Study On Molecular Mechanisms Of Staphylococcus Aureus Two-component Regulatory System VraSR Regulating Macrophage Autophagy And Promoting HVISA Immune Escape Escape

Staphylococcus aureus is widely distributed in nature.It can secrete a variety of virulence factors and surface adhesion proteins.It can cause serious infections such as bacteremia,pneumonia,endocarditis,septic arthritis,otitis media,deep abscess.S.aureus has strong viability and immune escape ability,often leading to chronic persistent infection.The immune escape mechanism is related to its virulence factors,such as toxins,enzymes and surface adhesion factors.On the other hand,it may reduce or inhibit host immune defense function through its interaction with host cells.In recent years,with the widespread use of vancomycin,heterogeneous vancomycin-mediated S.aureus(hVISA)strains have emerged.Research on hVISA strains shows that antibiotics are not effective for them and the body's immune system hard to eliminate them.But the molecular mechanism of hVISA immune escape is unclear.Therefore,an in-depth study of the interaction between hVISA and the host immune system will help us understand the molecular mechanism of S.aureus evading host immune response and achieving persistent infection.And it will provide us a theoretical basis for new therapeutic targets.Macrophages are an important part of the human body's innate immune system,with powerful functions of recognition,phagocytosis,removal of bacteria and foreign bodies.Autophagy is a self-stabilizing mechanism necessary for the survival,differentiation,development and metabolism of a body widely existed in eukaryotic cells.Autophagy is an important part of natural immunity and also acts as a physiological behavior for self-protection of the body.And it play a key role in infectious diseases and inflammation.But some pathogens have evolved mechanismsthat resist or escape autophagy clearance.Studies have shown that S.aureus can induce host bacteria to produce pro-bacterial autophagy,which will help S.aureus survive in host cells.Our team has been concerned about the regulation role of two component regulation system VraSR in S.aureus.VraSR is found that not only regulate vancomycin resistance but also bind the global regulator agr promoter region in S.aureus,indicating that VraSR has a wide range of regulatory functions.Our previous study found that VraSR can promote S.aureus adhere to epithelial cells,and enhance S.aureus against phagocytosis and sterilization of human neutrophils,and promote hVISA strains to escape the attack of human immune system.However,the mechanism of VraSR in the virulence regulatory network and immune escape mechanisms in S.aureus is unclear.This study will focus on the molecular mechanism of VraSR in the regulating the pathogenicity of hVISA and promoting immune escape by regulating macrophage autophagy from three aspects: bacteria,animals and cells.OBJECTIVE: In this study,to investigate the regulatory network of VraSR and its relationship with pathogenicity of S.aureus,hVISA standard strain(Mu3)and its vraSR knockout strain(Mu3?vraSR)and its complemented strain(Mu3?vraSR-C)were used by transcriptomics,intracellular survival experiments and animal experiments.The molecular mechanism of VraSR involved in the regulation of macrophages autophagy to promote the S.aureus immune escape was studied.The results will lay the theoretical foundation for finding the new method for the diagnosis and treatment of S.aureus infection.METHODS:(1)The purified rVraR protein and rabbit-derived VraR polyclonal antibody were obtained,and the expression levels of VraSR in primary strains of hVISA(Mu3),vraSR gene knockout strains(Mu3?vraSR)and their complemented strains(Mu3?vraSR-C)were identified by qRT-PCR and Western blotting.(2)RNA was extracted from Mu3 and Mu3?vraSR strain,and differentially expressed genes in Mu3 and Mu3?vraSR strain were screened by RNA-seq technique combined with bioinformatics analysis.The results were verified by qRT-PCR.The results will reveal the regulatory network and pathogenicity of VraSR in S.aureus.