Establishment Of Rapid Detection Technology For Mrsa Bacteria With Pvl And Study On Its Pathogenic Mechanism

Research background:In the world,Staphylococcus aureus is one of the important pathogens that cause invasive infection in hospitals such as severe pneumonia,and it is also one of the main pathogens of community-acquired infection.With the increasing use of antibiotics in clinical treatment,a large number of"super-bacteria"with multiple drug resistance have emerged,namely methicillin resistant Staphylococcus aureus（MRSA）.The large number of MRSA in clinical patients directly leads to its treatment more difficult.In recent years,it has been found that MRSA with Panton-Valentine leukocidin（PVL）is more virulent and more harmful,which can cause serious community infection and seriously endanger the lives of infected people.The rate of death necrotic pneumonia caused by it can reach to 75%.At present,there is no rapid identification method for MRSA with pvl gene,which is not conducive to the timely diagnosis and treatment of infected people,so it is of great clinical value to establish related rapid detection technology.At present,the pathogenic mechanism of MRSA with pvl gene is still unclear.The study of its pathogenic mechanism can provide new ideas for clinical treatment.Objective:1.Establish the rapidmethod for MRSA strain with PVL detection;2.To study the mechanism of MRSA with PVL on autophagy,apoptosis and pyroptosis;Methods:1.To establish a modified alkaline lysis method to extract the genomic DNA of S.aureus,using PCR to detect spa,mecA,femB and pvl genes,and detecting spa and mecA genes by LAMP technology,to establish a rapid detection technology of S.aureus.2.Recombinant and purify rPVL protein of E.coli DH5α,and then immunize rabbits to prepare polyclonal antibody.Western blotting method was used to detect the expression of PVL protein of S.aureus,to establish a rapid detection technology of pvl gene of S.aureus.3.The Spa,mecA and pvl genes were detected by PCR and Western blotting.A MRSA with PVL(MRSA^PVL+)and a MRSA without PVL(MRSA^PVL-)were screened for further experimental study.4.MRSA^PVL+and MRSA^PVL-co cultured with THP-1 cells after 1 h,3 h and 6 h,extracting the RNA to detect the expression of autophagy related genes（atg3,atg4B,atg5,atg7,atg12 and atg16L1）,inflammatory factor related genes（IL-4,IL-1β,IL-6,IL-10,IL-12,IFN-αand TGF-β）,inflammasome related genes（NLRP3,caspase-1 and IL-1β）and toll like receptor related genes（TLR-2 and TLR-4）by RT-PCR.Then extracting the proteins to detect the expression of autophagy related proteins beclin-1,LC-3,mTOR,PI3K,Akt,p-mTOR,p-PI3k,p-Akt,apoptosis related proteins caspase-3,cleaved Caspase-3 and pyroptosis related proteins GSDMD,NLRP3 and Caspase-1by Western blotting.5.THP-1 cells were transfected NLRP3 siRNA to inhibit the expression of NLRP3,and then co cultured with MRSA^PVL+and MRSA^PVL-for 1 h,3 h and 6 h,then extracting the proteins to detect the expression of autophagy related proteins beclin-1,LC-3B,apoptosis related proteins caspase-3,cleaved Caspase-3 and pyroptosis related proteins GSDMD,NLRP3 and Caspase-1 by Western blotting.6.MRSA^PVL+and MRSA^PVL-were co cultured with THP-1 cells for 1 h,3 h and 6 h,then the expression of ROS was detect by DCFH-DA fluorescence probe method and annexin V-FITC/PI method was used to detect apoptosis.7.After co culturing A549 cells with MRSA^PVL+and MRSA^PVL-for 1 h,3 h and 6 h,cell proteins were extracted and detected by Western blotting such as beclin-1,LC-3B,caspase-3 and cleaved caspase-3.8.MRSA^PVL+and MRSA^PVL-were co cultured with A549 cells for 1 h,3 h and 6 h,the expression of ROS was detected by DCFH-DA fluorescence probe method,and apoptosis was detected by annexin V-FITC/PI method.Results:1.Established a rapid detection method for detection of MRSA:PCR method to detect spa、mecA and pvl genes,lamp method to detect spa and mecA genes.2.Established a rapid detection method for detection of PVL:PCR method to detect pvl gene;Recombinant and purify PVL protein and then prepare multi antibody serum.Western blotting method was used to detect the expression of PVL protein of MRSA.3.One MRSA without PVL(MRSA^PVL-)which did not express PVL and one MRSA