Genetic interference of distinctive Mycobacterium tuberculosis peptidoglycan modifications enhances β-lactam susceptibility and reveals expression-sensitive host immune dynamics.

The high mortality associated with tuberculosis (TB), alongside the lack of efficient therapeutics against emerging multidrug-resistant Mycobacterium tuberculosis (Mtb) strains, emphasizes the need for novel antitubercular targets. Mycobacterial peptidoglycan (PG), displaying characteristic modifications comprising the amidation of D-iso-glutamate (D-iGlu) and the N-glycolylation of muramic acid, is therefore a promising therapeutic target. The genes encoding the enzymes mediating these modifications (murT/gatD and namH) were silenced in Mtb using CRISPR interference (CRISPRi) to investigate their impact on β-lactam susceptibility and host immune responses. First, qRT-PCR confirmed successful target mRNA knockdown and phenotyping assays corroborated the essentiality of D-iGlu amidation for mycobacterial growth, in contrast to muramic acid N-glycolylation. The susceptibility assays demonstrated that both PG modifications promote β-lactam resistance. Indeed, we observed reductions in the minimum fractional inhibitory concentration index (FICImin) value for AMX/MEM + CLA and EMB combinations following the depletion of both PG modifications. Furthermore, D-iGlu amidation was found to promote Mtb fitness within THP-1-derived macrophages 6 days post-infection. Infection with MurT/GatD-depleted Mtb was associated with increased IL-1β and decreased IL-10, whereas NamH depletion was linked to increased IL-1β and IL-10 levels. Altogether, our findings unveiled the potential of targeting these PG modifications for the development of innovative therapeutic regimens against TB.