EHEC的新型粘附因子研究进展

肠出血性大肠杆菌（enterohemorrhage E-coli,EHEC）是一种重要的人畜共患病,世界各地包括中国都有不同规模的暴发流行。EHEC有多种血清型,其中毒力最强血清型是0157：H7。EHEC0157：H7感染除可使人发生常规腹泻外,还可在5％-10％的病例中引发严重并发症,甚至死亡。该菌是重要的食源性致病菌,危害严重,缺乏有效的防治手段,而抗生素治疗可能会加剧溶血性尿毒症（haemoluticuraemicsyndrome,HUS）。由于以上特点EHEC0157：H7成为世界公共卫生问题,引起微生物学家和公共卫生工作者的广泛关注。目前,临床针对EHEC感染只是对症治疗和适当的抗茵治疗。粘附是EHEC感染宿主细胞的第一步,没有这一步,细菌和宿主肠道细胞之间不会发生任何的相互作用,而且对于许多病原菌来说,粘附具有宿主特异性。本文概述了EHEC的流行病学及粘附机理,并对近年在EHEC研究中的发现一些新型粘附因子和一些假设的定植因子的研究背詈及作用机理作一综述。     

大肠杆蓖O157：H7致病因子及其保护性免疫研究现状

大肠杆菌O157：H7是肠出因型大肠杆菌（EHEC）的主要病原血清型,它可产生特殊的粘附因子粘附靶细胞,产生Vero毒素和肠溶血素毒力因子。近几年对大肠杆菌O157：H7的毒力因子有了深入的了解,对致病机理作了一些探讨,用实验动物对保护性免疫进行了研究。本文对近几年来O157：H7大肠杆菌的致病因子及其主要的保护性免疫的研究作一简要综述。     

大肠杆菌O157:H7致病因子及其保护性免疫研究现状

大肠杆菌O157:H7是肠出血型大肠杆菌(EHEC)的主要病原血清型,它可产生特殊的粘附因子粘附靶细胞,产生Vero毒素和肠溶血素毒力因子.近几年对大肠杆菌O157:H7的毒力因子有了深入的了解,对致病机理作了一些探讨,用实验动物对保护性免疫进行了研究.本文对近几年来0157:H7大肠杆菌的致病因子及其主要的保护性免疫的研究作一简要综述.     

肠出血性大肠杆菌Ⅲ型分泌系统及其转位效应器蛋白质

肠出血性大肠杆菌(enterohemorrhagic Escherichia coli,EHEC)0157:H7是一种重要的肠道病原微生物,感染后可引发多种疾病,严重者可导致死亡.EHEC O157:H7通过Ⅲ型分泌系统(TTSS)将其转位效应器蛋白质转位至宿主细胞,经一系列的信号传导过程介导与宿主细胞的"黏附与擦拭"(attaching and effacing,A/E)损伤.对EHEC0157:H7 Ⅲ型分泌系统及其转位效应器蛋白质进行研究,可使我们进一步认识EHEC以及引起A/E损伤的病原菌的致病机理,丰富有关Ⅲ型分泌系统和致病岛的知识.     

用含10%胎牛血清的DMEM培养肠出血性大肠杆菌O157∶H7可增强其对HeLa细胞的黏附/擦拭损伤

目的:研究生长在不同培养基中的肠出血性大肠杆菌(EHEC)O157∶H7对宿主细胞造成的黏附/擦拭损伤是否存在差异。方法:分别用LB、DMEM、DMEM(含10%胎牛血清)、DMEM(含终浓度为25mmol/L的HEPES)等4种培养基培养O157∶H7,然后感染HeLa细胞,利用Giemsa染色观察细菌黏附差异,进行肌动蛋白荧光染色实验并观察宿主细胞肌动蛋白聚集差异。结果:在含10%胎牛血清的DMEM培养基中培养EHECO157∶H7,其黏附力增加,聚集细胞骨架肌动蛋白的能力明显增强。结论:为进一步研究EHECO157∶H7的致病性,探索O157∶H7新的致病因子奠定了基础。     

肠出血性大肠杆菌（EHEC）O157：H7紧密粘附素免疫保护性片段（Intimin-C）的克隆表达纯化及部分生物学活性研究

克隆表达并纯化肠出血性大肠杆菌(EHEC)O157:H7紧密粘附素免疫保护性片段(Intimin-C),并对其部分生物学活性进行研究.     

