Serine/threonine phosphatase Stp1 contributes to reduced susceptibility to vancomycin and virulence in Staphylococcus aureus

Abstract

The genetic mechanisms that contribute to reduced susceptibility to vancomycin in Staphylococcus aureus are complex and heterogeneous. In addition, debate is emerging as to the true effect of reduced susceptibility to vancomycin on staphylococcal virulence. To investigate this, comparative genomics was performed on a collection of vancomycin-exposed isogenic S. aureus pairs (14 strains in total). Previously described mutations were observed in genes such as vraG, agrA, yvqF, and rpoB; however, a new mechanism was identified involving a serine/threonine phosphatase, Stp1. After constructing an stp1 deletion mutant, we showed that stp1 is important in vancomycin susceptibility and cell wall biosynthesis. Gene expression studies showed that stp1 also regulates virulence genes, including a hemolysin, superantigen-like protein, and phenol-soluble modulin, and that the deletion mutant is attenuated in virulence in vivo. Stp1 provides a new link between vancomycin susceptibility and virulence in S. aureus.

Figure 1.

Deletion of stp1 results in reduced vancomycin susceptibility in Staphylococcus aureus. (A) Vancomycin minimum inhibitory concentrations (MICs) were determined by Etest for A5937, A5937Δstp1, the complemented strain A5937Δstp1-C, and the vancomycin nonsusceptible isolate A5940. Values in parentheses represent MICs based on broth dilution. (B) Determination of vancomycin heteroresistance was performed using population analysis profiling. The stp1 deletion mutant had subpopulations that grew in the nonsusceptible range (>2 µg/mL); this was not seen in the parent strain (A5937) but was more pronounced in the clinical vancomycin-intermediate S. aureus daughter strain (A5940). CFU, colony-forming unit. (C) Transmission electron microscopy was performed to determine cell wall thickness. Three representative cells from each strain are shown, and values represent mean cell wall thickness (95% confidence interval). A5940 had the thickest cell wall, followed by the A5937Δstp1 and finally A5937.

Figure 2.

Deletion of stp1 results in attenuated virulence in Staphylococcus aureus. (A) Six-week-old C57BL/6J mice were injected with ∼1.0 × 10⁸ bacterial cells. Kaplan-Meier curves indicate time to euthanasia for each strain. A5937 produced significantly more killing compared with both A5937Δstp1 and A5940 (P = .01; log-rank test). (B) Macroscopic and (C) histopathological analyses showed that the clinical vancomycin-susceptible parent strain (A5937) caused hepatic abscess formation and tissue necrosis, whereas this was not seen with the stp1 deletion mutant (A5937Δstp1) and the clinical vancomycin-intermediate S. aureus daughter strain (A5940). White arrows point to abscess formation in the liver; N represents severe necrosis; and I represents inflammatory infiltrate. Tissues were stained with hematoxylin and eosin.