Determining the importance of the stringent response for methicillin-resistant Staphylococcus aureus virulence using a zebrafish model of infection

Staphylococcus aureus is a bacterial pathogen that poses a major threat to human health. The ability of this bacterium to adapt to stresses encountered in the host is essential for disease. The stringent response is a signalling pathway utilised by all bacteria to alarm cells when stressed, and has been linked to the virulence of a number of species. This signalling pathway is controlled by the nucleotide alarmones guanosine tetra- (ppGpp) and pentaphosphate (pppGpp: collectively termed (p)ppGpp), produced in S. aureus by three synthetase enzymes: Rel, RelP and RelQ. Here, we used a triple (p)ppGpp synthetase mutant ((p)ppGpp0) to examine the importance of this signalling network for the survival and virulence of S. aureus in vivo. Using an established zebrafish larval infection model, we observed that infection with (p)ppGpp0 resulted in attenuated virulence, which was not due to a reduced ability of the mutant to replicate in vivo. Of the three (p)ppGpp synthetases, Rel was established as key during infection, but roles for RelP and RelQ were also observed. Zebrafish myeloid cell depletion restored the virulence of (p)ppGpp0 during systemic infection, indicating that (p)ppGpp is important for survival within host phagocytes. Primary macrophages infection studies, followed by in vitro tolerance assays to key innate immune effectors, demonstrated that (p)ppGpp0 was more susceptible to stressors found within the intracellular macrophage environment, with roles for all three synthetases implicated. Lastly, the absence of CodY, a transcription factor linked to the stringent response, significantly increased the tolerance of S. aureus to phagolysosomal-like stressors in vitro, but had no impact in vivo. Taken together, these results define the importance of the stringent response for S. aureus infection, revealing that (p)ppGpp produced by all three synthetases is required for bacterial survival within the host environment by mediating adaptation to the phagolysosome.