Effects of CwlM, a peptidoglycan synthesis regulator, on beta-lactam tolerance and host-pathogen interactions

TL;DR

This study explores how CwlM, a protein in bacteria, affects resistance to antibiotics and survival inside human cells, suggesting it could be a target for new treatments.

Contribution

The study reveals CwlM's role in beta-lactam tolerance and intracellular survival despite lacking peptidoglycan hydrolase activity.

Findings

CwlM contributes to increased tolerance to meropenem and cefotaxime in M. smegmatis.

CwlM supports M. smegmatis survival within THP-1-derived macrophages.

CwlMTB lacks peptidoglycan-hydrolytic activity, suggesting it regulates peptidoglycan biosynthesis.

Abstract

The emergence and spread of multidrug-resistant (MDR) strains of Mycobacterium tuberculosis (Mtb) urge the development of novel drugs and efficient therapeutic programs. A recent study aiming to uncover differential beta-lactam susceptibility phenotypes in clinical strains of Mtb found that the M237V substitution in cwlM (Rv3915) was associated with increased susceptibility to amoxicillin. Considering that Mycobacterium smegmatis (Msm) is a widely used surrogate model for Mtb, we constructed a cwlM knockdown mutant in Msm using CRISPR interference (CRISPRi) to elucidate the role of CwlM in beta-lactam susceptibility and intracellular survival.

Quantitative RT-PCR assays confirmed the successful repression of cwlM, while the phenotyping assays confirmed the essentiality of CwlM-related processes for mycobacterial growth. Collectively, the antibiotic susceptibility assays suggested that CwlMSMEG may contribute to increased tolerance to meropenem and cefotaxime. Moreover, CwlMSMEG was found to support M. smegmatis survival within THP-1-derived macrophages. To address conflicting reports regarding its predicted peptidoglycan (PG) hydrolase activity, we purified recombinant CwlMTB. The Micrococcus luteus-derived PG-based zymogram indicated that CwlMTB lacks PG-hydrolytic activity, suggesting it might act as a regulator of PG biosynthesis instead.

Our findings indicate that CwlM contributes to beta-lactam tolerance and intracellular survival, regardless of lacking detectable PG-hydrolytic activity. Overall, CwlM was found to be essential and highly vulnerable, highlighting its potential as a therapeutic target that warrants further investigation.

The online version contains supplementary material available at 10.1186/s12866-025-04548-6.

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