Research Interests:
My research interest for the past ten years has been on two major areas:
a) molecular pathogenesis of mycobacteria, and b) Mechanisms of cytadherence in Mycoplasma genitalium. Following are the summaries of research projects that are currently underway in my laboratory.

1. Role of Msrs in the intracellular survival of mycobacteria.  Pathogenic mycobacteria like M. tuberculosis and M. leprae have the ability to survive and replicate within mononuclear phagocytes including macrophages. The intracellular environment is very hostile since macrophages have an impressive battery of antimicrobial responses such as hydrolases, bacterial peptides and toxic reactive oxygen and nitrogen intermediates (ROI). Elucidation of the mechanism by which intracellular mycobacteria evade macrophage responses, particularly the bacterial factors associated with evasion, is central to the identification of novel vaccine and therapeutic targets. My laboratory investigates the role of methionine sulfoxide reductases (Msrs). Msrs are antioxidant repair enzymes which reduce oxidized methionine (Met-O) in proteins to methionine (Met). Since oxidation of methionine in proteins impairs the function of proteins, absence of Msr, particularly MsrA, leads to functional abnormalities in different organisms. We are investigating how absence of Msrs affects the intracellular survival of mycobacteria. We have disrupted the gene coding for MsrA in M. smegmatis, a mycobacterium which has the ability to survive inside macrophages but not to replicate, and demonstrated that MsrA is important for the survival of this species in macrophages. Using confocal microscopy and biochemical approaches we identified that phagosomes of msrA mutant M. smegmatis strain acquired the ROS generating enzymes like p67phox (component of NADPH oxidase) and iNOS (inducible nitric oxidase synthase) much earlier than the phagosomes of the wild-type strain. This indicated that some surface proteins of M. smegmatis that normally subvert the phagosomal trafficking of p67phox and iNOS have been affected in the msrA mutant strain of M. smegmatis. Currently we use proteomic approach to identify the affected proteins in this species. We are also working on the disruption of msrA in M. tuberculosis to further determine its role in the intracellular survival. We collaborate very closely with Dr. Jagannath UTHSC-Houston and Dr. Hart, UTHSC-San Antonio for cell biologic and crystallographic studies, respectively, in this project. It is expected that the project will shed light on the pathogenic mechanisms associated with surface proteins of mycobacteria.

2. Stress signaling in M. tuberculosis. During the infectious process, M. tuberculosis undergoes a variety of stress conditions. Of particular interests are the intracellular stress and the dormancy stress in the tubercle granulomas. The latter has been speculated to be due to deprivation of oxygen and nutrients. A sigma factor SigF and a two-component system DevS-DevR have been implicated in the dormancy of M. tuberculosis. However, how these regulators receive stress signals remain obscure. In my laboratory we investigate how SigF receives stress signals. Using protein database searches and protein-protein interaction studies (yeast two-hybrid system), we have identified six new molecules that have putative functions in stress signaling. Further characterization of these molecules in the activation of SigF is underway. We also investigate the role of Obg in stress signaling in M. tuberculosis. Obg is a Rho or Ras like signal molecule which plays critical role in the initiation of sporulation in Bacillus subtilis by affecting the phosphorelay system. Obg also plays critical role in events leading to aerial mycelium formation and subsequent sporulation of Streptomyces griseus and S. coelicolor, which are phylogenetically related to mycobacteria. We have demonstrated that M. tuberculosis Obg autophosphorylates similar to Ras and Rho proteins. We have also evidences that Obg physically interacts with RsbW kinase, which is an anti-sigma factor for SigF. Currently we investigate the effect of Obg on other proteins of M. tuberculosis using DNA microarray and 2D electrophoresis. It is expected that this study will identify the putative proteins which are under the regulatory control of Obg. Overall this project will lead to a better understanding of the mechanism of stress signaling in M. tuberculosis.

3. Mechanisms of cytadherence in Mycoplasma genitalium. Mycoplasma genitalium, the smallest known self-replicating organism, is a cell wall-less bacteria of the class Mollicutes and a causative agent for non-gonococcal urethritis in human. Pathogenic mycoplasmas, including M. genitalium, possess tip or terminal attachment organelle to adhere (attach) with eukaryotic cells. The tip structure has been best studied in M. pneumoniae. Spontaneous cytadherence-negative M. pneumoniae mutants not only display morphologically abnormal tip-structures but also lack, or cannot functionally mobilize, adhesins (P1 and P30) and cytadherence-accessory proteins (HMW1, HMW2, HMW3, B and C). HMW1, HMW2 and HMW3 maintain the integrity of the M. pneumoniae tip organelle and facilitate clustering and anchoring of adhesins at the tip. A spontaneous frame shift mutation or transposon insertion in hmw2 leads to proteolysis of HMW1, HMW3 and P65 proteins, further reinforcing critical structural and functional relationships among cytadherence-implicated proteins. M. genitalium protein homologues corresponding to P1 and P30 adhesins of M. pneumoniae have been identified and designated P140 and P32, respectively. As with M. pneumoniae, specific classes of spontaneous M. genitalium mutants deficient in cytadherence lack functional P140, thus reinforcing its fundamental importance in cytadherence. In addition, both P140 (140 kDa) and P32 (32 kDa) adhesins of M. genitalium exhibit structural and functional similarities to adhesins P1 (169 kDa) and P30 (30 kDa) of M. pneumoniae. Further, we recently reported that targeted disruption of protein MG218 (190 kDa) of M. genitalium, an orthologue of HMW2 of M. pneumoniae, resulted in increased turnover of adhesin P140 and its operon-related protein P110. The latter is encoded by gene mg192 located downstream of mg191 which encodes P140. Thus, disruption of mg218 in M. genitalium results in a phenotype similar, but not identical, to HMW2-deficient M. pneumoniae. One of the intriguing similarities between cytadherence-related genes of M. pneumoniae and M. genitalium is their existence in three clusters at three distinct chromosomal regions. In M. pneumoniae, gene mpn141, encoding P1 adhesin, is flanked by two genes, upstream mpn140 (28 kDa) and downstream mpn142 (130 kDa). Similarly, gene mg191, which encodes M. genitalium P140 adhesin (P1 homologue), is flanked by upstream gene mg190 (37 kDa) and downstream gene mg192 (114 kDa). The second cluster of cytadherence-related M. pneumoniae genes (4 genes including hmw2) and third cluster (9 genes including hmw1, p30 and hmw3) are also highly conserved in M. genitalium. We have evidences that the adherence related gene clusters constitute operons in both M. pneumoniae. and M. genitalium. We are now determining the role each gene in the operons in relation to cytadherence. We use targeted gene disruption technology to disrupt each gene in the operon. It is hoped that this project will ultimately enhance our knowledge about the mechanism of cytadherence in mycoplasma.