 |
Mechanisms of Physiological Adaptation in Mycobacterium tuberculosis
Summary: Valerie Mizrahi is studying the mechanisms of DNA metabolism, culturability, and resuscitation in Mycobacterium tuberculosis, the organism that causes human tuberculosis. Development of more effective tools for TB control require understanding the mechanisms underlying TB's remarkable ability to adapt to adverse conditions and persist in a dormant state from which it can reactivate to cause disease.
It is estimated that approximately two billion people worldwide are infected with Mycobacterium tuberculosis, the causative agent of tuberculosis (TB). This staggering burden of infection, combined with the lethal synergy with HIV and the emergence and global spread of drug-resistant (including multi-drug-resistant) strains of M. tuberculosis, underscores the urgent need for new methods for managing this devastating disease. Despite recent scientific advances, it is sobering to note that the functions of as many as 48 percent of the genes of M. tuberculosis cannot, as yet, be assigned. Moreover, more than half the genes identified as important for in vivo growth of M. tuberculosis are of unknown function and many of these have no obvious homologues outside mycobacteria and closely related Actinomycetes. This inadequacy of knowledge is reiterated in many areas of mycobacterial physiology. For instance, the mechanisms of physiological adaptation of M. tuberculosis are poorly understood; they provide this organism with its remarkable ability to persist in a dormant state in subclinically infected individuals, from which it can reactivate to cause clinical disease, and to persist in a drug tolerant form in the face of continuing TB drug therapy. In the context of DNA metabolism, which is one of my laboratory's main areas of interest, the regulation of DNA metabolic pathways during growth and persistence has not been investigated to any significant extent. Furthermore, although much has been learned about the sources of genome plasticity in mycobacteria by comparisons within and between species, the mechanisms underlying genome diversification, which has a profound impact on properties such as the virulence and drug susceptibility of strains of M. tuberculosis, remain largely unexplored.
For the past several years, we have been investigating specific aspects of mycobacterial metabolism with a view to gaining a deeper understanding of the molecular mechanisms underlying the ability of this organism to adapt physiologically to the complex environments encountered during the course of infection of a human host. Each of the biological functions under investigation—DNA metabolism, culturability, resuscitation, and phenotypic drug tolerance—is served by a multiplicity of genes, whose regulation and individual and collective functions are being explored using an integrated genetic and physiological approach.
Our work in the area of DNA metabolism is geared primarily towards understanding the processes that lead to genomic variation in M. tuberculosis. Recent work has shown that the environments encountered by M. tuberculosis during infection are significantly DNA-damaging (genotoxic), producing lesions in the chromosome that must be repaired for the organism to survive in the intracellular environment of its host phagocytic cell. In many organisms, mechanisms that are induced in response to genotoxic stress play a crucial role in assisting the organism to tolerate the damage. These mechanisms involve the action of specialized DNA polymerase enzymes that may catalyze error-prone DNA replication and provide the organism with the ability to genetically adapt to conditions of stress—a property that may be of particular importance in the case of M. tuberculosis. Previous work carried out in collaboration with Helena Boshoff and Clifton Barry at the NIH demonstrated that a novel specialized DNA polymerase, DnaE2, plays the dominant role in tolerance of DNA damage caused by UV irradiation and other damaging treatments in mycobacteria, even though Y-family DNA polymerases, which are responsible for performing this function in other bacteria, are also present in mycobacteria. These findings raised fundamental questions about the cellular functions of Y-family DNA polymerases in bacteria that also harbor DnaE2. To address these questions, we constructed a large panel of mutant strains of M. tuberculosis and its non-pathogenic relative, Mycobacterium smegmatis, with altered specialized polymerase gene complements and/or expression levels; we used these mutants in studies of damage tolerance, long-term survival and mutagenesis in vitro, and growth and survival in vivo, using a murine infection model. Our work has revealed both redundancy and differentiation of function of the various specialized DNA polymerases in mycobacteria. It has also provided new insights into the DnaE2-dependent pathway for damage-induced mutagenesis and into the functional and/or regulatory interdependence between and among the specialized DNA polymerases and other proteins, which are the subjects of ongoing investigation.
A second area of interest in our laboratory concerns mycobacterial culturability and resuscitation. Although poorly understood, the molecular mechanisms by which M. tuberculosis persists or remains dormant before reactivating are thought to be determined both by host immunity and by physiological characteristics of the organism itself. M. tuberculosis contains a family of five proteins with similarity to the resuscitation-promoting factor (Rpf) of Micrococcus luteus; the factor is a secreted protein essential for growth of this organism. Various lines of evidence suggest that the M. tuberculosis homologues may play similar roles in mycobacterial growth stimulation and resuscitation. However, the extent of functional redundancy, cross-talk, and/or functional differentiation within the M. tuberculosis Rpf-like protein family is unclear. To address these questions, we produced a collection of allelic exchange mutants of M. tuberculosis harboring deletions in one or more of the rpf-like genes and, in collaboration with colleagues in the U.S.A. (Gilla Kaplan) and Russia (Arseny Kaprelyants and Alex Apt), are using these mutants to investigate the effects of progressive depletion of rpf-like gene function on growth, long-term survival, culturability (ability to form colonies on agar-solidified media), and resuscitation from a non-culturable state in various models. In parallel studies, we are using a culturability model of M. smegmatis to investigate the physiologic changes that occur during the transition of the organism to a non-culturable state, on the one hand, and during resuscitation on the other.
We are also investigating the regulation, function, and interplay of M. tuberculosis genes encoding alternate ribonucleotide reductases and methionine synthase enzymes, which exist in both vitamin B12-dependent and -independent forms, as well as the function of large family of genes which, through work in other bacteria, have been implicated in responses to stress and in phenotypic drug tolerance. It is hoped that the insights gained from our studies on M. tuberculosis physiology will inform the development of novel drugs for the treatment of TB.
Our work is also supported by grants from the National Research Foundation of South Africa, the South African Medical Research Council, the University of the Witwatersrand, and the Columbia-Southern African Fogarty AIDS International Training and Research Program (5 D43 TW00231).
Last updated August 2008
|
|
|
|
