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 to identify novel antitubercular targets. Mycobacterial peptidoglycan, displaying characteristic modifications comprising the amidation of D- iso -glutamate (D- i Glu) and the N -glycolylation of muramic acid, is a promising therapeutic target. The genes encoding the enzymes mediating these PG 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, with variable repression efficiency based on the selected sgRNA, PAM strength, and target site. Phenotypic characterization through spotting dilution and growth curve assays corroborated the essentiality of D- i Glu amidation for mycobacterial survival, in contrast to the N -glycolylation of muramic acid. Moreover, susceptibility assays showed that both PG modifications contribute to β-lactam resistance, with sgRNA2-mediated murT knockdown substantially increasing β-lactam and isoniazid susceptibility. Furthermore, checkerboard assays showed reductions in the minimum fractional inhibitory concentration index (FICI _min ) value for AMX/MEM+CLA and EMB combinations following the depletion of both PG modifications, with significant differences observed upon namH knockdown. Additionally, D- i Glu amidation was uncovered as a determinant of Mtb survival within THP-1-derived macrophages at 6 days post-infection. Infection of THP-1-derived macrophages with MurT/GatD-depleted Mtb upregulated IL-1β and downregulated IL-10, whereas NamH depletion caused upregulation of both IL-1β and IL-10. Altogether, our findings unveiled the potential of targeting these PG modifications for the development of innovative therapeutic regimens against TB.