长春地区家禽(家畜)中E.coli O157∶H7调查

为了通过对宿主动物EHEC O157∶H7监测,了解长春地区肠出血性大肠埃希菌O157∶H7的污染状况,建立流行病学监测网,以便为疾病预防控制提供良好的科学依据。结果在采集的639份家禽、家畜粪便样品中共检出7株EHEC O157∶H7。     

长春地区家禽（家畜）中E．coli O157：H7调查

为了通过对宿主动物EHEC O157∶H7监测,了解长春地区肠出血性大肠埃希菌O157∶H7的污染状况,建立流行病学监测网,以便为疾病预防控制提供良好的科学依据.结果在采集的639份家禽、家畜粪便样品中共检出7株EHEC O157∶H7.     

白色念珠菌粘附的研究进展

本文论述了白色念珠菌（简称白念菌）粘附的分子机理和实验模型观察,从病原菌和宿主两方面探讨了白念菌感染的发病机理,并从阻断白念菌对宿主细胞的粘附着手,展望防治白念菌感染的新途径和措施。     

食源性致病性大肠杆菌O157:H7和O55:H7特异性噬菌体的分离与鉴定

【背景】肠出血性大肠杆菌(enterohemorrhagic Escherichia coli,EHEC) O157:H7和肠致病性大肠杆菌(enteropathogenic E. coli,EPEC) O55:H7是2株常见食源性致病菌,能导致腹泻及肠道外疾病,其特异性噬菌体具有制备新型抗菌制剂的应用前景。【目的】分离能特异裂解O157:H7和O55:H7的噬菌体,并分析其生物学特性和基因组特征,探索致病性大肠杆菌防控的抗生素替代方法。【方法】利用双层平板法从环境水样中分离噬菌体,对其形态、感染复数、宿主范围、一步曲线等生物学特性进行鉴定,使用Illumina MiSeq平台对其全基因组进行测序,利用RAST、Prokka、BLASTp等软件进行生物信息学分析。【结果】分别以E.coli O157:H7和O55:H7为宿主分离出2株特异性烈性噬菌体:vB＿EcoM＿P251和vB＿EcoM＿P255,均属于肌尾病毒科(Myoviridae)。最佳感染复数均为1,在培养15min内能以91.9%和90.8%的速率吸附到宿主细胞上,而且在37-60℃、pH4.0-11.0条件下保持高且稳...     

Avian- and mammalian-derived antibodies against adherence-associated proteins inhibit host cell colonization by Escherichia coli O157:H7

AIM: To evaluate the potential for polyclonal antibodies targeting enterohaemorrhagic Escherichia coli (EHEC) virulence determinants to prevent colonization of host cells by E. coli O157:H7. METHODS AND RESULTS: Rats and laying hens were immunized with recombinant proteins from E. coli O157:H7, EspA, C-terminal intimin or EscF. Rat antisera (IgG) or chicken egg powders (IgY) were assessed for their ability to inhibit growth and colonization-associated processes of E. coli O157:H7. Mammalian antisera with antibodies to intimin, EspA or EscF effectively reduced adherence of the pathogen to HeLa cells (P<0.05) and prevented type III secretion of Tir. Similarly, HeLa cells treated with chicken egg powder containing antibodies against intimin or EspA were protected from EHEC adherence (P<0.05). Neither egg nor rat antibody preparations had any antibacterial effect on the growth of EHEC (P>0.05). CONCLUSIONS: Antibody preparations targeting EHEC adherence-associated factors were effective at preventing adhesion and intimate colonization-associated events. SIGNIFICANCE AND IMPACT OF THE STUDY: This work indicates that immunotherapy with anti-adherence antibodies can reduce E. coli O157:H7 colonization of host cells. Passive immunization with specific antibodies may have the potential to reduce E. coli O157:H7 colonization in hosts such as cattle or humans.     