with PVL(MRSA^PVL+)which expressed PVL were screened and by PCR and Western blotting for further study.4.The MRSA^PVL-strain and MRSA^PVL+strain can induce THP-1 cells and A549 cells to express a large number of ROS,ROS expression was the highest at 6 h,and with the extension of time the ROS accumulation increased,and the ROS expression of MRSA^PVL+is stronger than that of MRSA^PVL-.5.The MRSA^PVL-strain and MRSA^PVL+strain can induce autophagy of THP-1 cells at1 h,and the autophagy of THP-1 cells is the most significant at 3 h,and the autophagy ability of MRSA^PVL+is significantly stronger than that of MRSA^PVL-（P<0.05）;both MRSA^PVL-and MRSA^PVL+can induce autophagy by inhibiting the phosphorylation of PI3K,Akt and mTOR proteins of THP-1 cells at 1 h,3 h and 6 h,while in 1 h and 3 h was more obvious,and the inhibition ability of MRSA^PVL+was stronger than that of MRSA^PVL-（P<0.05）.6.The MRSA^PVL-strain and MRSA^PVL+strain can induce THP-1 cells and A549 cells to have time-dependent apoptosis,and the apoptosis at 6h was very obvious,and MRSA^PVL+were more significant than MRSA^PVL-,in which THP-1 cells are mainly in early apoptosis,but A549 cells were mainly in late apoptosis.7.The MRSA^PVL-strain and MRSA^PVL+strain can induce THP-1 cells to release a large number of inflammatory factors and inflammatory complex related factors.The expression of IL-1β,IL-6,IL-12,IL-17A and IFN-αgenes were significantly increased after ifected by MRSA^PVL+for 1 h（P<0.01）.The expression of IL-4,IL-1βand TGF-βgenes were increased after ifected by MRSA^PVL+for 3 h（P<0.05）,but the amplitude was lower than 1 h.When infected for 6 h,the expression of IL-4 and IFN-αgenes were higher in MRSA^PVL-group（P<0.05）and the expression of IL-4,IL-1β,IL-10,TGF-β,IL-6,IL-17A and IFN-αgenes were increased in MRSA^PVL+group（P<0.01）.The inflammatory factor NLRP3 increased at 1 h,and at 6 h only increased in MRSA^PVL+group.The expression of caspase-1 and IL-1βin THP-1 cells induced by MRSA^PVL-and MRSA^PVL+increased when the time prolonged.Compared with the MRSA^PVL-,the MRSA^PVL+had a more significant effect on the expression of inflammatory related factors in THP-1（P<0.05）.MRSA^PVL-and MRSA^PVL+can promote the activation of NF-κB protein phosphorylation in THP-1 cells,and it can be seen that the phosphorylation of NF-κB protein was significantly increased at 1 h,and the effect of MRSA^PVL+was stronger.The MRSA^PVL-strain and MRSA^PVL+strain can stimulate THP-1 cells to produce NLRP3-related protein,the protein NLRP3 increased most obviously at 1 h and 6 h,not at 3 h;the caspsae-1 protein increased at 1 h and 3 h with the treatment of the MRSA^PVL-strain,and increased at 1 h,3 h and 6 h with the treatment of MRSA^PVL+strain,which was also stronger in MRSA^PVL+group.8.The MRSA^PVL-strain and MRSA^PVL+strain could induce the change of GSDMD expression in THP-1 cells in 3 h（P<0.05）.9.After transfected with the NLRP3-siRNA to inhibit the expression of NLRP3 in THP-1 cells,the MRSA^PVL-strain and MRSA^PVL+strain for 1 h,3 h and 6 h,the expression of NLRP3 and caspase-1 proteins were significantly lower than that in control group（P<0.05）,the expression of LC-3 and Beclin-1 were significant decreased at 1 h and 3 h（P<0.05）,the Caspase-3 expression was obviously lower than that in control group（P<0.05）at 1 h,3 h and 6 h,and the pyroptosis related proteins GSDMD was decreased at 3 h（P<0.05）.Conclusion:The MRSA strain can induce autophagy and apoptosis of THP-1 cells,and induce apoptosis of A549 cells.MRSA with PVL will induce the process of autophagy and apoptosis.MRSA with pvl gene can regulate autophagy and apoptosis of THP-1 cells through PI3K/Akt/mTOR signaling pathway mediated by toll like receptor.The process is accompanied by a large amount of ROS,activation of inflammatory complex and expression of a large number of pro-inflammatory cytokines,which eventually leads to autophagy,apoptosis and necrosis of THP-1 cells and A549 cells.MRSA can also induce pyroptosis in THP-1 cells,and MRSA with pvl gene induce more effectively.When inhibiting the expression of NLRP3,the apoptosis of THP-1 cells will also be inhibited,reduce the expression of pro-inflammatory cytokines,and reduce the inflammatory of patients.