| Introduction: Methicillin-resistant Staphylococcus aureus(MRSA)is a common pathogen in the clinic and can cause a variety of infectious diseases.The glycopeptide antibiotic vancomycin is the most commonly used antibiotic for clinical treatment of MRSA infection and is also considered to be the last line of defense against severe MRSA infection.However,with the extensive use of vancomycin,the detection rate of low-level vancomycin sensitive Staphylococcus aureus has increased,including vancomycin intermediate drug resistant Staphylococcus aureus(VISA)and heterogeneous VISA(hVISA).It is generally accepted that VISA/hVISA is an adaptive resistance produced by vancomycin-sensitive Staphylococcus aureus(VSSA)or its partial strains under the pressure selection of vancomycin.VISA has phenotypic changes in cell wall thickness,hemolysis,autolytic activity,biofilm formation,but the resolution of VISA resistance at the DNA or protein level is not sufficient.Staphylococcus aureus was originally thought to be an extracellular bacterium,but more and more studies have shown that it can invade host cells.Clinically VISA often causes chronic persistent infections,which may be related to the resistance to intracellular bacteria clearance of host cells.At present,although the Staphylococcus aureus completely resistant to vancomycin,is still rare,the detection rate of low-level vancomycin-resistant Staphylococcus aureus hVISA and VISA is still high.Antibiotics are still the most effective drugs for the treatment of bacterial infections.However,under the pressure selection of antibiotics,more and more bacteria have multi-drug resistance.Therefore,it is more urgent to seek new methods to control infection.The immune system is an important weapon for the body to fight infection.The innate immune system is able to quickly identify and remove pathogens that enter into the body,making it the first line of defense against infection.Macrophages are innate immune cells of the body,and capable of phagocytizing and clearing pathogens in cells,and secreting a variety of biologically active substances,playing an important role in the body’s immune defense.Autophagy is a catabolic process that relies on lysosomes to degrade proteins and organelles in cells.It not only plays an effective role in cell stress,but also plays an important role in the prevention ofintracellular infections.However,some pathogens can promote their own survival in host cells by interfering with cellular autophagosome maturation.Studies have shown that macrophages play an important role in autophagy to regulate immune and inflammatory responses.Therefore,this study first selected vancomycin-sensitive Staphylococcus aureus(VSSA)strain 11 isolated in clinic,simulating the clinical treatment to increase the amount of vancomycin,using multi-step vancomycin induction,the minimum inhibition of vancomycin.Infected mouse macrophage RAW264.7 with strains 11 Y and 11 to explore the macrophage intracellular strains survival,cellular inflammatory response and autophagy and its mechanisms,and resolved the drug-resistance characteristics of 11 Y.Methods: 1.MTT assay was used to detect the viability of RAW264.7 macrophage infected with 11 Y and 11 respectively for 30 min,1 h,2 h and 4 h.2.BHI agar plate colony counting method was used to detect the viability of phagocytic strains 11 Y or 11.3.The bilayer membrane around strain 11 Y or 11 in RAW264.7 macrophages was detected by transmission electron microscopy.4.LC3-II in RAW264.7 macrophages was detected by laser confocal microscopy.5.Real-time PCR was used to detect the mRNA expression of inflammatory cytokines IL-1β,IL-6,IFN-γ and TNF-α in RAW264.7 macrophages after infection by strains 11 Y or 11.6.ELISA was used to detect the changes of above cytokines in the supernatant of RAW264.7 macrophage after infection of strain 11 Y or 11.7.Fluorescence microplate reader was used to detect the ROS content in RAW264.7macrophage after infection of strain 11 Y or 11.8.Western Blot was used to detect the contents of NLRP3,mTOR/p-mTOR,LC3 and SQSTM1 in RAW264.7 macrophage after infection by strains 11 Y or 11.9.The ROS inhibitor NAC was used to remove ROS produced by RAW264.7 cells infected with strains 11 Y or 11.10.The cell wall thickness of strain 11 Y and 11 was observed by transmission electron microscopy.11.Blood agar plates were used to determine the δ-hemolytic activity of strain 11 Yand 11.12.Visible light spectrophotometer was used to detect the absorbance of strain 11 Y and 11 to determine the autolytic activity.13.The biofilm formation ability of strain 11 Y and 11 was examined by crystal violet staining.14.Tandem mass spectrometry tag(TMT)labeled total proteins of strain 11 Y and 11 and the differentially expressed proteins were analyzed by LC-MS/MS tandem mass spectrometry.Results: 1.Strain 11 Y and 11 infected RAW264.7 cells can cause autophagy and inflammation.(1)Transmission electron microscopy results showed that 11 Y and 11 were infected with RAW264.7 cells for 4 hours,and both of them had a representative double-layer membrane structure around the single bacterium in the cells,only a part of the bilayer membrane structure or no bilayer membrane structure was observed around the vesicles,indicating that the autophagy was inhibited with the division and proliferation of 11 Y and11 in RAW264.7 cells.(2)The results of laser confocal microscopy showed that 11 Y and 11 were able to observed the autophagy formed by LC3-II accumulation in the cells at 30 min,1h,2h and4 h after infection of RAW264.7 cells,indicating that both 11 Y and 11 can cause autophagy after they infect RAW264.7 cells.