Effects of CwlM, a peptidoglycan synthesis regulator, on beta-lactam resistance and host-pathogen interactions
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Background
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.
Results
Quantitative RT-PCR assays confirmed the successful repression of cwlM , while the phenotyping assays confirmed the essentiality of CwlM-related processes for mycobacterial viability. Collectively, the antibiotic susceptibility assays suggested that CwlM SMEG may promote beta-lactam resistance, particularly to meropenem and cefotaxime. Moreover, CwlM SMEG was found to support M. smegmatis intracellular survival within THP-1-derived macrophages. To address conflicting reports regarding its predicted peptidoglycan (PG) hydrolase activity, we purified recombinant CwlM TB . The Micrococcus luteus -derived PG-based zymogram indicated that CwlM TB lacks PG-hydrolytic activity, suggesting it might act as a regulator of PG biosynthesis instead.
Conclusions
Our findings indicate that CwlM contributes to beta-lactam resistance 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.
