Hyperproduction of alpha-hemolysin in a sigB mutant is associated with elevated SarA expression in Staphylococcus aureus

Abstract

To evaluate the role of SigB in modulating the expression of virulence determinants in Staphylococcus aureus, we constructed a sigB mutant of RN6390, a prototypic S. aureus strain. The mutation in the sigB gene was confirmed by the absence of the SigB protein in the mutant on an immunoblot as well as the failure of the mutant to activate sigmaB-dependent promoters (e.g., the sarC promoter) of S. aureus. Phenotypic analysis indicated that both alpha-hemolysin level and fibrinogen-binding capacity were up-regulated in the mutant strain compared with the parental strain. The increase in fibrinogen-binding capacity correlated with enhanced expression of clumping factor and coagulase on immunoblots. The effect of the sigB mutation on the enhanced expression of the alpha-hemolysin gene (hla) was primarily transcriptional. Upon complementation with a plasmid containing the sigB gene, hla expression returned to near parental levels in the mutant. Detailed immunoblot analysis as well as a competitive enzyme-linked immunosorbent assay of the cell extract of the sigB mutant with anti-SarA monoclonal antibody 1D1 revealed that the expression of SarA was higher in the mutant than in the parental control. Despite an elevated SarA level, the transcription of RNAII and RNAIII of the agr locus remained unaltered in the sigB mutant. Because of a lack of perturbation in agr, we hypothesize that inactivation of sigB leads to increased expression of SarA which, in turn, modulates target genes via an agr-independent but SarA-dependent pathway.

FIG. 1

Northern analysis of sar transcripts of S. aureus RN6390, its isogenic sigB mutant ALC1001, and complemented mutant ALC1497. Northern analysis revealed that the sigB gene was transcribed in ALC1497 (data not shown). Ten micrograms of total cellular RNA obtained at the stationary phase (OD₆₅₀, 1.7, as determined with an 18-mm borosilicate glass tube) was applied to each lane. The probe was a 730-bp sarA fragment (nucleotides 620 to 1349, based on the published sequence) (3). Because the transcription of sarB, the largest transcript within the sar locus, was minimal during the late log and stationary phases, only data for sarA and sarC transcripts are shown.

FIG. 2

Immunoblot of cell extracts of the parental strain, sigB mutant strain ALC1001, and complemented mutant strain ALC1497 probed with anti-sigB monoclonal antibody 2D7 (1:1,000 dilution). About 50 μg of cellular proteins was applied to each lane. The experiment was repeated two times, with essentially the same results. M. W., molecular weight. Numbers at left are in thousands.

FIG. 3

Lysis of platelets monitored on the basis of OD₆₀₀. The supernatants of 18-h bacterial cultures in TSB were mixed with platelets (10⁹ platelets/ml). Platelet lysis was monitored by measuring the decrease in the OD₆₀₀ over time (2). The medium and purified alpha-toxin served as the negative and positive controls, respectively. Symbols: diamonds, TSB control plus washed platelets; squares, RN6390 (supernatant) plus washed platelets; triangles, SigB (supernatant) plus washed platelets; circles, purified alpha-toxin (1 mg/ml) plus washed platelets.

FIG. 4

Western and Northern analyses of the alpha-hemolysin gene product. (A) An immunoblot of extracellular proteins of ALC1001, its isogenic parent RN6390, and complemented strain ALC1497 from the late log phase was probed with rabbit anti–alpha-hemolysin antibody (1:2,500 dilution). (B) Ten micrograms of total cellular RNA obtained from the late log to early stationary phases was applied to each lane. The probe was a 3-kb EcoRI-HindIII fragment of the alpha-hemolysin gene (7). Similar complementation results were obtained with another sigB mutant (RUSA168) and plasmid pALC1496. These experiments were repeated three times, with similar results. The results of a representative experiment are shown.

FIG. 5

Immunoblots of cell wall extracts of RN6390 and ALC1001 probed with fibrinogen (A), anti-ClfA antibody (B), and anti-Coa antibody (C). Equivalent amounts of cell extracts were applied to the lanes. The positive controls were fibrinogen (A) and purified coagulase (C). M.W., molecular weight. Numbers at left are in thousands.

FIG. 6

Immunoblot of cell extracts of RN6390 and ALC1001 probed with anti-SarA monoclonal antibody 1D1 (1:2,500 dilution). About 30 μg of protein was applied to each lane. The positive control was purified SarA protein (14.5 kDa). M.W., molecular weight. Numbers at left are in thousands.

FIG. 7

Organization of the sigB operon in B. subtilis and S. aureus. In contrast to those in B. subtilis, the functions of the putative rsbU, rsbV, and rsbW genes in S. aureus have not been demonstrated. Similarly, the putative ςA and ςB promoters in S. aureus have not been experimentally confirmed.