(3)Real-Time PCR results showed that mRNA expression levels of IL-1β,IL-6 and TNF-αwere significantly increased at 30 min,1 h,2 h and 4 h after 11 Y and 11 infection of RAW264.7 cells.The degree was positively correlated with the infection time.There was no significant difference between the 11 Y and 11 infection groups,and the expression level of IFN-γwas not significantly changed.The ELISA results showed that the protein expression levels of the above inflammation-related cytokines were basically consistent with the results of Real-time PCR.It is indicated that 11 Y and 11 infected RAW264.7 cells can cause inflammatory reaction,and there is no significant difference in the degree of inflammatory reaction caused by the two.2.Strains 11 Y and 11 regulate RAW264.7 autophagy via the ROS/NLRP3/mTOR pathway.(1)Western Blot results showed that the expression of autophagy-related protein LC3-II increased at 30 min,1 h,2 h,4 h after 11 Y and 11 infection of RAW264.7 cells,and the expression of LC3-II at various time points after 11 Y infection Both are higher than 11.At 30 min after 11 Y infection,the expression of SQSTM1 was first decreased,and then increased to a level comparable to that of the control group,while the expression level of SQSTM1 was decreased at each time point after infection of 11 cells.It is indicated that the infection caused by autophagy in RAW264.7 cells was smooth,while the upstream reaction of autophagy caused by 11 Y infection was normal,and the downstream autophagosome and lysosome fusion disorder.(2)Both strains 11 Y and 11 could cause NLRP3 and p-mTOR levels in RAW264.7cells to increase,and NLRP3 and p-mTOR increased more after 11 Y infection.The results of fluorescence microplate reader showed that 11 Y and 11 could cause the increase of ROS content in RAW264.7 cells.There was no significant difference in ROS content between the two cells at 30 min,1h,2h and 4h after infection.Both 11 and 11 Y may regulate autophagy in RAW264.7 cells via the ROS/NLRP3/mTOR pathway.(3)The expressions of NLRP3,p-mTOR and SQSTM1 in NAC pretreated RAW264.7 cells infected with 11 Y and 11 were significantly lower than control group,and the expression of LC3-II protein was significantly higher than that in control group.It further confirmed that both 11 and 11 Y regulate RAW264.7 autophagy via ROS/NLRP3/mTOR pathway.3.The viability of strain 11 Y in RAW264.7 cells was higher than strain 11,and the protective effect of NAC on the cells infected by strain 11 was obvious.(1)BHI agar plate colony count results showed that strains 11 Y and 11 were infected with RAW264.7 cells at MOI=25,and the number of strains 11 Y in the cells at 30 min,1h,2 h,and 4 h after infection was 10 times higher than that of strain 11.The RAW264.7cells pretreated with NAC for 24 h could almost eliminate all the strains 11 in the cells,but only clearance some strains 11 Y,not all.(2)Real-time PCR results showed that the mRNA expressions of IL-1β,IL-6 and TNF-α were significantly decreased in NAC pretreated RAW264.7 cells infected with11 Y and 11 after at 30 min,1 h,2 h and 4 h.The expression levels of the cytokines in the strain 11 infected group were more than those in the 11 Y infected group.The ELISAresults showed that the protein expression levels of the cytokines were consistent with the results of Real-time PCR.This indicates that NAC can significantly reduce the inflammatory response caused by strain 11 Y or 11 infection.4.Compared with strain 11,there were differences in drug resistance-related phenotypes and protein expression in strain 11 Y.(1)The results of transmission electron microscopy showed that the cell wall of 11 Y was significantly thicker than 11;the results of δ-hemolytic experiment showed that there was no hemolysis enhancement zone at the interface between 11 Y and strain RN4420;the results of autolysis experiment showed that the absorbance values of 11 Y at different detection time points were significantly higher than strain 11;the autolysis and biofilm formation of strain 11 Y decreased,indicating that the phenotypes of VISA strain 11 Y and VSSA strain 11 are different,the pathogenicity of strain 11 Y decreased.(2)TMT LC-MS/MS proteome tandem analysis showed that the expression of structural and functional proteins related to drug resistance was elevated in strain 11 Y.Compared with strain 11,there were 128 proteins with a higher expression than 2.0 times,and 21 proteins with less than 0.5 times in strain 11 Y.The cluster analysis of Kyoto Encyclopedia of Genes and Genomes(KEGG)showed that these differential proteins are mainly distributed in carbon metabolism,RNA degradation,tricarboxylic acid cycle,RNA synthesis,ribosome,pyrimidine metabolism,Sulfur transport system,peptidoglycan synthesis,lysine metabolism,Staphylococcus aureus infection and ABC transport.It is indicated that there is a difference in the expression of vancomycin resistance-related proteins in 11 Y compared with 11 and these proteins are present in various pathways of bacterial metabolism and infection.Conclusion: 1.Both strain 11 Y and 11 infected with RAW264.7 macrophage were able to cause autophagy and inflammatory responses.2.Strain 11 Y and 11 regulate RAW264.7 autophagy via the ROS/NLRP3/mTOR pathway.3.The drug resistance phenotype and protein expression of strain 11 Y were different from those of strain 11. |
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