Distinct Regulation of Host Defences by CRISPR-Cas in Typhoidal and Non-Typhoidal Salmonella serovars

CRISPR-Cas systems are best known for their role in adaptive immunity, but emerging evidence suggests broader regulatory functions. Here, we show that the CRISPR-Cas system acts as a serovar-specific regulator of stress adaptation in Salmonella enterica , exerting opposing effects in host-restricted (S. Typhi) and broad-host-range ( S. Typhimurium) serovars. In S. Typhi, CRISPR-Cas system deletion reduces acid and bile tolerance by impairing envelope integrity and repressing key stress-response regulators ( envZ, cadB, phoPQ, lexA, ruvB, wecD ), while increasing resistance to cationic antimicrobial peptides via pmr activation and reduced oxidative damage. Conversely, CRISPR-Cas system loss in S. Typhimurium enhances acid survival-partly through speF upregulation but increases sensitivity to antimicrobial peptides. Spacer-1 of S. Typhi CRISPR-I array as the main regulator of gene expression, and its reintroduction partially restored stress tolerance, supporting spacer-dependent control of physiological pathways. These findings establish the CRISPR-Cas system as a non-canonical, spacer-dependent regulator of stress response networks in S. enterica, revealing its contribution to the evolutionary divergence of survival strategies between S. Typhi and S. Typhimurium.