Abstract Staphylococcus aureus is a leading cause of both acute and chronic infections in humans. Its ability to persist within host cells is thought to play an important role in chronicity and treatment failures. The importance of the pentose phosphate pathway (PPP) during S. aureus chronic infection is currently largely unexplored. Here, we focused on one key PPP enzyme, transketolase. We showed that inactivation of the unique gene encoding transketolase activity in S. aureus USA300 (Δ tkt ) led to an impaired growth in broth. Using time-lapse video imaging, we correlated this phenotype with a defect in early intracellular proliferation compared to wild-type strain. As determined by metabolomic analysis, tkt inactivation also had an important impact on S. aureus metabolism. We then monitored long-term intracellular persistence over 10 days by counting of viable bacteria. Unexpectedly for such a slow-growing strain, the Δ tkt mutant was almost completely eliminated by endothelial cells after ten days, as opposed to a prototypical slow-growing Δ hemDBL mutant for which we recovered 1,000 fold more viable bacteria. We found that in infected cells, the transcriptional activity of the two master regulators Sigma B and RpiRc was drastically reduced in the Δ tkt mutant compared to wild-type strain. Concomitantly, RNAIII transcription was strongly increased. This transcriptional profile is likely to explain the inability of this slow-growing mutant to sustain long-term intracellular survival, suggesting that TKT -or a functional PPP-is required for intracellular bacteria to enable a transcriptional program geared towards persistence. Importance Staphylococcus aureus is a leading cause of severe bacterial infections. This bacterium is readily internalized by non-professional phagocytes and infected cells have been proposed to play an important role in chronic infections and treatment failures. Here, we show the importance of the unique transketolase TKT of S. aureus USA300 in bacterial adaptation during chronic intracellular infection. We show that TKT is mandatory for the metabolomic homeostasis of S. aureus during intracellular persistence. This work unravels the critical role of TKT in the transcriptional regulation of the master regulators Sigma B, RpiRc and RNAIII linking the pentose phosphate pathway to the control of chronic S. aureus infections.

Transketolase is involved in the control of Sigma B during chronic infection byStaphylococcus aureus