Identification of the Staphylococcus aureus vfrAB Operon, a Novel Virulence Factor Regulatory Locus

Abstract

b During a screen of the Nebraska Transposon Mutant Library, we identified 71 mutations in the Staphylococcus aureus genome 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, designated vfrAB, that had not been characterized previously. Interestingly, a ⌬vfrB mutant exhibited only minor effects on the transcription of the hla gene, encoding alphahemolysin, 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 ⌬vfrB mutant had increased aur and sspAB protease expression under these conditions. However, disruption of the known secreted proteases in the ⌬vfrB mutant did not restore hemolytic activity in the ⌬vfrB mutant on blood agar. Further analysis revealed that, in contrast to the minor effects of VfrB on hla transcription when strains were cultured in liquid media, the level of hla transcription 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, hla regulation is highly responsive to factors associated with growth on solid media. Intriguingly, the ⌬vfrB mutant displayed increased pathogenesis in a model of S. aureus dermonecrosis, 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 for S. aureus pathogenesis.

VfrB Is a Key Activator of the Staphylococcus aureus SaeRS Two-Component System

“…we observed previously (26). Furthermore, complementation with vfrB did not restore the ␣-hemolysin activity of the saeR mutant, demonstrating the need for SaeR for VfrB-dependent activation of Hla production.…”

“…Our group previously demonstrated that virulence factor production in a vfrB mutant is altered from that of the wild-type strain (26). Similar to saeRS mutants (28), vfrB mutants show a marked reduction in Hla-dependent hemolytic activity when grown on rabbit blood agar, demonstrating decreased ability to produce Hla, and have enhanced production of V8 protease (26). Hla is under the control of multiple transcriptional and posttranscriptional factors (29)(30)(31)(32)(33)(34).…”

Identification of a two-component fatty acid kinase responsible for host fatty acid incorporation by Staphylococcus aureus

“…We have named this gene fakA to reflect its biochemical function as a Fak uncovered in this research. This locus (first known as vfrB) most often exists in an operon with a gene encoding a protein with similarity to acid shock protein Asp23 (vfrA) (5). Genetic analysis shows vrfB (fakA) is a potent modifier of α-hemolysin production, but vfrA…”

“…does not impact this phenotype (5). FakA belongs to a family of Gram-positive bacterial proteins of unknown function (COG1461) that contain an N-terminal domain similar to the diMg 2+ ATP binding domain (Dak2) of dihydroxyacetone (Dha) kinase of Citrobacter freundii (6) (Fig.…”

Biochemical Roles for Conserved Residues in the Bacterial Fatty Acid-binding Protein Family

“…We did not detect any incorporation of [1-14 C]oleate into phospholipid in the ⌬fakB2 strain consistent with the absolute requirement of Fak for fatty acid activation and incorporation (Table 5). Strains expressing FakB2(T61A) or FakB2(H266A) showed 20 -40-fold lower incorporation of [1][2][3][4][5][6][7][8][9][10][11][12][13][14] C]oleic acid into membrane phospholipids illustrating these proteins had severely impaired Fak activity in vivo (Table 5), as they had in vitro ( Table 2). The FakB2(R170A) mutant was indistinguishable from the ⌬fakB2 deletion strain with no detectable exogenous fatty acid incorporation into phospholipid (Table 5) corroborating the essential nature of Arg-170 in Fak catalysis (Table 2).…”