Antagonistics Of Surface Layer Protein Of Enterococcus Faecium WEFA23 Against Internalization And Colonization Of Listeria Monocytogenes As Well As The Construction Of SLP-knockout Strain

Lactic acid bacteria(LAB)is an important part of probiotics,which possess many important physiological functions such as promoting the absorption of nutrients,antagonizing the adhesion and colonization of pathogenic bacteria,enhancing the immunity of the body and maintaining the intestinal homeostasis and so on.The basis of its functions is that LAB can adhere to intestinal epithelial cells and colonize in the intestinal tract,further antagonizing the internalization and colonization of pathogenic bacteria.However,the potential molecular mechanism of probiotic function of LAB is not clear.Surface layer protein(SLP)as a substance contact with outside directly,it obtains many physiological characteristics such as enhancing the adhesion of LAB to intestinal epithelial cells,antagonizing the adhesion and internalization of pathogenic bacteria,and regulating host immunity.Therefore,the study of the role of SLP of LAB in antagonizing the adhesion and colonization of pathogenic bacteria is beneficial to clarify the mechanism of interaction between probiotics and pathogenic bacteria.In this study,the molecular mechanism of surface layer protein of Enterococcus faecium WEFA23 on antagonizing the internalization and colonization of Listeria monocytogenes was systematically explored by using cell and animal models.And the gene-deficient strain of ornithine carbamoyl transferase(OTC)which as the main component of surface protein was constructed to provide basic materials for functional experiment verification.In chapter one,the functional characteristics of the SLP of LAB,the pathogenesis and research trends of L.monocytogenes and E.faecium against it,gene recombination technology and its application in the field of microorganism were summarized.In addition,the effect of the SLP of E.faecium WEFA23 against the internalization and colonization of L.monocytogenes was proposed.In chapter two,the mechanism of SLP of E.faecium WEFA23 against the internalization of L.monocytogenes to intestinal epithelial cells was investigated.The results showed that 50-200 ?g/mL SLP had no toxicity to Caco-2 cells but promote the proliferation of it.The SLP could significantly antagonize the internalization of L.monocytogenes to Caco-2 cells in the exclusion assay and the invasive number of pathogenic bacteria reduced by 100 times at the concentration of 200 ?g/mL;SLP could decrease the expression of proinflammatory factors(IL-6,IL-1?,IFN-y and TNF-?)and the virulence factors(inl A.inl B,plc A,plc B,act A and prf A)(P<0.05);SLP could reduce the damage of intestinal barrier caused by L.monocytogenes infection,which reflected by increasing transmembrane resistance(P<0.05),decreasing number of L.monocytogenes(P<0.05),decreasing FITC-Dextran content(P<0.05),and increasing related tight junction proteins(ZO-1,Claudin-1 and Occludin)expression(P<0.05).The above results indicated that the SLP could reduce the number of pathogenic bacteria by repairing the intestinal barrier damage,reducing the release of inflammatory mediators and the expression of virulence factors.In chapter three,the intervention of SLP of E.faecium WEFA23 on L.monocytogenes infected mice was studied.The results showed that SLP of E.faecium could effectively intervene L.monocytogenes infection in mice,in which the body weight of mice increased significantly(P<0.05),the mortality rate was reduced by about 15%,the number of L.monocytogenes transferred to mouse tissues was reduced;the number of goblet cells in colon was increased,the damage of intestinal tissue structure was alleviated(manifested as neat villi structure and complete crypt structure,etc.),and the expression t of tight junction protein(Claudin-1,Occludin and ZO-1)in colon increased;the volume of liver and spleen was reduced,the inflammatory infiltration in liver tissue was reduced,the number of CD4+and CD8+T cells was reduced,the structure of spleen was repaired,the number and volume of white pulp were normal,the boundary of lymph sheath around border area and artery was clear,the spleen cord was hypertrophic,and the pro-inflammatory factors IL-6,IL-1?(P<0.05),IFN-y and TNF-? decreased,indicating that the immune disorders of mice were effectively regulated.These results suggested that might recovering the body weight and reducing the death of colonized mice of the SLP from E.faecium WEFA23 might through modulating the damage of intestinal barrier and the host immunity.In chapter four,the SLP of E.faecium WEFA23 was analyzed by LC-MS/MS,and the major protein,ornithine carbamoyltransferase(OTC)was selected as its representative component.The OTC knockout strain was successfully constructed by homologous recombination,which provided experimental materials for further exploration of the molecular mechanism.In chapter five,the primary mechanism of SLP of E.faecium WEFA23 against internalization and colonization of L.monocytogenes was summarized and prospected.In a word,the SLP of E.faecium WEFA23 could alleviate the damage of intestinal barrier and immune function caused by L.monocytogenes infection,inhibit the internalization of L.monocytogenes to intestinal epithelial cells,reduce the infection of it,reduce the tissue colonization of pathogen in mice,and thus reduce the mortality of mice and restore the weight of mice.The results of this study provide a thinking to elucidate the interaction mechanism between probiotics and pathogens,and provide a scientific basis for the application of E.faecium WEFA23 as a probiotic candidate strain in microecological preparations.