Adhesion and colonization of enterohemorrhagic Escherichia coli O157:H7 in cecum of mice

Infectious diseases due to enterohemorrhagic Escherichia coli (EHEC) are characterized by diarrhea, hemorrhagic colitis and hemolytic uremic syndrome. The adherence of EHEC on intestinal epithelial cells is a first step for developing these diseases. In the present study, we examined whether EHEC O157:H7 adhere to intestinal epithelial cells of mice and cause F-actin accumulation in the epithelial cells following the intragastric inoculation of the pathogen. Fecal shedding of the EHEC O157:H7 strain was observed in ICR mice up to 3 weeks. Fecal shedding periods of the type III secretion system-related gene (espA and sepL) deletion mutants were clearly shorter than that of the wild-type EHEC O157:H7 strain. The EHEC O157:H7 colonies were found on the epithelial surfaces of the ceca in association with F-actin accumulation beneath the attached bacteria.     

Surface-layer protein extracts from Lactobacillus helveticus inhibit enterohaemorrhagic Escherichia coli O157:H7 adhesion to epithelial cells

Adherence of intestinal pathogens, including Escherichia coli O157:H7, to human intestinal epithelial cells is a key step in pathogenesis. Probiotic bacteria, including Lactobacillus helveticus R0052 inhibit the adhesion of E. coli O157:H7 to epithelial cells, a process which may be related to specific components of the bacterial surface. Surface-layer proteins (Slps) are located in a paracrystalline layer outside the bacterial cell wall and are thought to play a role in tissue adherence. However, the ability of S-layer protein extract derived from probiotic bacteria to block adherence of enteric pathogens has not been investigated. Human epithelial (HEp-2 and T84) cells were treated with S-layer protein extract alone, infected with E. coli O157:H7, or pretreated with S-layer protein extract prior to infection to determine their importance in the inhibition of pathogen adherence. The effects of S-layer protein extracts were characterized by phase-contrast and immunofluorescence microscopy and measurement of the transepithelial electrical resistance of polarized monolayers. Pre-treatment of host epithelial cells with S-layer protein extracts prior to E. coli O157:H7 infection decreased pathogen adherence and attaching-effacing lesions in addition to preserving the barrier function of monolayers. These in vitro studies indicate that a non-viable constituent derived from a probiotic strain may prove effective in interrupting the infectious process of an intestinal pathogen.     

Heparin blocks the adhesion of E. coli O157:H7 to human colonic epithelial cells

Adhesion of Shiga toxin-producing Enterohemorrhagic Escherichia coli (EHEC) O157:H7 to human colonic epithelium is a critical step for infection by this type of bacteria. Here, we demonstrate that adherence of EHEC O157:H7 to cultured human colonic T84 epithelial monolayers can be blocked by heparin and heparan sulfate in a dose-dependent fashion. In doing this, heparin and heparan sulfate also prevent dysfunction of the T84 barrier and disorganization of epithelial tight junction protein ZO-1 caused by EHEC O157:H7. This inhibition by heparin and heparan sulfate seems to result from a block in the binding interactions of bacteria intimin with epithelial β₁ integrins. This study provides evidence, for the first time, that heparin and heparan sulfate can serve as novel effective blockers in preventing EHEC O157:H7 infection.     

Cell surface-localized nucleolin is a eukaryotic receptor for the adhesin intimin-gamma of enterohemorrhagic Escherichia coli O157:H7.

Intimin-gamma is an outer membrane protein of enterohemorrhagic Escherichia coli (EHEC) O157:H7 that is required for the organism to adhere tightly to HEp-2 cells and to colonize experimental animals. Another EHEC O157:H7 protein, the Transferred intimin receptor (Tir), is considered the primary receptor for intimin-gamma. Nevertheless, Tir-independent binding of intimin-gamma to HEp-2 cells has been reported. This observation suggests the existence of a eukaryotic receptor(s) for intimin-gamma. In this study, we sought to identify that receptor(s). First, we determined by equilibrium binding titration that the association of purified intimin-gamma with HEp-2 cells was specific and consistent with a single host cell receptor. Second, we isolated a protein from lysates of HEp-2 cells that bound intimin-gamma and subsequently identified this molecule as nucleolin, a protein involved in cell growth regulation that can be cell surface-expressed. Third, we established that purified intimin-gamma and nucleolin were co-localized on the surface of HEp-2 cells and that the site of EHEC O157:H7 attachment was associated with regions of nucleolin expression. Finally, we demonstrated that mouse anti-nucleolin sera significantly decreased the adherence of EHEC O157:H7 to HEp-2 cells. From this, we conclude that nucleolin is the HEp-2 cell receptor for intimin-gamma expressed by EHEC O157:H7.     

