Regulation of exoprotein gene expression by the Staphylococcus aureus cvfB gene

Abstract

We previously reported that the cvfB gene (SA1223) of Staphylococcus aureus is responsible for the virulence of this pathogenic bacterium. We show here that the cvfB gene regulates exoprotein gene expression. In a cvfB gene deletion mutant, hemolysin, DNase, and protease production were decreased, whereas protein A expression was increased. The amount of RNAIII, the transcript from the P3 promoter in the agr locus that regulates the expression of various virulence factors, was also reduced in the cvfB mutant. In addition, P2 and P3 promoter activity in the agr locus was decreased in the mutant. Under the genetic background of the agr-null mutation, cvfB gene disruption decreased the production levels of DNase and protease. Moreover, the cvfB and agr double mutant was less virulent than the agr mutant in silkworms. These results suggest that the cvfB gene product contributes to the expression of virulence factors and to pathogenicity via both agr-dependent and agr-independent pathways.

FIG. 1.

Changed exoprotein profile and protein A expression in the cvfB mutant. Extracellular proteins at the late log phase, early stationary phase, and stationary phase were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and stained with Coomassie brilliant blue. Green arrowheads indicate proteins with increased amounts in the cvfB mutant. The magenta arrowhead represent proteins with decreased amounts in the cvfB mutant. Extracellular proteins at the late log phase and early stationary phase were subjected to Western blot analysis using an anti-protein A monoclonal antibody and are shown in the bottom panel. The signals were normalized with a 14-kDa band stained with Coomassie brilliant blue and correspond to the band indicated by green arrowheads.

FIG. 2.

Increased expression of the spa gene in the cvfB mutant. (A) The transcript of the spa gene was detected by Northern blot analysis. Total RNA (2.5 μg) was electrophoresed and transferred to a nitrocellulose membrane. The membrane was probed with a ³²P-labeled DNA fragment that is a part of the spa gene. The amount of 23S rRNA in the sample was determined by staining the gel with ethidium bromide as shown in the bottom panel. (B) The parent strain (RN4220) and the cvfB mutant (M1223) were transformed with the plasmid harboring the spa::luc fusion (pCK5004). Luciferase activity during growth was measured. Means ± thestandard deviations from three independent experiments are shown. *, P < 0.01.

FIG. 3.

Decreased activity of the hla promoter in the cvfB mutant. The parent strain (RN4220) and the cvfB mutant (M1223) were transformed with the plasmid harboring the hla::luc fusion (pCK5003). The luciferase activity during growth was measured. Means ± the standard deviations from three independent experiments are shown. *, P < 0.01.

FIG. 4.

Reduced expression of agr in the cvfB mutant. (A) The amount of RNAIII was determined by Northern blot analysis. Total RNA (5 μg) was isolated from cultures at the mid-log phase (OD₆₀₀ = 2.5) and early stationary phase (OD₆₀₀ = 7). The amount of 23S rRNA in the sample was determined by staining the gel with ethidium bromide as shown in the bottom panel. (B and C) represent P3 activity and P2 activity, respectively. The parent strain (RN4220) and the cvfB mutant (M1223) were transformed with the plasmid harboring the P2::luc fusion (pCK5001) or the plasmid harboring the P3::luc fusion (pCK5002). The luciferase activity at the mid-log phase (OD₆₀₀ = 2.5) was measured. Means ± the standard deviations of three independent experiments are presented. *, P < 0.01.

FIG. 5.

Attenuated virulence caused by the cvfB disruption under the agr-null background in silkworm infection model. Ten silkworms were injected with 10-fold-diluted overnight cultures (50 μl, 3.5 × 10⁷ CFU) of the parent strain (RN4220) and the cvfB mutant (M1223), the agr mutant (CK3), and the cvfB agr double mutant (CK5), and larval survival was monitored. The curves are representative of at least three independent experiments. P values between RN4220 and M1223 and between CK3 and CK5 were <0.05, respectively.

FIG. 6.

Attenuated virulence caused by the cvfB disruption in an agr-null background in mouse infection model. Five or six CD-1 mice were injected via the tail vein with bacterial suspensions (100 μl, 8 × 10⁸ CFU) of parent strain (RN4220), the cvfB mutant (M1223), the agr mutant (CK3), and the cvfB agr double mutant (CK5). The numbers of bacteria in the spleens were determined at 24 h after infection. The horizontal bar shows the group mean. Student t test P values between RN4220 and M1223 and between CK3 and CK5 are shown.