DESCRIPTION (provided by applicant): The cell envelope of Mycobacterium tuberculosis (Mtb) is the basis of many of the physiological and pathogenic features of this bacterial pathogen and the site of susceptibility and resistance to many anti- tuberculosis drugs. In spite of being Gram-positive bacteria, mycobacteria are unique in having a cell wall devoid of (lipo)teichoic acids and instead containing a heteropolysaccharide known as the arabinogalactan (AG) covalently attached to peptidoglycan (PG). To this date, the cell wall ligase(s) responsible for the covalent attachment of these two macromolecules has/have defied definition. Despite the fundamental structural differences that exist between AG and wall teichoic acids (WTA), the structure of the unit linking AG to PG in mycobacteria shares similarities with the linker involved in the covalent attachment of WTA to PG in many Gram-positive bacteria. Enzymes of the widespread LytR-Cps2A-Psr (LCP) family were recently identified as the likely ligases mediating WTA-PG attachment in Bacillus subtilis and Staphylococcus aureus. We identified three LCP-like proteins in the genome of Mtb H37Rv, one of them mapping to an AG biosynthetic gene cluster. We here propose to use a combination of genetic and biochemical approaches to determine whether these three LCP homologs are the long sought mycobacterial cell wall ligases and to define their therapeutic potential. In particula, we will test whether two novel antibacterial compounds, caprazamycin B and CPZEN-45, products of a collaboration with the Institute of Microbial Chemistry (BIKAKEN, Tokyo, Japan) that inhibit mechanistically similar enzymes in mycobacterial cell wall assembly (MraY and WecA, respectively) may represent promising scaffolds for the future development of inhibitors targeting the assembly of the mycobacterial cell wall. Similar to the situation with WTA ligases, i is likely that the ligase(s) of Mtb interact(s) with other wall proteins to coordinate cell wall synthesis with cell elongation and cell division. The characterization of Mtb's ligase(s) therefore also represents an important first step toward the elucidation of this key aspect of the physiology of mycobacteria and the future design of innovative therapeutic strategies aimed at targeting cell elongation and division.

PUBLIC HEALTH RELEVANCE: In the context of the increasing incidence of multi-drug-resistant strains of Mycobacterium tuberculosis (Mtb), understanding the key physiological processes allowing this bacterium to assemble its unique cell wall and coordinate its synthesis with cell elongation and division may prove useful in designing innovative therapeutic strategies. We here propose to functionally characterize three Mtb proteins that we recently identified as the likely ligases responsible for the assembly of the cell wall core of this bacterium, and to provide the molecular bases required for the development of innovative strategies aimed at targeting cell wall assembly and cell elongation and division in mycobacteria.