(3)We infected BALB/c mice by subcutaneous inoculation with Mu3,Mu3?vraSR and Mu3?vraSR-C and observe the effect of VraSR on mouse skin abscess area and the bacterial survival rate.The pathological changes of skin tissue inflammation were observed by H&E staining.(4)We infected BALB/c mice with Mu3,Mu3?vraSR and Mu3?vraSR-C by tail vein injection respectively.The effect of VraSR on the survival of S.aureus in vivo was calculated by CFU in mouse liver and kidney.And the pathological changes of liver and kidney inflammation were observed by H&E staining.(5)Mu3,Mu3?vraSR and Mu3?vraSR-C were respectively infected with mouse macrophage cell line RAW 264.7and establish infection model in vitro,and RAW 264.7 cells were collected at 0h,3h,6h,12 h and 34 h after infection.Calculate intracellular bacterial colony forming units(CFU)in different infection groups by plate colony counting method.(6)After infected RAW264.7 cells with Mu3,Mu3?vraSR and Mu3?vraSR-C respectively,we observed the ultrastructure of cells after S.aureus infection with RAW 264.7 by transmission electron microscope,and compare the autophagocytic vesicles in different infection groups.The S.aureus-infected cells were collected at 0h,1.5h,3h,4.5h and 6h.And the transcription analysis of three key genes Ulk1,Becn1 and Atg5 was detected by qRT-PCR.The cells were collected at 3 hours after infection,and the expression level of LC3 and P62 were detected by Western blotting.RAW 264.7 cells were transfected with mRFP-GFP-LC3 adenovirus,and S.aureus infected with the mRFP-GFP-LC3 cells for3 hours.We observed that intracellular LC3 dots after S.aureus infection by laser confocal microscopy.And after pretreatment with autophagy inhibitors 3-Methyl adenine(3-MA)and Bafilomycin A1(Baf A1),the growth rate of different strains was compared to exclude the effect of them on the growth rate of S.aureus.The growth rate and the expression level of LC3 and P62 in different infection groups were compared.(7)To investigate the specific molecular mechanism of VraSR involved in the regulation of autophagy in macrophages,we examined the AKT/mTOR/P70S6K and p38 signaling pathways and verified.RESULTS:(1)The expression levels of VraSR gene in Mu3,Mu3?vraSR and Mu3?vraSR-C strains were detected by qRT-PCR and Western blotting.It was found that vraSR was deleted in Mu3?vraSR strain,and Mu3?vraSR-C strain restored vraSR gene expression.It was confirmed that the knockout strain and complemented strain were successful.(2)The transcriptome analysis showed that 1210 genes were changed in the Mu3?vraSR strain compared with the Mu3 strain,421 genes were significantly down-regulated and 789 genes was up-regulated in the Mu3?vraSR strain.14 TCSs genes and 23 virulence-related genes changed significantly.GO analysis showed that VraSR regulated genes were mainly enriched in cell processes,colonization,metabolism.KEGG pathway analysis showed that VraSR regulated genes are mainly enriched in pathways such as metabolic pathways,amino acid biosynthesis,and ABC transport.It was shown that the saeR,clfA,hla,hlb,tst,essA,dltb,efb,sodA and sbi genes were down-regulated in Mu3?vraSR strain,while agrA,sdrE,coa and spa genes were up-regulated,confirming that VraSR is involved in the regulation of the pathogenesis of S.aureus.This leads to a pleiotropic alteration in the virulence of Mu3?vraSR strain.(3)The results of skin abscess in mice showed that compared with Mu3 strain,the area of abscess in Mu3?vraSR infected groups was reduced,the survival of Mu3?vraSR strains in abscess was reduced,the leukocyte infiltration,inflammatory reaction and tissue damage were also lighter than Mu3 infected groups.After VraSR gene was complemented,the area of abscess,colony count and tissue inflammation of Mu3?vraSR-C strain was similar to that of Mu3 strain,indicating that VraSR could helpS.aureus to form abscess in mouse skin and to survive in mouse skin.(4)Colony count and histopathological results showed that the colony count of Mu3?vraSR strain in liver and kidney was significantly lower than that of Mu3 strain,leukocyte infiltration and tissue abscess and necrosis were lighter than that of Mu3 strian.After complemented vraSR gene,the colony count of Mu3?vraSR-C strains was significantly restored,and the pathological damage was similar to that of Mu3 strain,indicating that VraSR can help S.aureus to from abscesses in liver and kidney of mice,and help it survive and reproduce in vivo.