Investigating the role of CwlM interaction with MviN and peptidoglycan in mycobacterial growth and stress response

Mycobacterium tuberculosis is one of the major disease-causing agents worldwide; it claims over one million lives annually. The complex lipid rich cell wall is a critical component that makes this pathogen very successful. Biosynthesis and remodelling of the cell wall are regulated by essential serine/threonine protein kinases, PknA and PknB. This project was focused on the investigation of main PknB substrates, CwlM and MviN. CwlM, previously annotated as N-acetylmuramoyl-L-alanine amidase, regulates peptidoglycan biosynthesis. CwlM is a non-secreted cytoplasmic protein which has two peptidoglycan binding domains and an amidase domain. However, its enzymatic activity has been disputed. MviN is the proposed flippase of peptidoglycan precursors. It is a transmembrane protein with an intracellular domain of unknown function that is only present in Actinobacteria. Previous studies have demonstrated interaction between non-phosphorylated CwlM and the intracellular domain of MviN (MviNicd) using immunoprecipitation assays and mycobacterial two-hybrid system. However, the CwlM-MviNicd complex has not been characterised and its role in peptidoglycan biosynthesis has not been established.

In this study, the interaction between CwlM and MviNicd was further examined using several approaches such as label transfer and size exclusion chromatography. Cryo-EM and crystallography studies were attempted to resolve the structure of the complex. Structural predictions and results of pilot experiments indicated that the N-terminal part of CwlM, containing the peptidoglycan binding domains, interacted with MviNicd. Bioinformatic approaches identified amino acid residues involved in binding of CwlM with peptidoglycan. The importance of these residues for the CwlM-peptidoglycan interaction was confirmed by biochemical experiments and complementation studies using a previously described conditional cwlM mutant of M. smegmatis. Additionally, the role of putative enzymatic activity of CwlM in mycobacterial stress response was investigated. M. smegmatis strains expressing CwlM variants with mutated catalytic residues, E217A or D339A, had growth defects in rich media, and impaired survival under nitrosative and oxidative stresses. Findings in this study provide further mechanistic details about CwlM function in cell wall biosynthesis and mycobacterial survival in stressful conditions.