Cell wall carbohydrate recycling in Mycobacterium tuberculosis

Project Summary A typical course of antibiotics for tuberculosis (TB) lasts for six months. Subpopulations of non/slow-replicating Mycobacterium tuberculosis have long been hypothesized to be tolerant to antibiotics and contribute to lengthy treatment. Pathways that are active in quiescent M. tuberculosis are high value targets for drug discovery, particularly extracellular components that are freely accessible to small molecules. Cell envelope recycling is an example of a vulnerable pathway in stressed, non/slow-replicating M. tuberculosis. Recycling of the trehalose component of the outer mycomembrane, which occurs in several in vitro models of stress, promotes M. tuberculosis survival in macrophages and in vivo as well as antibiotic tolerance. While the mycomembrane is a distinctive attribute of the Mycobacteriales cell envelope, these organisms also have a more-conserved peptidoglycan cell wall. Very little is known about cell wall turnover and reuse in the Mycobacteriales. In other bacteria, cell wall recycling genes usually do not contribute to fitness in standard laboratory conditions but can support survival in stationary phase and tolerance to certain cell wall-acting antibiotics. Using bioorthogonal metabolic labeling, the investigators serendipitously discovered that M. tuberculosis, M. smegmatis, and Corynebacterium glutamicum recycle the peptidoglycan sugar N-acetylmuramic acid in a manner that is not explained by their known repertoire of cell wall recycling genes. The central hypothesis of this application is that Mycobacteriales MurNAc recycling occurs via a novel mechanism and promotes survival in stressed, non/slow-replicating M. tuberculosis. To test, the investigators will identify and characterize MurNAc recycling genes in M. tuberculosis and determine the phylogenetic distribution of the pathway within and outside of the Mycobacteriales. Successful execution of the proposed work 1) will reveal fundamental mechanistic insight into cell envelope homeostasis not previously defined in standard model organisms and 2) uncover new vulnerabilities in non/slow-replicating M. tuberculosis, including inner- and mycomembrane transporters with good accessibility to small molecules. The results will also form the basis for future investigation of MurNAc recycling in vivo and as a potential antibiotic target.