaccines.

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