Evolution of Resistance to Phenazine Antibiotics in Staphylococcus aureus and Its Role During Coinfection with Pseudomonas aeruginosa

In the niches that Staphylococcus aureus and Pseudomonas aeruginosa coinhabit, the later pathogen produces phenazine antibiotics to inhibit the growth of S. aureus. Recently, a group of halogenated phenazines (HPs) has been shown to have potent antimicrobial activities against Staphylococci; however, no HP-resistant mutant has been reported. Here, we demonstrate that S. aureus develops HP-resistance via single amino acid change (Arg116Cys) in a transcriptional repressor TetR21. RNA-seq analysis showed that the TetR21(R116C) variation caused drastic up-regulation of an adjacent gene hprS (halogenated phenazine resistance protein of S. aureus). Deletion of the hprS in the TetR21(R116C) background restored bacterial susceptibility to HP, while hprS overexpression in S. aureus conferred HP-resistance. The expression of HprS is under tight transcriptional control of the TetR21 via direct binding to the promoter region of hprS. The R116C mutation in TetR21 significantly reduced its DNA binding affinity. Moreover, natural phenazine antibiotics (phenazine-l-carboxylic acid and pyocyanin) and a HP analog (HP-22) are ligands for the TetR21, regulating its repressor activity. Combining homology analysis and LC-MS/MS assay we demonstrated that HprS is a phenazine efflux pump. To the best of our knowledge, we provide the first report of phenazine efflux pump in S. aureus. Interestingly, the TetR21(R116C) variation has been found in some clinical S. aureus isolates, and a laboratory strain of S. aureus with TetR21(R116C) variation showed enhanced growth competitiveness toward P. aeruginosa and promoted coinfection with P. aeruginosa in the host environment, demonstrating significance of the mutation in host infections.