Role of WalK(S221P) mutation on virulence of vancomycin-intermediate Staphylococcus aureus and underlying mechanism

Abstract

Objective To explore the role and mechanism of WalK(S221P) mutation affecting the virulence of vancomycin-intermediate Staphylococcus aureus (VISA). Methods RNA sequencing (RNA-seq) was used to compare the differentially expressed genes (DEGs) between VISA strain XN108 and its isogenic WalK(S221P) mutation cured strain K65. The expression of selected DEGs in strains of interest were detected by RT-qPCR. Hemolytic experiment was employed to detect the hemolytic activities of S. aureus strains. WalK(S221P) mutation carried strains (K-USA300 and K-USA300ΔagrA) were constructed by homologous recombination technologies. E-test was applied to detect the vancomycin sensibilities of S. aureus strains. The electrophoretic mobility shift assay (EMSA) was performed to detect the binding ability of WalKR on the promoter regions of target gene. Results RNA-seq revealed that the expression levels of several important virulence factors were upregulated in WalK(S221P) mutation cured K65 when compared with those in the wild-type XN108 (P < 0.01). The activity of Agr and the hemolytic activity in K65 were also enhanced. Introduction of WalK(S221P) mutation into MRSA strain USA300 resulted in the reduced vancomycin susceptibility of K-USA300 than that of the wild-type USA300, however, K-USA300 presented decrease in the expression levels of these important virulence factors, and the hemolytic activity of K-USA300 was also reduced. EMSA showed that WalK-phosphorylated WalR could directly bind to the agr promoter region. Introduction of WalK(S221P) into USA300ΔagrA reduced vancomycin susceptibility of K-USA300ΔagrA, while the expression levels of selected virulence genes and hemolytic activities were comparable between K-USA300ΔagrA and USA300ΔagrA. Conclusion WalK(S221P) mutation affects the virulence of VISA by directly contacting the agr promoter region. WalK(S221P) mutation induces decreased WalKR activity and lowered activation to Agr, and thus affects the virulence of VISA.

Keywords

• vancomycin-intermediate staphylococcus aureus
• two-component system
• walkr
• walk(s221p) mutation
• virulence