Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is one of the most remarkably well-adapted and successful human pathogens known. It has infected 2 billion people worldwide, leading to complex clinical disease that ranges from asymptomatic, latent TB infection (LTBI) to chronic granulomatous disease, the latter resulting in severe lung damage and respiratory insufficiency. This disease complexity reveals an inherent metabolic flexibility in the tubercle bacillus, which allows for the establishment of phenotypically heterogeneous bacterial subpopulations during infection. It is our hypothesis that these bacterial subpopulations grow at different rates, and in the case of latent infection, it has been postulated that the infecting organisms adopt a nonreplicating dormant state, thus allowing for subversion of the immune response. Additionally, these nonreplicating bacteria are very difficult to eradicate because they are inherently tolerant to most antibiotics, which are usually effective only on replicating organisms. The presence of these residual organisms during infection is problematic because they have the potential to reactivate growth, leading to bacterial proliferation and full-blown disease. It is important to note that the majority of infected individuals develop LTBI and carry a defined lifetime risk of developing active TB, a risk that is significantly higher in the presence of HIV coinfection. Consequently, the ability to switch between active growth and dormancy will have dramatic consequences on disease progression and treatment outcome.
Because of the limited understanding of mycobacterial physiology during dormancy, few antibiotics have been developed to treat LTBI or prevent reactivation of disease in latently infected individuals. My previous work focused on trying to understand the molecular events that lead to reactivation of dormant bacteria. In this regard, I studied the biological functions of a group of proteins termed resuscitation promoting factors (Rpfs), which have been implicated in reactivation from dormancy in other bacterial species. Rpfs are small, secreted cell wall-associated proteins that have the ability to stimulate the growth of dormant bacteria. M. tuberculosis encodes five distinct Rpfs, and our studies revealed that these proteins are collectively dispensable for growth and survival in vitro. We also demonstrated that Rpfs are essential for the reactivation of bacteria from dormancy and confirmed that they are collectively required for pathogenesis in the mouse model of TB infection.
Research by other groups in the field revealed that Rpfs cleave peptidoglycan (PG) in the bacterial cell wall; however, the specific mechanism through which these proteins mediate reactivation of growth remains elusive. The PG in bacterial cell walls is a polymer made up of cross-linked sugar molecules, which comprises the glycan component, and stem peptides consisting of amino acids with various modifications. Rpfs are predicted to cleave the glycan, which could directly result in reactivation of bacterial growth or, alternatively, lead to the production of PG breakdown products that may serve as growth stimulatory molecules. Deletion of multiple rpf genes in M. tuberculosis also results in increased susceptibility to antibiotics such as vancomycin, suggesting significant alterations in PG structure or stability. The emerging picture from these studies is that remodeling of the PG in the cell wall of M. tuberculosis is an essential process for pathogenesis, reactivation, and drug tolerance.
Biosynthesis and remodeling of the PG are complex processes that require the coordinated activity of many different enzymes during bacterial growth and reactivation from the dormant state. Indeed, it has been shown that two of the five Rpfs in M. tuberculosis interact with an essential cell wall peptidase resulting in synergistic PG degradation during cell division. These data suggest that in addition to Rpfs, other PG-degrading enzymes may also be important for PG remodeling and reactivation from dormancy. Therefore, my current research focuses on the role of PG-degrading amidases and carboxypeptidases in PG synthesis, degradation, and remodeling. My lab will further assess the role of these enzymes in pathogenesis and reactivation from the dormant state. We will also study the metabolic consequences of removing these PG-degrading enzymes, specifically focusing on PG assembly and amino acid biosynthesis. The underlying hypothesis for these studies is that PG degradation and remodeling are critical for growth, virulence, and reactivation of disease.
The presence of bacterial subpopulations in differential growth states, within one infected individual, has been recently confirmed. Sputum samples from patients with active TB, before the initiation of treatment, contain a significant amount of organisms that are dependent on Rpfs for growth. This finding has significant consequences for the diagnosis of active disease and determination of the sterilizing activity of drugs. These data further suggest that Rpfs could play a critical role in TB transmission and subsequent disease. We intend to study these phenomena further in a defined patient cohort from South Africa.
Collectively, our studies are aimed at further describing microbial physiology during the different stages of TB infection. We anticipate that our work will lead to the identification and validation of new drug targets, with novel modes of action, which could form the basis of a new treatment regimen to finally eradicate TB from human society.
Grants from the South African Medical Research Council, University of the Witwatersrand, National Health Laboratory Service, and National Research Foundation of South Africa supported this work.
As of January 17, 2012