Phosphorylation and acetylation of secreted bacterial proteins: a new regulatory

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DESCRIPTION (provided by applicant): 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.