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

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

RESULTS: Quantitative RT-PCR assays confirmed the successful repression of, while the phenotyping assays confirmed the essentiality of CwlM-related processes for mycobacterial growth. Collectively, the antibiotic susceptibility assays suggested that CwlMmay contribute to increased tolerance to meropenem and cefotaxime. Moreover, CwlMwas found to supportsurvival within THP-1-derived macrophages. To address conflicting reports regarding its predicted peptidoglycan (PG) hydrolase activity, we purified recombinant CwlM. The-derived PG-based zymogram indicated that CwlMlacks PG-hydrolytic activity, suggesting it might act as a regulator of PG biosynthesis instead.

CONCLUSIONS: 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.

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