Investigating the impact of acid adaptation on cross-protection in Salmonella Typhimurium and its regulatory mechanisms based on the PmrA/B two-component system

• pmrA deletion impaired Salmonella resistance to acid, heat, salt, and H 2 O 2 stress. • Genes for LPS modification and peptidoglycan synthesis were downregulated in Δ pmrA . • Membrane modification via LPS genes contributes to PmrA-mediated cross-stress protection. This study investigated whether the slightly acidic environment in the meat industry activates the Salmonella PmrA/B two-component regulatory system (TCS), thereby enhancing acid tolerance and cross-protection, by constructing a mutant strain lacking the response regulator PmrA. Results showed that deletion of pmrA not only reduced the acid tolerance induced by S . Typhimurium at pH 5.4 (the ultimate pH for beef cattle after slaughter), but also further reduced its cross-stress resistance to heat (55°C), osmotic (8% NaCl), oxidative (5 mM H 2 O 2 ), and most the antibiotic stresses. Transcriptomics data revealed that down-regulation of genes mediating lipopolysaccharide modification and peptidoglycan synthesis led to reduced resistance to cationic antimicrobial peptides. Down-regulation of glycerophospholipid and lysine metabolic pathways, together with limited activation of genes in the glutathione metabolism pathway in the Δ pmrA mutant, was associated with reduced cross-resistance to osmotic, acid, and oxidative stresses. Furthermore, the down-regulation of several TCS genes related to envelope modification and the electron respiratory chain may have further impaired Salmonella cross-stress resistance and intestinal colonization. These findings highlight the critical role of PmrA/B TCS in Salmonella cross-protection and provide mechanistic insights for controlling pathogen persistence in meat processing environments.