Contribution of hla Regulation by SaeR to Staphylococcus aureus USA300 Pathogenesis

Abstract

The SaeRS two-component system in Staphylococcus aureus is critical for regulation of many virulence genes, including hla, which encodes alpha-toxin. However, the impact of regulation of alpha-toxin by Sae on S. aureus pathogenesis has not been directly addressed. Here, we mutated the SaeR-binding sequences in the hla regulatory region and determined the contribution of this mutation to hla expression and pathogenesis in strain USA300 JE2. Western blot analyses revealed drastic reduction of alpha-toxin levels in the culture supernatants of SaeR-binding mutant in contrast to the marked alpha-toxin production in the wild type. The SaeR-binding mutation had no significant effect on alpha-toxin regulation by Agr, MgrA, and CcpA. In animal studies, we found that the SaeR-binding mutation did not contribute to USA300 JE2 pathogenesis using a rat infective endocarditis model. However, in a rat skin and soft tissue infection model, the abscesses on rats infected with the mutant were significantly smaller than the abscesses on those infected with the wild type but similar to the abscesses on those infected with a saeR mutant. These studies indicated that there is a direct effect of hla regulation by SaeR on pathogenesis but that the effect depends on the animal model used.

Keywords: Sae; Staphylococcus aureus; alpha-toxin; virulence regulation.

FIG 1

Construction of the sbm SaeR-binding mutation. (A) The promoter region containing the SaeR-binding consensus is in bold. The nucleotide changes in the sbm mutant are in red. (B) EMSA was performed using 40 fmol DIG-labeled wild-type 44-mer with increasing amounts of SaeR-His₆. Competition was done with 250-fold excess unlabeled sbm mutant 44-mer (mt) or with unlabeled wild-type 44-mer (wt). (C) EMSA was performed using 40 fmol DIG-labeled wild-type 44-mer or sbm 44-mer with increasing amounts of SaeR-His₆.

FIG 2

Western blot analysis of alpha-toxin production. Supernatants of 6-h cultures were analyzed by SDS-PAGE and blotted against anti-alpha-toxin antibody.

FIG 3

Effect of the sbm SaeR-binding mutation on hla regulation by (A) Agr or MgrA (B) CcpA. Mean relative hla expression levels determined by qRT-PCR on early-stationary-phase cultures are presented with standard errors. Data represent results from three independent experiments. Statistical significance was analyzed by Student's t test. *, P < 0.05; ns, not significant.

FIG 4

DNA binding of MgrA to hla regulatory region. EMSA was performed using 40 fmol DIG-labeled wild-type (wt) 44-mer or sbm 44-mer with increasing amounts of MgrA-His₆.

FIG 5

Contribution of hla regulation by SaeR to pathogenesis of USA300 SSTI in rats. Mean values of abscess sizes (measured daily) are presented with standard errors. *, P < 0.05 (versus wild-type JE2 using two-way ANOVA and Bonferroni’s post hoc comparisons).

FIG 6

Bacterial loads of SSTI postinfection at day 12 (A) and day 2 (B). Results are expressed as mean values with standard errors. Statistical significance was analyzed by Student's t test. ns, not significant.