A vancomycin resistance-associated WalK(S221P) mutation attenuates the virulence of vancomycin-intermediate Staphylococcus aureus

INTRODUCTION: Vancomycin-intermediate Staphylococcus aureus (VISA) is typically associated with a decline in virulence. We previously reported a WalK(S221P) mutation that plays an important role in mediating vancomycin resistance in VISA XN108. Whether this mutation is implicated in bacterial virulence remains unknown. OBJECTIVES: This study aimed to investigate the effect of WalK(S221P) mutation on the virulence of VISA and the underlying mechanism of this effect. METHODS: The influence of WalK(S221P) mutation on VISA virulence and its underlying mechanism were explored using animal models, RNA-seq analysis, RT-qPCR, hemolytic assay, slide coagulase test, Western blot, β-galactosidase assay, and electrophoresis mobility shift assay (EMSA). RESULTS: Compared with XN108, WalK(S221P)-reverted strain XN108-R exacerbated cutaneous infections with increased lesion size and extensive inflammatory infiltration in mouse models. The bacterial loads of S. aureus XN108-R in murine kidney increased compared with those of XN108. RNA-seq analysis showed upregulation of a set of virulence genes in XN108-R, which exhibited greater hemolytic and stronger coagulase activities compared with XN108. Introduction of WalK(S221P) to methicillin-resistant S. aureus USA300 and methicillin-susceptible strain Newman increased the vancomycin resistance of the mutants, which exhibited reduced hemolytic activities and decreased expression levels of many virulence factors compared with their progenitors. WalK(S221P) mutation weakened agr promoter-controlled β-galactosidase activity. EMSA results showed that WalK-phosphorylated WalR could directly bind to the agr promoter region, whereas WalK(S221P)-activated WalR reduced binding to the target promoter. Inactivation of agr in S. aureus did not affect their vancomycin susceptibility but mitigated the virulence alterations caused by WalK(S221P) mutation. CONCLUSION: The results of our study indicate that WalK(S221P) mutation can enhance vancomycin resistance in S. aureus of diverse genetic backgrounds. WalK(S221P)- bearing S. aureus strains exhibit reduced virulence. WalK(S221P) mutation may directly impair the activation of the agr system by WalR, thereby decreasing the expression of virulence factors in VISA.