The Role Of Two-component Regulatory System Vra Sr In Bacterial Pathogenicity Of Staphylococcus Aureus With Reduced Vancomycin Susceptibility

Objectives Many studies have reported that h VISA/VISA is usually accompanied by changes in biological phenotype such as virulence and biofilm.These changes can cause vancomycin osmotic barriers,bacteria are difficult to remove,and infection symptoms persist,making treatment extremely difficult.Vra SR is a vancomycin resistance associated TCRS that is significantly upregulated in VISA/h VISA strains.We previously demonstrated that Vra SR directly regulated Agr,indicating that Vra SR has a wide range of regulatory functions.This study focused on the role of Vra SR in the h VISA/VISA pathogenicity in order to provide a theoretical basis for the diagnosis and clinical treatment of h VISA/VISA infection based on Vra SR.Methods(1)Using gene knockout and gene complementation technology to construct heterogeneous vancomycin-mediated Staphylococcus aureus(Mu3)vra SR gene deletion mutant(Δvra SR)and gene complement strain(CΔvra SR),and to verify whether the construction is success though RT-PCR.(2)Biological phenotypic study: the growth of Staphylococcus aureus was measured by spectrophotometer,and the growth curve was drawn;the MIC value of vancomycin was detected by E-test method;The hemolysin activity and coagulase activity were quantitatively detected by dilution method;the bacterial cell wall was observed by electron microscopy.Co-culture with human cervical cancer epithelial cells to observe the bacterial adhesion ability;in vitro neutrophil phagocytosis and killing detection of bacterial anti-phagocytosis and anti-bactericidal ability.The q RT-PCR method detects the transcription levels of adhesion-related genes(fnb A,fnb B,clf A,ebps,sbi)and cell wall synthesis-related genes(cap5K,cap5 N,nan A,tag A,mur D).Results 1.We have constructed vra SR gene deletion mutant strain and gene complement strain successfully.The transcription of vra SR gene in Δvra SR was completely deleted,and the transcription of vra SR gene was restored by plasmid replenishment of Δvra SR.2.The effect of the deletion of vra SR gene in Mu3 on the biological phenotype:(1)Compared with Mu3,Δvra SR had no significant effect on the growth of S.aureus;(2)The vancomycin MIC values ??of Mu3,Δvra SR and CΔvra SR were 1.5 μg/ml,0.38 μg/ml,and 1.5 μg/ml,the vancomycin MIC value of theΔvra SR strain was decreased 3.95 fold compared with Mu3.The CΔvra SR strain can restore the vancomycin MIC value;(3)There was no significant difference in hemolysin activity and coagulase activity between Δvra SR and Mu3;(4)The cell wall synthesis ability of Δvra SR strain was decreased,and the average cell wall thickness of Mu3 andΔvra SR were 18.53 ± 2.84 nm and 26.64 ± 3.07nm(p < 0.01).CΔvra SR strain can restore cell wall synthesis ability,the average cell wall thickness is 23.32 ± 3.59 nm(p <0.01);(5)Compared with Mu3,Δvra SR has decreased the ability to adhere to He La cells(LOG10 CFU/ml,5.75±0.43 Vs 3.53±0.71,p<0.01),CΔvra SR can regain adhesion to He La cells(LOG10 CFU/ml 5.20±0.36,p<0.05).The CΔvra SR strain can restore the expression of adhesion genes.(6)The biofilm synthesis ability of Δvra SR strain was decreased,and the average thickness of Mu3 and Δvra SR biofilms was7.38±1.4 μm Vs 14.44±1.65 μm,respectively(p < 0.001).CΔvra SR can restore the biofilm synthesis ability(9.35±1.37μm,p < 0.01).(7)Compared with Mu3,Δvra SR is more susceptible to phagocytosis and killing by neutrophils.Mu3 and Δvra SR phagocytosis rates are 47.75 ± 2.56% and 71.29 ± 2.55%,respectively(p<0.001),the phagocytic index was 1.65±0.16 and 3.11±0.32,respectively(p<0.01),and the survival rate of bacteria in neutrophils was 5.56±0.63 Vs 2.73±0.50(LOG10 CFU/ml)respectively(p<0.001).(8)The expression of adhesion-related genes fnb A,fnb B,clf A,ebps,sbi and cell wall synthesis-related genes cap5 K,cap5N,nan A,tag A,mur D were all down-regulated,and the difference was significant(p<0.05).Conclusions The Vra SR system plays an important role in the pathogenicity of h VISA/VISA.The vra SR gene can promote the resistance of Staphylococcus aureus to vancomycin,increase adhesion,cell-wall synthesis and biofilm-forming ability.The vra SR gene can also enhance phagocytosis and bactericidal ability of Staphylococcus aureus against human neutrophils,and assist Staphylococcus aureus to escape the attack of human immune system,which is conducive to its survival in the host.