The Apoptosis Function Induced By Espf Protein Of Enterohaemorrhagic Escherichia Coli O157:H7

1. Background and ObjectiveEnterohaemorrhagic Escherichia coli (EHEC) O157:H7is a new intestinal bacterium which is able to cause hemorrhagic colitis (HC), hemolytic uremic syndrome (HUS) and thrombotic thrombocytopenic purpura (TTP) in human. Riley noticed a serious food-borne hemorrhagic colitis caused by EHEC O157:H7in America for the first time in1983, whereas EHEC O157:H7was acquired firstly in China in1986, more than10provinces had isolated the bacteria from food, poultry and livestock and diarrhea patients. Nowadays the prevalance of EHEC O157:H7frequently breaks out all over the world and threatens human public health, which is becoming a global public health problem.The pathogenic role of EHEC O157:H7was completed mainly through the Shiga-like toxins (STX) and the attaching and effacing lesions (A/E lesions) of epithelial cells for bacteria adhesion. O157:H7can produce deadly shiga toxin (Stx), which encoded by slt gene on a prophage, was the remarkable characteristic that Vcrocytotoxin-producing E.coli (VTEC) different from other E.coli. The capacity to form A/E lesions is encoded mainly by the locus of enterocyte effacement (LEE) which consisted of5operons. The LEE encodes a type III secretion system (TTSS), translocators (EspA, EspB, and EspD), effectors (Tir, EspG, EspF, Map, and EspH), intimin, chaperones (CesAB, CesD, CesD2, CesF, and CesT), and regulators (Ler, GrlA, and GrlR). The espF gene is located in LEE4operon which encodes EHEC-secreted protein F. At present, pathogenic effect protein of EHEC O157:H7infection has increased to more than20. EspF is one of many effector proteins exclusive to the attaching and effacing pathogen family and the most multifunctional effector proteins. EspF coordinated with mitochondria, nucleolus, many intracellular protein molecules to disrupt the epithelial tight junction, efface microvillus, remodel cytoskeleton, assemble host actin, recruit pedestal and finally activate apoptosis. Academics think that effect protein EspF is holding the balance for researching the molecular mechanism of pathogenicity of A/E pathogens, even saw the EspF effector as a Bacterial Pathogen’s Swiss Army Knife.Although the role of EspF protein of EPEC was researched in cells specific A/E lesions by different laboratories, the molecular foundation of EspF induced host cell apoptosis is unknown and remaining a lot of questions:Is EspF competent to disrupt epithelial barrier and tight junction and induce host apoptosis in EHEC O157:H7? After adhering to the intestinal epithelium, does EHEC O157:H7impair mucouslayer and cause A/E lesions? Which region of EspF does it take effect?In previous study of this laboratory,48-204bp sequence in N terminal of espF gene was selected as knockout object. The espF gene defect mutant in EHEC had be constructed successfully by OL-PCR and homologous recombination. The experiments of bacterial adhesion and cytotoxicity had shown that defecting espF gene chould weaken adhesion ability and cytotoxicity effect of EHEC, but not influence the biological characteristics of EHEC. This research focuses on the basis of espF gene defect mutant. Firstly, we will detect Caspase-family protease-9/-3activity through western blot and detect infected cell apoptosis in vitro by TUNEL assay. Secondly, we will analyze rats mortality rate after which infected by wt strain and mutant strain. Finally, we will compare the colon pattern, weight, the number of adhered bacterial and observe intestinal mucosa lesion and bacteria adhesion. We attempt to reveal the role of EspF protein during the infectious process and the A/E lesions molecular mechanism on the basis of espF gene defect mutant, comparing the properties of wide type infecting intestinal epithelial cells to mutant from the molecules, cells and animal infection model. Besides, The virulence appraisal of18EHEC O157:H7popular strains were identified by PCR technology, bacteria adhesion experiment and cells LDH release experiment, which provide the theory basis for the subsequent EHEC pathogenic mechanism research screening the poisonous strains.2. Methods2.1The research of apoptosis induced by EspF protein of EHEC O157:H7(1) Detection of caspase-3/9apoptotic protein in Lovo cells.According to Western Blot experiment, Lovo cells were infected by EHEC O157:H7wt strain, EHEC O157:H7espF mutant strain and negative strain for3h. Caspase-family protease was activated and SDS-PAGE isolated it after lysing and turned film, subsequent Caspase-3/9was detected by immunoblotting. Microtubules protein Tubulin was choosed as internal reference controls.(2) TUNEL cell apoptosis assay. Lovo cells were infected by EHEC O157:H7wt strain, EHEC O157:H7espF mutant strain and negative strain for3h. Cells were stained with TUNEL cell apoptosis in situ test kit, counterstained with DAPI, then cell apoptosis and TUNEL positive cells were examined by fluorescent microscopy.