CvfA protein and polynucleotide phosphorylase act in an opposing manner to regulate Staphylococcus aureus virulence

We previously identified CvfA (SA1129) as a Staphylococcus aureus virulence factor using a silkworm infection model. S. aureus cvfA-deleted mutants exhibit decreased expression of the agr locus encoding a positive regulator of hemolysin genes and decreased hemolysin production. CvfA protein hydrolyzes a 2',3'-cyclic phosphodiester bond at the RNA 3' terminus, producing RNA with a 3'-phosphate (3'-phosphorylated RNA, RNA with a 3'-phosphate). Here, we report that the cvfA-deleted mutant phenotype (decreased agr expression and hemolysin production) was suppressed by disrupting pnpA-encoding polynucleotide phosphorylase (PNPase) with 3'- to 5'-exonuclease activity. The suppression was blocked by introducing a pnpA-encoding PNPase with exonuclease activity but not by a pnpA-encoding mutant PNPase without exonuclease activity. Therefore, loss of PNPase exonuclease activity suppressed the cvfA-deleted mutant phenotype. Purified PNPase efficiently degraded RNA with 2',3'-cyclic phosphate at the 3' terminus (2',3'-cyclic RNA), but it inefficiently degraded 3'-phosphorylated RNA. These findings indicate that 3'-phosphorylated RNA production from 2',3'-cyclic RNA by CvfA prevents RNA degradation by PNPase and contributes to the expression of agr and hemolysin genes. We speculate that in the cvfA-deleted mutant, 2',3'-cyclic RNA is not converted to the 3'-phosphorylated form and is efficiently degraded by PNPase, resulting in the loss of RNA essential for expressing agr and hemolysin genes, whereas in the cvfA/pnpA double-disrupted mutant, 2',3'-cyclic RNA is not degraded by PNPase, leading to hemolysin production. These findings suggest that CvfA and PNPase competitively regulate RNA degradation essential for S. aureus virulence.