Lipopolysaccharide O-antigen of enterohemorrhagic Escherichia coli O157:H7 is required for killing both insects and mammals

Studies of enterohemorrhagic Escherichia coli (EHEC) infection mechanisms using mammals require large numbers of animals and are both costly and associated with ethical problems. Here, we evaluated the pathogenic mechanisms of EHEC in the silkworm model. Injection of a clinically isolated EHEC O157:H7 Sakai into either the silkworm hemolymph or intraperitoneal fluid of mice killed the host animals. EHEC O157:H7 Sakai deletion mutants of the rfbE gene, which encodes perosamine synthetase, a monosaccharide component synthetase of the O-antigen, or deletion mutants of the waaL gene, which encodes O-antigen ligase against the lipid A-core region of lipopolysaccharide (LPS), had attenuated killing ability in both silkworms and mice. Introduction of the rfbE gene or the waaL gene into the respective mutants restored the killing ability in silkworms. Growth of both mutants was inhibited by a major antimicrobial peptide in the silkworm hemolymph, moricin. The viability of both mutants was decreased in swine serum. The bactericidal effect of swine serum against both mutants was inactivated by heat treatment. These findings suggest that the LPS O-antigen of EHEC O157:H7 plays an important defensive role against antimicrobial factors in the host body fluid and is thus essential to the lethal effects of EHEC in animals.     

Encoded protein from ycbR gene of enterohemorrhagic Escherichia coli O157:H7 associated with adherence to HEp-2 cells

Adhesion is one of the significant virulence factors in enterohemorrhagic Escherichia coli (EHEC) O157:H7 pathogenesis. It is regulated by specific loci in the genome sequence. This study mainly focused on investigating the influence of ycbR gene and its encoded YCBR protein on the adhesion of EHEC O157:H7 to HEp-2 cells. In the first part, mutants of EHEC O157:H7 were constructed through TnphoA mutagenesis and assayed for adherent ability. Six mutant strains with lost adherence to HEp-2 cells were isolated and then sequenced using a primer that hybridized to phoA sequence downstream of the fusion joint. The sequencing results indicated that the gene of eae and ycbR, between the initiation codon and the −10 sequence of Z4182, yciI, ARAC-type regulator protein, and high-affinity gluconate transporter of EHEC were all possibly related to adhesion. Of the six genes, the ycbR gene was cloned to the pET28a vector to analyze its function further. Recombinant YCBR protein fused to a His tag (YCBR-His) was expressed under IPTG induction and purified by Ni-NTA column. The purified protein was subcutaneously injected to rabbits to prepare antisera. The results of an adherence assay in the presence of anti-YCBR-His antibodies indicated that antibodies blocked the adherence of EHEC O157:H7 to HEp-2 cells. These observations suggested that ycbR encoded a novel adherence-associated determinant of EHEC O157:H7, which could contribute to the adhesive capacity of the bacteria.     

A carboxy-terminal domain of Tir from enterohemorrhagic Escherichia coli O157:H7 (EHEC O157:H7) required for efficient type III secretion

The type III secreted protein Tir from Enterohemorrhagic Escherichia coli (EHEC O157:H7) plays a central role in adherence and pedestal formation during infection. Little is known about how Tir domains outside of the amino-terminus contribute to efficient Tir secretion and translocation. We found a 6 amino acid (519-524) carboxy-terminal region which was required for efficient Tir secretion and translocation. Interestingly, EHEC O157:H7 Tir(Delta)519-524 was efficiently secreted when expressed in the related pathogen enteropathogenic E. coli. These data suggest that this region may play a role in maintaining EHEC O157:H7 Tir in a secretion-competent conformation.