(3) EHEC strains infected mice and tissue lesions. Gastric lavage infection of EHEC O157:H7WT strain, espF mutant strain and negative strain was performed to mice which then were feeding20d to observe Growth and motality rate. At7-days postinfection of other group, these mice were sacrificed and5cm of distal colon from the rectum were cut vertically along the colon, which was researched for comparison of the colon form and pathologic characteristics of enteritis and edema, colon weight and the number of cfu per mouse were calculated. Colon frozen sections were cut and immunostained with EHEC O157:H7antibody, rodamine-phalloidin and DAPI. Immunostained samples were examined by fluorescent microscopy for intestinal mucosa lesion and bacteria adhesion.2.2The virulence identify of EHEC O157:H7popular strains.According to E-hlyA, stx2, stxl gene sequences design primer, PCR identified the virulence genes of EHEC O157:H7strains that were outbreaked in different region. Vero cells were infected with different strains for3h, to observe bacteria adhesion of Vero cells by Giemsa dyeing; GENMED L-lactate dehydrogenase (LDH) test kit tested cell LDH release infected with different bacterial, through calculation LDH release rate, comparing different strains cytotoxicity strength.2.3Statistical analysis. TUNEL positive cell and colon weight were counted by One-Way ANOVA; Bacterial adhesion of colon were counted by independent samples T test; LDH release rate was counted by independent sample nonparametric test; The repeatability of FQ-PCR was evaluated by using coefficient of variation of Ct value(SD/Mean of repeat samples).3. Results(1) The capability of apoptosis induced by espF mutant strains was declined compared to wild type. Postinfection Lovo cells, Caspase-9and Caspase-3protein strips were visible clear in EHEC wild type induced group (WT), which indicated Lovo cells had produced Caspase-9/3apoptosis proteins, and caspase-9/3protein strip in espF gene defective mutant induced group (ΔespF) were light color or invisible, which explained the produced apoptosis proteins content was lower than WT group, but higher than control. Second, Caspase-3protein strip exceeded caspase-9in each group, indicated that infected cells may enter the late apoptosis. The protein strip of apoptosis inducer actinomycin D (Act-D) treatment group was similar to WT group. The protein strip of normal control group (Normal) was rather weak.Lovo cells apoptosis were detected by TUNEL apoptosis assay. TUNEL assay showed that cells infected with WT appeared strong green fluorescence and apoptosis while that of ΔespF appeared weak green fluorescence and mild apoptosis. In contrast, the untreated group no found green fluorescence and not or rarely apoptosis.(2) The A/E lesions degree of mutant induced was ease compared to wild type in mice colon. The WT, ΔespF and control group by corresponding bacterial postinfection, Mice infected with WT showed30%mortality, whereas mice infected with ΔespF survived through day20. Although intestines from the mice inoculated with LB were healthy with solidified feces, those from mice infected with WT were mild edematous without solidified feces and were typical findings of bacterially induced intestinal colitis. The intestines from mice inoculated with ΔespF remained healthy with solidified feces, albeit some portions of the intestine were slightly swollen. Colon weight and adhesion bacteria number of mice infected with WT strains are higher than mice infected with espF strains. Colon frozen sections were also stained using EHEC O157:H7antibodies and counterstained with rhodamine-phalloidin and DAPI, then observed by fluorescence microscopy. More green fluorescence were visible over the epithelial surfaces including intestinal crypts with WT infection but not ΔespF infection, indicated that more bacteria adhered to mice intestinal mucosa infected with WT than mice infected with espF strains.(3) The virulence of EHEC O157:H7popular strains were different. There were14strains including hemolysin E-hlyA gene,14strains including stx2genes, and only eight strains including stxl genes among18bacteria strains. Cell adhesion test and cytotoxicity test found that the virulence of stx2gene expression protein higher than stxl gene expression protein. Vero cytotoxicity of Chinese popular strain882364,01H112and Japanese strain Cua9closed to international strain933W/O cytotoxicity.4. Conclusion(1) The bacteria adhesion, cytotoxicity and induced apoptosis ability were reduced after the deletion of N-terminal sequence of espF gene, which caused intestinal epithelium cells damage and A/E lesion degree to relieve. These results from this series of experiments further support the notion that the ability of EspF is critical for bacterial colonization of the intestinal epithelium and the initiation of disease processes, and confirm N terminal as functional areas of EspF.(2) The poisonous force genes and Vero cell toxicity were different among different EHEC strains. The virulence of strains882364,01H112,933W/O and Cua9were quite powerful that could screen out ideal strong strain for further EHEC pathogenic mechanism research.