Using a zebrafish model to examine the importance of the stringent response for Staphylococcus aureus pathogenesis

Staphylococcus aureus is a human commensal organism with the potential to become opportunistic given the right conditions. During infection, bacteria are exposed to stresses like nutrient limitation, which they respond to by inducing the stringent response. This is a conserved bacterial reaction to stress that ultimately shuts down macromolecular processes to promote bacterial survival, achieved by the production of the nucleotide signalling molecule guanosine penta-/tetraphosphate ((p)ppGpp). The S. aureus genome encodes three (p)ppGpp synthetases: Rel, RelP and RelQ. (p)ppGpp produced by these synthetases contributes to bacterial pathogenesis in a number of species. In this study, a toolbox of S. aureus strains was constructed to improve understanding of how the stringent response is important for the survival and virulence of S. aureus in vitro and in vivo. To elucidate the mechanism behind the requirement of (p)ppGpp for intracellular survival in vitro, the S. aureus strains were exposed to stress conditions typically found in a phagolysosome. A (p)ppGpp0 mutant was observed to be more susceptible to hydrogen peroxide, itaconic acid and hypochlorous acid whereas an overproduction of (p)ppGpp increased the tolerance of S. aureus to these stressors. RelP alone could complement itaconic acid stress while both Rel and RelP could have a role in the tolerance of S. aureus to hydrogen peroxide. Systemic infection of zebrafish embryos with a (p)ppGpp0 mutant revealed increased survival of embryos when compared to embryos infected with the wildtype, which is likely due to a virulence defect as the (p)ppGpp0 mutant replicated at a similar rate to the wildtype in vivo. (p)ppGpp overproduction also resulted in an increased survival of embryos. Complementation experiments revealed that Rel is a key (p)ppGpp synthetase during infection, but that RelP and RelQ may be sufficient in the absence of Rel. Taken together, these results demonstrate a role of (p)ppGpp for S. aureus pathogenesis within a zebrafish embryo systemic infection model, with in vitro experiments showing that the stringent response is required for survival during stress.