Uncovering Virulence Factors in Cronobacter Sakazakii: Insights from Genetic Screening and Proteomic Profiling.

The increasing problem of antibiotic resistance has driven the search for virulence factors in pathogenic bacteria, which can serve as targets for the development of new antibiotics. Although whole-genome Tn5 transposon mutagenesis combined with phenotypic assays has been a widely used approach, its efficiency remains low due to labor-intensive processes. In this study, we aimed to identify specific genes and proteins associated with the virulence of Cronobacter sakazakii, a pathogenic bacterium known for causing severe infections, particularly in infants and immunocompromised individuals. By employing a combination of genetic screening, comparative proteomics, and in vivo validation using zebrafish and rat models, we rapidly screened highly virulent strains and identified two genes, rcsA and treR, as potential regulators of C. sakazakii toxicity toward zebrafish and rats. Proteomic profiling revealed upregulated proteins upon knockout of rcsA and treR, including FabH, GshA, GppA, GcvH, IhfB, RfaC, MsyB, and three unknown proteins. Knockout of their genes significantly weakened bacterial virulence, confirming their role as potential virulence factors. Our findings contribute to understanding the pathogenicity of C. sakazakii and provide insights into the development of targeted interventions and therapies against this bacterium.