Staphylococcus aureus PhoU Homologs Regulate Persister Formation and Virulence

Abstract

PhoU homologs are one of the determinant factors in the regulation of persister formation and phosphate metabolism in many bacterial species; however, the functions of PhoU homologs exhibit species-specific characteristics. The pathogenesis of <i>Staphylococcus aureus</i> is closely correlated with persister formation and virulence factors. The functions of two PhoU homologs, PhoU1 and PhoU2, in <i>S. aureus</i> are unclear yet. In this study, single- and double-deletion mutants of <i>phoU1</i> and <i>phoU2</i> were generated in strain USA500 2395. The Δ<i>phoU1</i> or Δ<i>phoU2</i> mutants displayed a change in persister formation and virulence compared to the parent strain; the persisters to vancomycin and levofloxacin were decreased at least 1,000-fold, and the number of intracellular bacteria surviving in the A549 cells for 24 h decreased to 82 or 85%. The α-hemolysin expression and activity were increased in the Δ<i>phoU2</i> mutants. Transcriptome analysis revealed that 573 or 285 genes were differentially expressed by at least 2.0-fold in the Δ<i>phoU1</i> or Δ<i>phoU2</i> mutant vs. the wild type. Genes involved in carbon and pyruvate metabolism were up-regulated, and virulence genes and virulence regulatory genes were down-regulated, including type VII secretion system, serine protease, leukocidin, global regulator (<i>sarA</i>, <i>rot</i>), and the two-component signal transduction system (<i>saeS</i>). Correspondingly, the deletion of the <i>phoU1</i> or <i>phoU2</i> resulted in increased levels of intracellular pyruvate and ATP. Deletion of the <i>phoU2</i>, but not the <i>phoU1</i>, resulted in the up-regulation of inorganic phosphate transport genes and increased levels of intracellular inorganic polyphosphate. In conclusion, both PhoU1 and PhoU2 in <i>S. aureus</i> regulate virulence by the down-regulation of multiple virulence factors (type VII secretion system, serine protease, and leucocidin) and the persister generation by hyperactive carbon metabolism accompanied by increasing intracellular ATP. The results in <i>S. aureus</i> are different from what we have previously found in <i>Staphylococcus epidermis</i>, where only PhoU2 regulates biofilm and persister formation. The different functions of PhoU homologs between the two species of <i>Staphylococcus</i> warrant further investigation.