Role Of Three Membrane Proteins And A Novel Secreted Protein EsxX In The ESAT-6 Secretion System Of Community-associated Staphylococcus Aureus Sequence Type(ST)398

Objective:To investigate the role of three membrane proteins EsaA,EssA,EssC and the novel secreted protein EsxX in the ESS of CA-SA ST398.Methods:A representative clinical isolate of CA-SA ST398 was selected as wildtype.The isogenic gene deletion and genetic complementation of esaA,essA and essC genes were constructed.Growth condition and capacity of hemolysis and biofilm formation were detected.Neutrophil lysis and murine models of skin and blood infections were performed to evaluate the impact of three membrane proteins on virulence of CA-SA ST398.The secretion of EsxA was studied by Western blot assay.Furthermore,we compared the ess locus between ST398 and USA300 strain,choosing a novel protein of unknown function in ST398 ESS for investigation,we named the protein EsxX.We performed sequence alignment of EsxX to analyze its conservation,and then we constructed esxX deletion and genetic complementation.The secretion and subcellular localization of EsxX were demonstrated by Western blot assay.We used quantitative RT-PCR to study whether esxX gene expression was regulated by agr system.Experiments were carried out to investigate the effects of EsxX on growth,hemolysis and biofilm formation.In vitro neutrophil lysis and phagocytosis were performed to evaluate role of EsxX in virulence and host innate defense.In vivo murine models of skin and blood infections were performed to investigate contribution of EsxX in virulence and pathogenesis of CA-SA ST398.Results:Protein EsaA,EssA and EssC did not influence the growth,hemolysis or biofilm formation of ST398.The essC mutant showed significantly decreased neutrophil lysis compared with the wild-type strain(P<0.01).In the murine skin abscess model,esaA,essA,essC three mutants all resulted in significantly smaller abscess area compared with ST398 wildtype(P<0.01),especially essC mutant(P<0.0001).In a low-dose(10~8CFU)bacteremia model,the mice infected with essA and essC mutant showed notably lower CFU counts in the kidneys 4 days after infection(P<0.01)when compared with the ST398 infected mice.Histological examination also showed decreased infiltration of inflammatory cells and tissue structure destruction in the kidneys of essA and essC mutant-infected mice.Furthermore,the Western blot suggested that esaA,essA,essC three mutants all led to EsxA secretion deficiency.By comparing the ess locus between ST398 and USA300strain,we found a novel WXG family protein in ST398 ESS,downstream of EsxB,and we named it EsxX.There was no homologous protein of EsxX in other S.aureus strains,and EsxX was highly conserved in ST398.EsxX was demonstrated as a secreted protein and its secretion was controlled by ESS.The expression of esxX gene was not under regulation of agr system,and also EsxX protein did not influence growth,hemolysis or biofilm formation of ST398.The esxX mutant showed significantly decreased neutrophil lysis compared with the wild-type strain(P<0.01),and the esxX mutant was more likely to be eliminated by neutrohpil than the wildtype strain(P<0.01).In murine skin abscess model,the esxX mutant caused significantly smaller abscess area than ST398 wildtype.In murine bacteremia model,the mice infected with the esxX mutant showed notably decreased CFU counts in the kidneys 4days after infection than the mice infected with the ST398 wildtype.Besides,the histological examinations of skin and kidneys showed decreased infiltration of inflammatory cells and tissue destruction in the mice infected with the esxX mutant than the ST398 wildtype.Conclusion:The EsaA,EssA,EssC proteins play important role in pathogenicity of CA-SA ST398.They contribute to virulence of ST398,of which the EssC protein is the most remarkable contributor.The three membrane proteins all controlled the secretion of EsxA,and this might be one of the mechanisms involved in the virulence of CA-SA ST398.EsxX is a novel secreted protein of ST398 ESS,which is highly conserved in ST398.EsxX promotes ST398 evade the innate host defense and contributes to virulence.Protein EsxX might as a virulence factor which plays an important role in pathogenesis of CA-SA ST398.