Functional and Proteomic Dissection of the Contributions of CodY, SigB and the Hibernation Promoting Factor HPF to Interactions of <i>Staphylococcus aureus</i> USA300 with Human Lung Epithelial Cells

<i>Staphylococcus aureus</i> is a leading cause of severe pneumonia. Our recent proteomic investigations into <i>S. aureus</i> invasion of human lung epithelial cells revealed three key adaptive responses: activation of the SigB and CodY regulons and upregulation of the hibernation-promoting factor SaHPF. Therefore, our present study aimed at a functional and proteomic dissection of the contributions of CodY, SigB and SaHPF to host invasion using transposon mutants of the methicillin-resistant <i>S. aureus</i> USA300. Interestingly, disruption of <i>codY</i> resulted in a "small colony variant" phenotype and redirected the bacteria from (phago)lysosomes into the host cell cytoplasm. Furthermore, we show that CodY, SigB and SaHPF contribute differentially to host cell adhesion, invasion, intracellular survival and cytotoxicity. CodY- or SigB-deficient bacteria experienced faster intracellular clearance than the parental strain, underscoring the importance of these regulators for intracellular persistence. We also show an unprecedented role of SaHPF in host cell adhesion and invasion. Proteomic analysis of the different mutants focuses attention on the CodY-perceived metabolic state of the bacteria and the SigB-perceived environmental cues in bacterial decision-making prior and during infection. Additionally, it underscores the impact of the nutritional status and bacterial stress on the initiation and progression of staphylococcal lung infections.