<p>RT-qPCR primer sequences used in this study.</p>

Herein, through transposon-insertion sequencing, we identify <i>nlpD</i>, a peptidoglycan hydrolase activator essential for maintaining membrane stability and facilitating bacterial division, as a critical gene for <i>Salmonella</i> infection. <i>nlpD</i>-deficient <i>Salmonella</i> exhibits significantly diminished survival in both <i>in vitro</i> and <i>in vivo</i> settings.

Mechanistically, <i>nlpD</i> deletion disrupts peptidoglycan and lipopolysaccharide homeostasis, rendering <i>Salmonella</i> more vulnerable to oxidative and acidic stresses within phagosomes. Accumulated cell wall components robustly activate the toll-like receptor-mediated NF-κB signaling pathway, driving M1 macrophage polarization and autophagy-lysosomal activation to eliminate intracellular bacteria. An FDA-approved drug screen identifies ketoconazole as an NlpD-targeting compound that reduces bacterial loads in murine models.