The long-term goal of this work is to understand how the cell wall of M. tuberculosis (Mtb) is regulated during cell growth and dormancy, and to identify drug candidates that will target cell wall enzymes in both of these conditions, thus making effective antibiotics. The objective of the research described in this proposal is to characterize, in Mtb and a related species, a previously-undescribed regulatory mechanism for secreted bacterial proteins. Work to date has shown that an essential cell elongation factor, CwlM, is phosphorylated and that this phosphorylation alters its activity in regulating cell elongation. The proposed work will probe the cellular and molecular function of CwlM by analyzing peptidoglycan structure and the localization of other elongation factors when CwlM is depleted, and will identify the regulatory targets of CwlM by using co-immunoprecipitation to identify protein interactors. A phosphoablative allele of CwlM that impairs cell elongation has been identified. The proposed work will measure the protein stability, and secretion of this mutant protein, and will analyze peptidoglycan structure and elongation factor localization in the mutant strain in order to understand how phosphorylation affects the activity of CwlM. In addition, the cognate kinase for CwlM will be identified. In an effort to understand how stress changes cell wall structure and growth dynamics, this work will study how stressful conditions alter the phosphorylation and activity of CwlM. This work will further the understanding of how secreted proteins - and especially those that remodel the cell wall - are regulated and how their activity is coordinated with cytoplasmic events. Understanding these processes is critical to building a model for how the cell wall is remodeled during both vegetative growth and the stress that leads to dormancy, and for identifying cell wall drug targets.
Antibiotic resistance in the important pathogen M. tuberculosis is an increasing problem, and identifying potential drug targets is a crucial step in the development of new drugs. Mycobacterial cell wall enzymes make good drug targets, but these proteins and their regulation must be more thoroughly understood in order to design drugs that will be bacteriocidal even in stressful conditions, when the activity of these enzymes and the structure of the cell wall changes. This work will describe a novel regulatory mechanism by which cell wall proteins are controlled, leading to improved predictions of which proteins will make good drug targets in varied conditions.