Transposon-insertion sequencing reveals NlpD as a vulnerability in intracellular survival and pathogenesis of Salmonella 4,[5],12:i:-

Salmonella 4,[5],12:i:- has emerged as a leading cause of non-typhoidal salmonellosis globally, yet the mechanisms governing its intracellular survival and pathogenic adaptation remain unclear. Herein, through transposon-insertion sequencing, we identify nlpD, a peptidoglycan hydrolase activator essential for maintaining membrane stability and facilitating bacterial division, as a critical gene for Salmonella infection. nlpD-deficient Salmonella exhibits significantly diminished survival in both in vitro and in vivo settings. Mechanistically, nlpD deletion disrupts peptidoglycan and lipopolysaccharide homeostasis, rendering Salmonella 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. Our findings establish NlpD as a critical regulator that limits M1-polarized inflammatory responses, highlighting its potential as a therapeutic vulnerability against Salmonella 4,[5],12:i:- infection.Author summary: Salmonella 4,[5],12:i:- is a rapidly spreading global pathogen, but the mechanisms by how it promotes intracellular survival and evades host immune defenses remain elusive. In this study, we identified nlpD, a gene required for bacterial cell wall remodeling, as a key factor for Salmonella survival within the host. We found that loss of nlpD disrupts peptidoglycan and lipopolysaccharide homeostasis, making the bacteria highly sensitive to oxidative and acidic stresses. These cell wall defects activate the host TLR/NF-κB signaling pathway, which shifts macrophages from a permissive M2 state to an activated M1 state. This shift further enhances autophagy-lysosomal degradation to eliminate the intracellular bacteria. We also discovered that ketoconazole can inhibit NlpD and effectively reduce bacterial loads in infected animals. Collectively, our work shows that targeting bacterial cell wall integrity restores host immune clearance and provides a potential therapeutic strategy against Salmonella infections.