| Acinetobacter baumannii(AB)is an aerobic,non-fermentative,Gram-negative conditioned pathogen,which is widely found in nature,especially in hospital environment and human skin,mucous membrane,respiratory tract and urinary tract.As an conditioned pathogen,it easily causes infection in hypoimmunity people.Outer membrane protein A(OmpA)is the main cytotoxic factor of Acinetobacter baumannii.In this study,the clinical isolated AB strain,the standard strain ATCC17978 strain and the OmpA protein knockout mutant Acinetobacter baumannii(omp A-/-AB strain)were used as the research objects.The bioinformatics characteristics of the outer membrane protein OmpA and its role in inducing autophagy and apoptosis of lung epithelial cells were studied.The results are as follows:1.Bioinformatics characteristics of Acinetobacter baumannii OmpAIn this study,twelve clinically isolated Acinetobacter baumanni strains were selected,DNA was extracted,and the Polymerase chain reaction(PCR)technique was used to amplify the OmpA gene.The results showed that the twelve clinically isolated AB strains belonged to six ST types by MLST analysis,and all of them could detect the OmpA gene.Total RNA was extracted and reverse transered into c DNA.Real-time fluorescent polymerase chain reaction(RT-PCR)was used to analyze the relative expression levels of OmpA in AB strains.There was no significant difference in RNA expression level of the twelve clinical isolates of Acinetobacter baumannii.The OmpA gene was amplified by Polymerase chain reaction(PCR)with twenty-four SNP sites extracted from DNA.Compared with the tertiary spatial structure and transmembrane structure of OmpA protein in Pubmed,OmpA protein has six tertiary structures,all of them were composed of ?-barrel structure conserved domain,which was a transmembrane protein.The results showed that OmpA gene was highly conserved in Acinetobacter baumannii,and there was no significant difference in the expression level of OmpA among the twelve clinical isolates.2.Preparation and morphological observation in OmpA knockout strain of Acinetobacter baumanniiBy using the method of homologous recombination we get OmpA gene replaced by Kn resistance gene cloning genes strains,named ATCC17978 / ? omp A: : Kn(omp A-/-AB).The wild-type AB strain and omp A-/-AB strain were inoculated on LB medium respectively,and the total RNA of wild-type AB strain and omp A-/-AB strain were extracted,and the reverse transcription was used as c DNA.RT-PCR was used to detect the relative expression level of OmpA,and the relative expression level of OmpA in the wild-type AB strain was significantly higher than that of omp A-/-AB strain(P < 0.05).Morphological observations showed that the wild-type AB colonies grew well,which were round,milky white,moist,smooth,opaque and slightly convex in the middle.The omp A-/-AB colonies had medium growth,smaller shape than the wild strain,and the same color as the wild type strain.The edge was neat,the surface was moist,and the middle was slightly bulge.The colony count of the wild type AB strain was significantly higher than that of the omp A-/-AB strain.The results indicated that the OmpA gene played an important role in the growth activity of Acinetobacter baumannii,and it was an important component protein in Acinetobacter baumannii.3.The effect of OmpA on the pathology and ultrastructure of WTRL1 cellsThe WTRL1 cells were infected by wild-type AB strain,which could result to increase cell contraction and solubility,and could decrease the cell viability significantly.However,when omp A-/-AB strain infected WTRL1 cells,the degree of cell contraction was reduced,the cell solubility was lower than that of the wild-type AB strain group,and the cell viability was significantly higher than that of wild-type AB strain group(P < 0.05).By transmission electron microscope,a large number of bacteria adhered to invaded into the cells,and autophagosomes with double membrane vacuoles were formed in the cells.Typical autophagy features such as endoplasmic reticulum dilation and mitochondrial ridge rupture were observed.At the same time,apoptotic bodies were observed.When OmpA-/-AB strain infected WTRL1 cells,the bacterial adhesion and invasion of cells were reduced,the the damage degree of WTRL1 cells was reduced,and the formation of autophagosomes was less(P <0.05).The results indicated that OmpA protein could play an important role in bacterial adhesion and invasion of host cells.At the same time,OmpA protein could induce the occurrence of autophagy and apoptosis in host cells and could aggravate cell destruction.4.Effect of OmpA on lung histopathology and ultrastructure in SD ratsWild-type AB strain infected the lung tissue of SD rats.Pathological observation of HE staining showed that the alveolar structure of rats was obviously damaged,inflammatory exudate was observed in the alveolar cavity,alveolar septum was obviously thickened and ruptured,blood vessels dilated and bleeding,a large number of inflammatory cells were observed around all levels of bronchus,and ciliary adhesion and lodgings were observed.However,in the lung group of SD rats infected with omp A-/-AB strain,pathological observation of HE staining showed that the alveolar structure of rats was reduced,and the alveolar septum was slightly thickened and ruptured,and a few inflammatory cells could be seen around all levels of bronchus.The pathological score was significantly lower than that of wild-type AB strain group(P<0.05).The transmission electron microscope observation showed a large number of bacteria adhered and invaded into the lung tissue cells of rats when the wild-type AB strain infected the