The Role of graRS in Regulating Virulence and Antimicrobial Resistance in Methicillin-Resistant Staphylococcus aureus.

Le Chen,Xianhui Li,Zihui Wang,Yang Wu,Hongfei Ge,Tao Xu,Keqing Zhao,Chunquan Zheng,Di Qu,Fangyue Zhou,Xiaoyi Zhu

doi:10.3389/fmicb.2021.727104

Abstract

Methicillin-resistant Staphylococcus aureus (MRSA) is a common cause of both community- and hospital-associated infections. The antibiotic resistance and virulence characteristics of MRSA are largely regulated by two-component signal transduction systems (TCS) including the graRS TCS. To make a relatively comprehensive insight into graRS TCS in MRSA, the bioinformatics analysis of dataset GSE26016 (a S. aureus HG001 WT strain vs. the ΔgraRS mutant) from Gene Expression Omnibus (GEO) database was performed, and a total of 563 differentially expressed genes (DEGs) were identified. GO analysis revealed that the DEGs were mainly enriched in the “de novo” IMP biosynthetic process, lysine biosynthetic process via diaminopimelate, and pathogenesis; and they were mainly enriched in purine metabolism, lysine biosynthesis, and monobactam biosynthesis in KEGG analysis. WGCNA suggested that the turquoise module was related to the blue module, and the genes in these two modules were associated with S. aureus virulence and infection. To investigate the role of graRS in bacterial virulence, a graRS knockout mutant (ΔgraRS) was constructed using MRSA USA500 2,395 strain as a parent strain. Compared to the wild-type strain, the USA500ΔgraRS showed reduced staphyloxanthin production, retarded coagulation, weaker hemolysis on blood agar plates, and a decreased biofilm formation. These altered phenotypes were restored by the complementation of a plasmid-expressed graRS. Meanwhile, an expression of the virulence-associated genes (coa, hla, hlb, agrA, and mgrA) was downregulated in the ΔgraRS mutant. Consistently, the A549 epithelial cells invasion of the ΔgraRS mutant was 4-fold lower than that of the USA500 wild-type strain. Moreover, on the Galleria mellonella infection model, the survival rate at day 5 post infection in the USA500ΔgraRS group (55%) was obviously higher than that in the USA500 group (20%), indicating graRS knockout leads to a decreased virulence in vivo. In addition, the deletion of the graRS in the MRSA USA500 strain resulted in its increased susceptibilities to ampicillin, oxacillin, vancomycin, and gentamicin. Our work suggests that the graRS TCS plays an important role in regulating S. aureus virulence in vitro and in vivo and modulate bacterial resistance to various antibiotics.

Highlights

Staphylococcus aureus (S. aureus) is a major Gram-positive pathogen causing both community-acquired and hospitalacquired infections (Tong et al, 2015)
The results indicated that the transcriptional levels of the genes encoding penicillin-binding proteins PBP1, PBP2, and PBP4, the PBP2a encoding gene mecA, peptidoglycan synthesis genes femA, autolytic activity related gene llm, global regulator genes mgrA, spx, agrA, and sigB, the cationic antimicrobial peptide (CAMP) resistance-related genes dltX, dltA, mprF, vraF, and vraG, were significantly downregulated in USA500ΔgraRS mutant strain (Figures 3A,B; Supplementary Figure S2)
The two-component system graRS of S. aureus belongs to the IM-HK family and is conserved within the firmicutes

Summary

Introduction

Staphylococcus aureus (S. aureus) is a major Gram-positive pathogen causing both community-acquired and hospitalacquired infections (Tong et al, 2015). The prevalence of methicillin-resistant Staphylococcus aureus (MRSA) has aroused more concerns over the past decades because of their antibiotic resistance and virulence (Otto, 2013). Staphylococcus aureus has 16 two-component systems (TCSs), which are significant for bacteria and commonly used to sense and respond to environmental changes (Capra and Laub, 2012; Burgui et al, 2018; Villanueva et al, 2018; Yan et al, 2019). The graRS is involved in regulating the susceptibility to vancomycin and daptomycin in MRSA (Doddangoudar et al, 2011; Cafiso et al, 2012; Mensa et al, 2014; Muller et al, 2018). The role of graRS in the regulation of bacterial virulence remains to be determined

Methods

Results

Conclusion