The SaeRS Two-Component System Controls Survival of Staphylococcus aureus in Human Blood through Regulation of Coagulase

Abstract

The SaeRS two-component system plays important roles in regulation of key virulence factors and pathogenicity. In this study, however, we found that the deletion mutation of saeRS enhanced bacterial survival in human blood, whereas complementation of the mutant with SaeRS returned survival to wild-type levels. Moreover, these phenomena were observed in different MRSA genetic background isolates, including HA-MRSA WCUH29, CA-MRSA 923, and MW2. To elucidate which gene(s) regulated by SaeRS contribute to the effect, we conducted a series of complementation studies with selected known SaeRS target genes in trans. We found coagulase complementation abolished the enhanced survival of the SaeRS mutant in human blood. The coa and saeRS deletion mutants exhibited a similar survival phenotype in blood. Intriguingly, heterologous expression of coagulase decreased survival of S. epidermidis in human blood. Further, the addition of recombinant coagulase to blood significantly decreased the survival of S. aureus. Further, analysis revealed staphylococcal resistance to killing by hydrogen peroxide was partially dependent on the presence or absence of coagulase. Furthermore, complementation with coagulase, but not SaeRS, returned saeRS/coa double mutant survival in blood to wild-type levels. These data indicate SaeRS modulates bacterial survival in blood in coagulase-dependent manner. Our results provide new insights into the role of staphylococcal SaeRS and coagulase on bacterial survival in human blood.

Keywords: S. aureus; SaeRS; coagulase; survival; two-component system.

Figure 1

Effect of saeS or saeRS null mutation and complementation on survival of S. aureus in human blood. Percent survival of wild-type S. aureus HA-MRSA WCUH29 (A), USA300 CA-MRSA 923 (B), USA400 CA-MRSA MW2 (C), and saeRS null mutants in freshly collected heparinized human blood with appropriate antibiotics. Bacteria were cultured overnight, diluted, inoculated into blood, and incubated at 37°C in a rotisserie incubator. Percent survival = (#CFU_final/#CFU_input)^*100. The data represents the mean ± SEM of at least six independent experiments.

Figure 2

Impact of sa1000, efb, fnbAB, or coa expression in trans on enhanced survival of saeRS null mutants. Percent survival of HA-MRSA WCUH29 (A), USA300 CA-MRSA 923 (B), USA400 CA-MRSA MW2 (C), and the sa1000, efb, fnbAB, or coa expression strain, and saeRS complementation strain in freshly collected heparinized human blood with appropriate antibiotics. Data is the mean and standard error of at least six experiments per strain.

Figure 3

Effect of coa deletion mutation and complementation on survival of S. aureus in human blood. Percent survival of wild-type S. aureus USA300 CA-MRSA 923 (A), USA400 CA-MRSA MW2 (B), and coa deletion mutants and its complementation in freshly collected heparinized human blood with appropriate antibiotics. Bacteria were cultured overnight, diluted, inoculated into blood, and incubated at 37°C in a rotisserie incubator. Percent survival = (#CFU_final/#CFU_input)^*100. The data represents the mean ± SEM of at least six independent experiments.

Figure 4

Effect of coa heteroexpression or addition of recombinant coagulase on bacterial survival. (A) Percent survival and (B) bacterial load of wild-type S. epidermidis and coa expression in trans in human blood during induction. (C) SDS-PAGE analysis of purified recombinant Coa (coagulase) and control protein SarZ from E. coli. M, ColorPlus Prestained protein Marker (NEB), the unit of size is kDa. (D) Coagulation activity of purified recombinant Coa (rCoa) from E. coli in human blood. CT, negative control without addition of rCoa. (E) Percent survival of wild-type USA300 CA-MRSA 923 and addition of different concentrations of purified rCoa and control rSarZ protein in human blood. Data is the mean and standard error of at least four experiments per strain.

Figure 5

Effect of Coa on bacterial resistance to H₂O₂. (A) Percent survival of wild-type USA300 CA-MRSA 923, coa deletion mutant and complementation strain after exposure of H₂O₂. (B) Percent survival of wild-type S. epidermiditis and coa expression strain in trans after exposure of H₂O₂. The bacterial strains were cultured with appropriate antibiotics. Approximately 5 × 10⁸ CFU were incubated in sterile PBS with 1.5% hydrogen peroxide at 37°C for 1 h. The percent survival was calculated as (#CFU_final/#CFU_input)^*100. The data represents the mean ± SEM of at least four independent experiments.

Figure 6

Effect of saeRS and coa double mutation, and complementation on survival of S. aureus in blood. Survival of wild-type USA300 CA-MRSA 923, saeRS/coa double deletion mutant, and coa or saeRS complementation strain in human blood. Data is the mean and standard error of at least four experiments per strain.