Study On The Function Of Outer Membrane Protein A Of Pathogenic Yersinia Enterocolitica

Object:Yersinia entercolitica is a Gram-negative budless bacillus,which belongs to Yersinia genus,Enterobacteriaceae.It is one of the three pathogenic bacteria of Yersinia genus,mainly infects human to cause gastrointestinal disease by contaminated food or water.The omp A gene of Yersinia entercolitica is highly conserved and has the crossimmunogenicity effect,and it is a potential candidate protein for vaccine research of pathogenic,including Yersinia pestis.Among other gram negative bacteria,Omp A has been proved to play important roles in bacterial adhesion,invasion,antimicrobial resistance and viability.In this study,we explore the role of the Y.entercolitica Omp A in pathogenicity,drug resistance,survivability and the immune protection,which provided a theoretical basis and technical basis for further basic research and vaccine development.Method:1.The HEp-2 cell model was used to identify the differency of 105.5R(r),105.5R(r)?omp A,105.5R(r)?ail and Y40 in cell adhesion,invasion,and cytotoxicity.2.The MIC value of 105.5R(r),105.5R(r)?omp A,105.5R(r)?ail in ?-lactams,furutain and polymyxin were detected by broth microdilution method.3.The amp C expression level and the hydrolytic activity of Amp C beta lactamase in 105.5R(r),105.5R(r)?omp A and 105.5R(r)?ail were detected using the luminescence report plasmid p LUXamp C and nitrocefin method,using the ?-lactam antibiotic resistant mutant strain 105.5R(r)?amp D123 as control.4.High pathogenic enterocolitis YE92010 was used to attack the Omp A immune group and the control group.The physical signs,bacteria exclusion time and histopathological changes in both group were to observe to evaluate the immunoprotection effect of Omp A on the rats.Result:1.The adhesion ability of all strains to HEp-2 cells was observed.The results showed that the adhesion and invasion ability of ?omp A strain and ?ail strain were lower than wild-type strain105.5R(r),but still higher than non-pathogenic strain Y40.The toxicity of 105.5R(r)?omp A and 105.5R(r)?ail strains to HEp-2 cells are also decreased significantly.2.Firstly,the amp C expression level and the total ?-lactamase activity of ?omp A strains were as same as the wild-type strains.Besides,the growth rate of these two strains at high oxidative pressure,low osmotic pressure and iron deficiency was also in the same level.However,the antimicrobial susceptibility test of these two strains showed that the MIC value of ampicillin,ampicillin-Shubatan,piperacillin-tazobactam and cefazolin,cefazolin and ceftazidime had a obviously decreased.3.In animal experiments,Omp A showed good immunoprotection effect.In both group,the symptoms of anorexia and hair disordered were occurred.However,the pathological change degree of liver,spleen and intestinal tissue,in Omp A immunization group were significantly lower than those in control group.Conclution:1.Omp A plays an important role in cell adhesion,invasion and cytotoxicity of pathogenic Yersinia enterocolitica.2.The viability of ?omp A strains in high oxidative pressure,low osmotic pressure and iron deficiency was as same as wild-type strain,indicating the deletion of Omp A did not significantly change the membrane permeability of bacteria.However,the resistance of 105.5R(r)?omp A to a variety of ?-lactam antibiotics is obviously weakened,this phenomenon is not caused by the decrease of the expression of beta lactamase,this mechanism remains needs to be further understood.3.Omp A has a good immunoprotection effect,the immuned group can effectively resist the high pathogenic bacteria to colonization in the intestinal,and the histopathological changes are also relatively light,reflects the good values in pathogenic Yersinia vaccine reseearch.