Date Submitted
2017
Research Mentor and Department
Jennifer A. Philips, MD, PhD; Division of Infectious Diseases, Department of Medicine, Washington University School of Medicine
Restricted/Unrestricted
Unrestricted
Abstract
Mycobacterium tuberculosis infects one-third of the world’s population and causes an estimated 2 million deaths per year, more than any other single bacterial pathogen. The inadequacies of existing tuberculosis therapies demand the discovery of novel agents to treat M. tuberculosis infection, which requires mechanistic insight into the pathways involved in mycobacterial pathogenesis. We identified an unanticipated role of the M. tuberculosis protein CpsA in preventing phago-lysosome fusion by host macrophages, resulting in enhanced intracellular survival of bacteria within the host. Strains of M. tuberculosis lacking cpsA are severely attenuated in both macrophage and mouse models, thus establishing CpsA as a crucial virulence factor in M. tuberculosis infection. CpsA is a yet uncharacterized secreted protein, belonging to the LytR–CpsA–PsR family, that we found interacts with host proteins NDP52 and TAX1BP1, which are involved in intracellular trafficking. Here, we sought to (1) examine whether CpsA is sufficient to confer virulence to Mycobacterium smegmatis, a rapidly-growing, non-pathogenic bacterium that does not contain CpsA naturally, and (2) determine which domains of CpsA are required for interacting with host proteins and contributing to virulence. We infected RAW264.7 macrophages with M. smegmatis expressing M. tuberculosis cpsA and vector control and scored for intracellular survival of bacteria. Our preliminary data suggest that CpsA confers enhanced intracellular survival to M. smegmatis, as compared to vector control. To characterize CpsA interaction with TAX1BP1 and NDP52, we generated deletion constructs of CpsA and, using a yeast two-hybrid system, investigated which domains of the CpsA protein are required for the interaction with these host proteins. Follow up work involves determining which domains are also required for virulence. Overall, we hope that by studying the molecular mechanisms by which M. tuberculosis CpsA sabotages cellular functions, we can better understand both the host immunity and mycobacterial pathogenesis.