Control of cell wall synthesis and antibiotic tolerance in mycobacteria

There is a critical need to understand the regulation of cell wall metabolism in Mycobacterium tuberculosis
because it contributes to antibiotic tolerance, which exacerbates tuberculosis outcomes. The objective of this
proposal is to build a molecular model for how environmental information flows through phosphorylation of three
cell wall regulators to dynamically control cell wall metabolism in mycobacteria. The central hypothesis of this
proposal is that the central regulators of the cell wall during growth also regulate it in stress. This hypothesis is
based on our data that the phosphatase PstP controls growth as well as stress responses, and that the
phosphorylated regulators CwlM and DivIVA are required for growth and antibiotic survival. The rationale for this
research is that a molecular understanding of cell wall regulation will pave the way for better TB drugs.
Aim 1: Determine how the phosphatase PstP orchestrates cell wall metabolism. Our working hypothesis
is that phosphorylation of PstP regulates its activity against cell wall factors and helps coordinate the transition
from growth to stasis. We will: a) determine how PstP phosphorylation is affected by stresses in Mtb; b) identify
the key substrates of PstP and determine the activity of different PstP phospho-isoforms on each substrate; and
c) determine how PstP contributes to antibiotic tolerance in Mtb.
Aim 2: Determine how CwlM regulates multiple peptidoglycan enzymes. Our working hypothesis is that
CwlM is regulated by phosphorylation and recycled peptidoglycan, and in turn regulates peptidoglycan synthesis
at multiple steps. We will: a) identify conditions that alter CwlM’s phosphorylation in Mtb; b) characterize the
effects of CwlM and CwlM~P on the binding and activity of its interaction partner enzymes; and c) Measure and
characterize the function and regulation of the catalytic activity of CwlM.
Aim 3: Determine how DivIVA coordinates polar cell wall metabolism. Our working hypothesis is that DivIVA
activates cell wall precursor enzymes to promote growth, and is regulated by phosphorylation. We will: a)
determine how DivIVA’s phosphorylation is affected by growth and stress conditions in Mtb; b) identify sites on
DivIVA required for its protein interactions, and characterize the phospho-dependence of the interactions; and
c) measure the effects of DivIVA and DivIV~P on the activity of their interaction partners.
Upon completion of this work we expect to have a multi-level molecular model of the signaling pathways
that control cell wall precursor synthesis in mycobacteria. We will characterize the regulation of the phosphatase
PstP, which is a master regulator and a candidate drug target for both antibiotics and anti-tolerance drugs. We
will describe the signaling role of the protein interactions between the intermediate regulators CwlM and DivIVA
and their enzymatic regulatory targets; these interactions are potential targets for anti-tolerance drugs. This work
will lay the groundwork for novel drug screens.

Grant Number: 5R01AI148917-05
NIH Institute/Center: NIH
Principal Investigator: Cara Boutte