(5)The results of intracellular survival experiments showed that the Mu3?vraSR strain survived less in RAW 264.7 cells than the Mu3 strain,and the survival rate of Mu3?vraSR strains continued to decrease throughout the whole infection process.When VraSR was complemented,the survival rate of Mu3?vraSR-C strain in RAW264.7 cells was similar to that of Mu3 strain,indicating that VraSR contributes to the survival and proliferation of S.aureus in macrophages.(6)Transmission electron microscopy showed that only a small amount of autophagosome-like vesicles(8±1.5/50 cells)were observed in the Mu3?vraSR strain infection group(P<0.01),while Mu3 strains(27±2.5/50 cells)and Mu3?vraSR-C strains(23±1.5/50 cells)have more autophagosome-like vesicles,indicating that VraSR contributes to the formation of autophagosome-like vesicles after infection of macrophages by S.aureus.The results of qRT-PCR showed that compared with Mu3 strain infected-cells,the Becn1 and Atg5 transcription levels were significantly decreased in Mu3?vraSR strain infected-cells at 3 h(P<0.01),while Mu3?vraSR-C infected-cells were returned to the level of Mu3,indicating that VraSR contributes to the expression of autophagy genes.Western blot analysis showed that the expression level of LC3-II and p62 in Mu3?vraSR infected-cells was significantly lower than that of Mu3 and Mu3?vraSR-C infection group(P < 0.01),indicating that VraSR is involved in the regulation of autophagic flow in S.aureus infected-RAW 264.7 cells.The results oflaser confocal microscopy showed that the Mu3?vraSR infection group had less LC3 yellow dot-like aggregation at 3h compared with the Mu3 infection group(P < 0.01),while the Mu3?vraSR-C infection group was similar to the Mu3 infection group.The results indicated that VraSR promoted the formation of autophagosomes in S.aureus-infected cells.The Mu3?vraSR infection group had more single-red LC3 punctate aggregation than the other two groups(P < 0.01).It indicated that VraSR can block or delay the fusion of autophagosomes and lysosomes in RAW 264.7 cells.The results of autophagy inhibitor treatment showed that 3-MA can decrease the expression of LC3-II in RAW 264.7 cells and significantly decrease the intracellular survival rate of Mu3 and Mu3?vraSR-C strains(P < 0.01).Baf A1 can significantly increase the expression of p62 protein in all infected groups,and the bacterial survival rate in the Mu3?vraSR infected group was significantly increased.The above results indicated that VraSR can increase the intracellular survival rate of S.aureus by inhibiting macrophage autophagic flow.(7)Western blot analysis showed that there was no significant difference in the expression levels of AKT,mTOR and P70S6K proteins and their phosphorylation levels in the three groups of infected cells,but the expression level of p38 protein phosphorylation in Mu3?vraSR infection group was significantly decreased.After VraSR was complemented,Mu3?vraSR-C infected group can restore p38 phosphorylation expression level.When the p38 phosphorylation level inhibitor was added,the expression level of p62 protein in the Mu3 infected group and the Mu3?vraSR-C infected group decreased significantly similar to that in the Mu3?vraSR infected group.These results indicate that VraSR can inhibit autophagosome maturation by activating p38 phosphorylation,preventing autophagosome fusion with lysosomes,and not related to AKT/mTOR/p70S6K signaling pathway.CONCLUSION:(1)VraSR is involved in the regulation of the pathogenesis ofhVISA strain,resulting in pleiotropic changes in virulence.(2)VraSR helps hVISA to survive in vivo and in vitro.(3)hVISA strain can use VraSR to induce autophagy activity of RAW 264.7 and promote hVISA survival in cells.(4)hVISA strain can use VraSR to activate p38 phosphorylation to inhibit autophagosome maturation and prevent autophagosome fusion with lysosome,but not related to AKT/mTOR/p70S6K signaling pathway.