The CRISPR–Cas system functions a regulatory module in diverse cellular processes in Salmonella enterica serovar Typhi and related Enterobacteriaceae

The CRISPR-Cas systems have been traditionally viewed as adaptive immune mechanisms; however, increasing evidence indicates that they also perform broader regulatory functions. Here, we demonstrate that the Type I-E CRISPR-Cas system in Salmonella enterica serovar Typhi modulates the expression of key transcriptional regulators, including OmpR and TviA. Furthermore, the CRISPR-Cas system controls critical virulence-associated phenotypes including intracellular replication within human macrophages, bile salt resistance, biofilm formation, and motility. Comparative analyses across members of the Enterobacteriaceae family reveal species-specific contributions of CRISPR-Cas to bacterial physiology and virulence. In Salmonella Typhimurium, its role is largely restricted to bile salt resistance, whereas in enterohemorrhagic Escherichia coli (EHEC) it additionally regulates, bile resistance, biofilm formation and motility. In contrast, no detectable CRISPR-Cas–dependent regulatory function was observed in Citrobacter rodentium or Escherichia coli K-12 under the conditions tested. Despite these differences, CRISPR-Cas systems consistently influence core physiological processes associated with host adaptation and pathogenicity. Collectively, our findings support a model in which CRISPR-Cas systems function as global regulatory module that enhance bacterial fitness and pathogenic potential by fine-tuning traits required for host colonization and environmental adaptation. This expanded functional paradigm underscores the importance of CRISPR-Cas in enteric pathogens and suggests that its contribution to virulence may be more widespread than previously appreciated.