Tweaking TB bacteria’s cell wall makes drugs work better

Tuberculosis remains a leading cause of death worldwide, made worse by the rise of multidrug-resistant Mycobacterium tuberculosis (Mtb) strains and a shortage of new therapies. Seeking fresh targets, researchers focused on distinctive chemical changes in the mycobacterial cell wall polymer called peptidoglycan (PG). Two such modifications are the amidation of D-iso-glutamate (D-iGlu) and the N-glycolylation of muramic acid. In work led by Maria João Catalão, the team used CRISPR interference (CRISPRi) to silence the genes that carry out these changes: murT/gatD, which mediates D-iGlu amidation, and namH, which mediates N-glycolylation. The study tested how turning down these genes affects bacterial survival, drug sensitivity and the way infected immune cells respond. qRT-PCR measurements confirmed successful knockdown of target mRNA, although repression varied with the chosen sgRNA, PAM strength, and target site. Phenotypic tests, including spotting dilution and growth curve assays, showed that D-iGlu amidation is essential for mycobacterial survival, while N-glycolylation of muramic acid is not, setting the stage to explore how each modification influences drug response and host immunity.

To probe drug susceptibility, the researchers performed susceptibility assays and checkerboard assays, and measured effects on infected immune cells using THP-1-derived macrophages. They found that both PG modifications help Mtb resist β-lactam antibiotics, but effects differed by gene and guide RNA. Notably, sgRNA2-mediated murT knockdown substantially increased susceptibility to β-lactam drugs and to isoniazid. Checkerboard assays revealed reductions in the minimum fractional inhibitory concentration index (FICI min) for AMX/MEM+CLA and EMB combinations after depletion of both PG modifications, with significant differences observed when namH was knocked down. Inside THP-1-derived macrophages, D-iGlu amidation proved important for Mtb survival at 6 days post-infection. Infection with MurT/GatD-depleted Mtb led to upregulation of IL-1β and downregulation of IL-10, while NamH depletion caused upregulation of both IL-1β and IL-10. These tests used established molecular and phenotypic tools—CRISPRi, qRT-PCR, spotting dilution, growth curve assays, susceptibility assays, and checkerboard assays—to link specific PG modifications to drug response and immune signaling.

The study highlights peptidoglycan chemistry as a promising avenue for new TB strategies. By showing that disrupting murT/gatD or namH changes how Mtb responds to β-lactams and other drugs, the work suggests that targeting these modifications could make existing antibiotics more effective against resistant strains. The contrasting roles of D-iGlu amidation and N-glycolylation—one being essential for survival and strongly influencing macrophage cytokine responses, the other shaping drug interaction effects and immune signaling differently—reveal that the bacterial expression level of these enzymes matters for both therapy and host-pathogen interactions. The observed shifts in IL-1β and IL-10 after specific gene knockdowns point to expression-sensitive immune dynamics that could inform adjunct therapies or host-directed approaches. Altogether, these findings support developing therapeutic regimens that combine drugs like AMX/MEM+CLA, EMB, and isoniazid with strategies to inhibit murT/gatD or namH activity, opening a path toward more effective treatments for TB.