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 , a pathogenic bacterium known for causing severe infections, particularly in infants and immunocompromised individuals. By employing a combination of genetic screening, comparative proteomics, and validation using zebrafish and rat models, we rapidly screened highly virulent strains and identified two genes, and , as potential regulators of toxicity toward zebrafish and rats. Proteomic profiling revealed upregulated proteins upon knockout of and , 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 and provide insights into the development of targeted interventions and therapies against this bacterium. IMPORTANCE The emergence of antibiotic resistance in pathogenic bacteria has become a critical global health concern, necessitating the identification of virulence factors as potential targets for the development of new antibiotics. This study addresses the limitations of conventional approaches by employing a combination of genetic screening, comparative proteomics, and validation to rapidly identify specific genes and proteins associated with the virulence of , a highly pathogenic bacterium responsible for severe infections in vulnerable populations. The identification of two genes, and , as potential regulators of toxicity toward zebrafish and rats and the proteomic profiling upon knockout of and provides novel insights into the mechanisms underlying bacterial virulence. The findings contribute to our understanding of 's pathogenicity, shed light on the regulatory pathways involved in bacterial virulence, and offer potential targets for the development of novel interventions against this highly virulent bacterium.