There is a fundamental gap in understanding how lipid components are supplied in a spatially coordinated fashion to support the polar growth of Mycobacterium tuberculosis (Mtb). This gap represents an important problem because the elongation of the cell envelope, an essential process of bacterial growth, is incomprehensive without understanding the precise mechanism of spatially coordinated lipid synthesis, transport and cell envelope integration. The long-term goal is to elucidate the contribution of finely controlled spatiotemporal control of cell envelope elongation to Mtb infection. As the next step to achieve this goal, the overall objective of this proposal is to characterize the dynamic response of the recently discovered Intracellular Membrane Domain (IMD) during exposure to stress. The central hypothesis is that: 1) the IMD is present in pathogenic mycobacteria in discrete regions within the plasma membrane; and 2) the IMD undergoes spatial and metabolic changes in response to stress conditions. The rationale is that characterizing the dynamic nature of the IMD during the stress response will lay the foundation for understanding the role of the IMD in the regulation of cell envelope elongation, thereby beginning to understand how the robust pathogen Mtb maintains its highly complex and impermeable cell envelope. Guided by published studies and preliminary data from the applicant?s laboratory, this hypothesis will be tested by pursuing two specific aims: 1) Examine the effect of stress on the subcellular organization of Mtb IMD; 2) Determine how the proteome of Mtb IMD changes during stress exposure. Under the first aim, the working hypothesis, that Mtb spatially rearranges the IMD during exposure to known stress conditions, will be addressed by live cell fluorescence imaging. Under the second aim, how the metabolic capacity of the Mtb IMD changes when the cells are exposed to stress conditions, will be determined by the approaches of comparative proteomics. The project is conceptually innovative because it introduces a novel subcellular structure to the understanding of cell envelope biosynthesis in mycobacteria. The proposed research is significant because it reveals the IMD as a subcellular membrane structure with defined spatial arrangement and metabolic roles in Mtb. The key preliminary data obtained from this study will form the basis to further investigate the role of the IMD in cell envelope elongation of Mtb, a central feature of mycobacterial cell physiology directly relevant to the intrinsic drug resistance and pathogenesis of Mtb.
The proposed research is relevant to public health because characterizing the nature of a membrane domain in Mycobacterium tuberculosis is a step toward understanding the spatiotemporal regulation of cell envelope biogenesis and its importance in the pathogenesis of the pathogen. Thus, the proposed research is expected to provide fundamental knowledge in mycobacterial cell biology, likely revealing novel drug targets in the future in line with NIH's mission to provide therapeutic strategies against tuberculosis.
