Identification of theStaphylococcus aureus vfrABOperon, a Novel Virulence Factor Regulatory Locus

Abstract

ABSTRACTDuring a screen of the Nebraska Transposon Mutant Library, we identified 71 mutations in theStaphylococcus aureusgenome that altered hemolysis on blood agar medium. Although many of these mutations disrupted genes known to affect the production of alpha-hemolysin, two of them were associated with an apparent operon, designatedvfrAB, that had not been characterized previously. Interestingly, a ΔvfrBmutant exhibited only minor effects on the transcription of thehlagene, encoding alpha-hemolysin, when grown in broth, as well as on RNAIII, a posttranscriptional regulatory RNA important for alpha-hemolysin translation, suggesting that VfrB may function at the posttranscriptional level. Indeed, a ΔvfrBmutant had increasedaurandsspABprotease expression under these conditions. However, disruption of the known secreted proteases in the ΔvfrBmutant did not restore hemolytic activity in the ΔvfrBmutant on blood agar. Further analysis revealed that, in contrast to the minor effects of VfrB onhlatranscription when strains were cultured in liquid media, the level ofhlatranscription was decreased 50-fold in the absence of VfrB on solid media. These results demonstrate that while VfrB represses protease expression when strains are grown in broth,hlaregulation is highly responsive to factors associated with growth on solid media. Intriguingly, the ΔvfrBmutant displayed increased pathogenesis in a model ofS. aureusdermonecrosis, further highlighting the complexity of VfrB-dependent virulence regulation. The results of this study describe a phenotype associated with a class of highly conserved yet uncharacterized proteins found in Gram-positive bacteria, and they shed new light on the regulation of virulence factors necessary forS. aureuspathogenesis.