Structural characterization of the elongasome and divisome in mycobacteria

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), is one of the leading causes of death due to infectious disease. Antibiotic treatment of Mtb is arduous and multidrug-resistant strains are on the rise, thus fueling the need for novel therapeutics. During infection, subpopulations of Mtb cells are phenotypically heterogeneous and can survive extensive chemical regimens, thus preventing the cure of disease. Cellular heterogeneity arises from the asymmetric growth and division of the bacterium, resulting in daughter cells with varying characteristics. As mycobacteria replicate, they must coordinate the division complex (divisome) and elongation machinery (elongasome) spatially and temporally for proper cell division. These protein systems share some similarities with the systems found in Escherichia coli and Bacillus subtilis, which grow and divide symmetrically. However, mycobacteria lack several major elongasome and divisome components that are found in these model bacteria, and components that are shared contain substantial N- and C-terminal expansions, which may interact with other co-factors. Immunoprecipitation and light microscopy suggest that the mycobacterial elongasome and divisome components potentially crosstalk with one another and form large multi- subunit protein complexes. PonA1 and CrgA are critical proteins of both the elongasome and divisome and are thought to make a multitude of interactions with other proteins in these systems. However, how these components coordinate and interact to support asymmetric growth and division is not well understood at a structural and mechanistic level. This proposal is focused on the biochemical and structural characterization of the elongasome and divisome from Mtb and the non-pathogenic model mycobacterium, Mycobacterium smegmatis. Bacterial genetics and endogenous protein purifications will be employed to purify PonA1, CrgA, and their associated complexes directly from mycobacterial cells, and single-particle cryo-electron microscopy will be used to determine their 3D structures (Aim 1). Novel proximity labeling will be utilized to define the protein- protein interaction networks of divisome and elongasome components and to decipher the roles of their N- and C-terminal expansions (Aim 2). Cutting-edge cryo-electron tomography will be implemented to capture 3D snapshots of dividing mycobacteria to determine how the elongasome and divisome proteins are structured and organized in situ (Aim 3). This proposal is innovative in that it applies emerging techniques to provide a more complete picture of the protein complexes that constitute the elongasome and divisome and how they interact to facilitate asymmetric growth and division in mycobacteria. This work will provide important insights into the structures of these multi-protein complexes that can be used for the design of new therapeutics for TB. Project Number: 1DP2AI184740-01 | Fiscal Year: 2025 | NIH Institute/Center: National Institute of Allergy and Infectious Diseases (NIAID) | Principal Investigator: James Chen | Institution: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO, SAN FRANCISCO, CA | Award Amount: $492,000 | Activity Code: DP2 | Study Section: Special Emphasis Panel[ZAI1 VSR-D (M1)] View on NIH RePORTER: https://reporter.nih.gov/project-details/1DP2AI18474001