Lab Research Focus
M. tuberculosis, the etiological agent of TB is one of the most effective human pathogens and is responsible for 2.2 million deaths every year (1 death every 10 seconds). It is estimated that a total of 225 million new cases and 79 million deaths will occur between 1998 and 2030. Tuberculosis (TB) continues to pose a significant threat to mankind that cannot be conquered without an effective vaccine strategy, which remains unavailable to date. The prophylactic vaccine BCG (Bacille Calmette-Guerin), fails to protect against the most common form of disease, adult pulmonary tuberculosis, and its efficacy varies dramatically e.g., from 0% in South India to 80% in the UK. Since the lungs of an infected patient contain more than a billion bacilli, poor treatment compliance can easily lead to multi drug resistant (MDR) strains. The cost for treating a patient infected with MDR-TB (currently designated as Category C Priority pathogens) can approach $250,000 – about 10-15 times the cost for treating a drug-sensitive case. One of the greatest risk factors for TB is HIV, which increases the risk of developing tuberculosis 30-fold.
Our long-term goal is to understand the mechanisms of M. tuberculosis virulence. The first project involves a gene, whiB3, which appears to play a unique role in the bacterium-host interaction. We established that WhiB3 interacts with the 4.2 domain of the principal sigma factor, RpoV in virulent M. tuberculosis, but not with RpoV of an attenuated strain containing a single point mutation (Arg515-His) in the 4.2 domain. Our studies showed that the M. tuberculosis whiB3 mutant behaved identical to the wild-type strain with respect to its ability to replicate in mice, but was attenuated in terms of host survival. In addition, the whiB3 mutant strain showed much reduced lung pathology, compared to wild type infected mice. Intriguingly, we showed that a whiB3 mutant of virulent Mycobacterium bovis was completely impaired for growth in guinea pigs. These mutants define a new class of virulence genes in M. tuberculosis and M. bovis. M. tuberculosis contains seven WhiB homologues that show strong similarity to Streptomyces spp. proteins that are required for sporulation. We hypothesize that WhiB3 controls a subset of genes required for virulence. It is notable that this virulence gene would not have been detected using conventional screens such as signature mutagenesis, which screen primarily for mutants defective in growth and not virulence.
A second project in the laboratory involves the development and application of "in vivo expression technology (IVET)" to rapidly identify M. tuberculosis genes that are specifically induced during infection in vivo. Since many bacterial virulence determinants share a unique phenotype - induction in the host; the development of such a system for mycobacteria would be of significance. Subsequently, we have successfully developed a genetic system, which uses the animal as a selective medium to identify M. tuberculosis genes specifically induced during infection. These in vivo induced genes are poorly expressed on laboratory medium, but exhibit elevated levels of expression in host tissues and suggest that they contribute to growth in restricted host tissues and thus enhance pathogenicity.
A third project in the laboratory is studying signal transduction pathways in M. tuberculosis. In most bacterial two-component systems, the signals or components of the two-component signaling pathways are mostly uncharacterized. We hypothesized that accessory proteins communicate directly, through direct protein-protein communication with the M. tuberculosis two component histidine kinases to modulate gene expression. Subsequently, we have shown that the sensing module of the M. tuberculosis histidine kinase KdpD specifically interacts with two membrane proteins, and that the N-terminal sensing module of KdpD and the histidine kinase domain of KdpD form a ternary complex with these membrane proteins. Our results suggest that the membrane proteins function as accessory or ligand-binding proteins that communicate directly with the sensing domain of KdpD to modulate kdp expression.
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