Abstract

Staphylococcus aureus causes a variety of infections, such as skin and soft tissue infections (SSTI), infectious endocarditis, bacteremia, osteomyelitis, and pneumonia. The virulence of S. aureus can be partially explained by its ability to acquire resistance to a wide range of antibiotics and innate immune effectors. In this study, a second messenger pathway involving the compound diadenosine tetraphosphate (Ap₄A) was disrupted to determine its role in S. aureus stress response. Ap₄A exists in both prokaryotes and eukaryotes; however, in bacteria, its primary role is believed to be a second messenger signaling molecule. In S. aureus, Ap₄A is synthesized from AMP and ATP when aminoacyl tRNA synthetases lack tRNA substrate, and it is degraded by the enzyme YqeK into two ADP. To determine the role of Ap₄A in S. aureus, we deleted the gene yqeK to create a strain with excess Ap4A. We then characterized the phenotype of this mutant during  Kanamycin, nitric oxide (NO·), and acid stress. Wild-type S. aureus, the deletion mutant, a complemented mutant, and an overexpression mutant were grown in increasing concentrations of Kanamycin, 20 mM of an NO· donor, or acidified (pH 5.5) media for 24-hour periods. The deletion of yqeK significantly lowered the minimum inhibitory concentration of Kanamycin and showed a general trend of delayed growth with  NO· or acid stress. To investigate the mechanism of how yqeK is linked to S. aureus stress responses, RNA-Seq was performed on WT and ∆yqeK strains during NO· and acid stress.  The yqeK deletion mutant exhibited reduced expression of genes involved in stress response pathways, which likely contribute to the observed phenotypes. Surprisingly, deletion of yqeK also resulted in reduced expression of several genes encoding toxins and virulence factors, while expression genes suggesting the initiation of prophage induction was increased. These results suggest that increased levels of Ap₄A in S. aureus may be detrimental to cell growth, antibiotic resistance, evasion of the human immune response, and virulence factor production. Future research will involve quantifying the concentration of Ap₄A in wild-type and mutant S. aureus when exposed to various stressors and using qRT-PCR to confirm RNA-Seq results.

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