lung tissue of SD rats.In the cells,there were autophagosomes with double-membrane vacuolation,endoplasmic reticulum dilation,mitochondrial spinal rupture,mitochondrial vacuolation formation,and apoptosis in the wild-type AB strain group.But the omp A-/-AB strain group had less bacterial adhesion and invasion into the cells,less damage to lung tissue cells,and less autophagosome formation(P<0.05).The results indicated that the OmpA protein could play an important role in the bacterial adhesion,invasion and induction of autophagy and apoptosis when the lung tissue cells were infected with Acinetobacter baumannii.5.The effect of OmpA on the expression levels of P62 and LC3 in lung tissue cellsWe used the immunofluorescence,immunohistochemistry and Western blotting analysis,when the wild-type AB strain and omp A-/-AB strain infected WTRL1 cells and the SD rat lung tissue respectively.The wild-type AB strain could cause the expression level of P62 protein to be lower than that of omp A-/-AB strain which were infected group of WTRL1 cells and lung tissue of SD rats(P < 0.05),while the LC3 protein expression level was higher than that of omp A-/-AB strain infected group(P < 0.05).These results indicated that when the host was infected with Acinetobacter baumannii,OmpA could decrease the expression of autophagy related protein P62,could increase the expression of LC3,which showed autophagy occuring in the lung tissue cells.6.Effects of OmpA on m TOR and pm TOR expression levels in the lung tissue cellsBy immunofluorescence,immunohistochemistry and Western blotting analysis,wild-type AB strain and omp A-/-AB strain were used to infect WTRL1 cells and lung tissue of SD rats respectively.The expression levels of m TOR and pm TOR protein in WTRL1 cells and lung tissue of SD rats were lower than those in omp A-/-AB strain group(P < 0.05).After the intervention of rapamycin,an important m TOR inhibitor in autophagy pathway,the expression levels of m TOR and pm TOR were further decreased,the expression level of P62 was decreased,LC3 expression was increased,and the autophagy response was enhanced.After the intervention of m TOR promoter chloroquine(CQ),the expression levels of m TOR and pm TOR were further increased,the expression level of P62 was increased,the expression level of LC3 was decreased,and the autophagy reaction was weakened,which was consistent with the trend of omp A-/-AB infection group(P < 0.05).The results indicated that OmpA induced autophagy in the lung tissue cells through m TOR pathway.7.The effect of OmpA on the expression levels of p AMPK and AMPK in lung tissue cellsImmunofluorescence,immunohistochemistry and Western blotting analysis showed that the expression levels of p AMPK protein and AMPK protein in WTRL1 cells and lung tissues of SD rats were significantly higher in the wild-type AB strain group than that of the omp A-/-AB strain group(P < 0.05).These results indicated that OmpA could induce to autophagy in lung tissue cells,which may play a role through the increased expression of AMPK and PAPMK genes upstream of m TOR signaling pathway.8.The effect of OmpA on the expression level of poptosisWTRL1 cells were infected with wild-type AB,and apoptosis level of SD WTLRs cells was detected by Flow Cytometry.The results showed that the apoptosis of WTRLs increased gradually at 1h,3h and 5 h,and the apoptosis rate were the highest at the 3h.group compared with the normal group.Apoptosis of WTRL1 cells in omp A-/-AB infected group and AB + CQ group for the 3h were lower than that of the wild-type AB infected group.9.Effect of OmpA on the expression level of apoptosis-related proteins in lung tissue cellsThe WTRL1 cells and lung tissues of SD rats were infected by wild-type AB strain and omp A-/-AB strain.Immunofluorescence,immunohistochemistry and Western blotting analysis showed that the protein expression levels of Beclin-1,JKN1,Drp-1,Caspase-3 and Caspase-9 in WTRL1 cells and lung tissues of SD rats in wild-type AB strain infection group were significantly higher than those of omp A-/-AB strain infection group(P < 0.05).The expression level of Bcl-2 was significantly lower than that of omp A-/-AB infection group(P <0.05).The results suggested that when Acinetobacter baumannii infected to the host,OmpA could increase the key autophagy related protein Beclin-1,meanwhile OmpA could increase the apoptosis-related proteins JNK1,DRP-1,Caspase-3 and Caspase-9,but could decrease the level of Bcl-2.These results suggested that OmpA could lead to damage in the mitochondria of lung tissue cells and induce apoptosis.10.OmpA changes the expression level of inflammatory cytokines in serum of SD ratsThe lung tissues of SD rats were infected by wild-type AB strain and omp A-/-AB strain.By ELISA test,the expression levels of IL-1?,GM-CSF,INF-? and TNF-? in serum of SD rats in wild-type AB strain group were significantly higher than those of omp A-/-AB strain group(P< 0.05).The results indicated that when Acinetobacter baumannii was infected with lung tissue of SD rats,OmpA could increase the level of inflammatory cytokines in serum.In summary,this study analyzed the bioinformatics characteristics of OmpA protein,and the microstructure of infected cells,the molecular regulation process of inducing autophagy and apoptosis in the lung tissue cell when the wild-type AB strain and omp A-/-AB strain infected the lung tissue of SD rats.The results showed that OmpA protein could play an important role in the process of Acinetobacter baumannii infection in host cells.This study provides a theoretical basis for the clinical treatment of Acinetobacter baumannii infection. |
PDF Full Text Request