Protection against Escherichia coli O157:H7 challenge by immunization of mice with purified Tir proteins

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 infections cause serious public health problems worldwide. The translocation intimin receptor (Tir) is responsible for adhesion and attaching and effacing lesions. In the current study, we used a mitomycin-treated mouse model to evaluate the efficacy of subcutaneous vs intranasal administration of the recombinant Tir as vaccine. Following immunization, mice were infected with E. coli O157:H7 and faces were monitored for shedding. Mice immunized intrasally with purified Tir proteins produced higher IgG and IgA titers in serum and feces, resulting in significant reductions in fecal shedding of EHEC O157 and higher a survival rate (92.9%), compared with subcutaneous or control immunizations. These results demonstrate the potential for the use of Tir proteins in mucosal vaccine formulations to prevent colonization and shedding of E. coli O157:H7. Therefore, purified Tir protects mice against EHEC challenge after intranasal immunization and is worth further clinical development as a vaccine candidate.     

The effect of probiotic treatment with Clostridium butyricum on enterohemorrhagic Escherichia coli O157:H7 infection in mice

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 has been considered as an agent responsible for outbreak of hemorrhagic colitis and the hemolytic uremic syndrome. We examined the effect of the probiotic agent Clostridium butyricum MIYAIRI strain 588 on EHEC O157:H7 infections in vitro and in vivo using gnotobiotic mice. The growth of EHEC O157:H7 and the production of Shiga-like toxins in broth cultures were inhibited by co-incubation with C. butyricum. The antibacterial effects of butyric and lactic acid were demonstrated in a dose-dependent manner. In addition, the inhibitory effect of butyric acid on the viability of EHEC was demonstrated not only at low pH, but also at neutral pH adjusted to 7.0. Flowcytometric analysis showed that pre-incubation of Caco-2 cells with C. butyricum and E. coli K12 inhibited the adhesion of EHEC O157:H7. However, the effect of C. butyricum on adhesion of EHEC to Caco-2 cells was more inhibitory than that of E. coli K12. Gnotobiotic mice mono-associated with EHEC O157:H7 died within 4-7 days after the infection. On the other hand, all gnotobiotic mice prophylactically pre-treated with C. butyricum survived exposure to EHEC O157:H7 and of the gnotobiotic mice therapeutically post-treated with C. butyricum, 50% survived. Both counts of EHEC O157:H7 and the amounts of shiga-like toxins (Stx1 and Stx2) in fecal contents of gnotobiotic mice di-associated with EHEC O157:H7 and C. butyricum were less than those of gnotobiotic mice mono-associated with EHEC O157:H7. These results indicated that the probiotic bacterium C. butyricum MIYAIRI strain 588 has preventive and therapeutic effects on EHEC O157:H7 infection in gnotobiotic mice.     

Oral immunization with attenuated Salmonella vaccine expressing Escherichia coli O157:H7 intimin gamma triggers both systemic and mucosal humoral immunity in mice

Human infections with EHEC such as O157:H7 have been a great concern for worldwide food-industry surveillance. This pathogen is commonly associated with bloody diarrhea that can evolve to the life-threatening hemolytic uremic syndrome. Animals are the natural reservoir where this pathogen remains asymptomatically, in steps of ingestion and colonization of the bowel. The bacterium is shed in the feces, contaminating the surroundings, including water and food that are directed for human consumption. A major player in this colonization process is intimin, an outer membrane adhesion molecule encoded by the E. coli attachment and effacement (eae) gene that has been shown to be essential for intimate bacterial attachment to eukaryotic host cells. In an attempt to reduce the colonization of animal reservoirs with EHEC O157:H7, we designed a vaccine model to induce an immune response against intimin gamma. The model is based on its recombinant expression in attenuated Salmonella, used as a suitable vaccine vector because of its recognized ability to deliver recombinant antigens and to elicit all forms of immunity: mucosal, systemic, and humoral responses. To test this model, mice were orally immunized with a S. enterica serovar Typhimurium strain carrying the pYA3137eaeA vector, and challenged with E. coli O157:H7. Here we show that immunization induced the production of high levels of specific IgG and IgA antibodies and promoted reduction in the fecal shedding of EHEC after challenge. The live recombinant vaccine reported herein may contribute to the efforts of reducing animal intestinal mucosa colonization.     

出血性大肠杆菌O157基因缺失疫苗株的构建及其免疫

出血性大肠杆菌O157感染是重要的新发食物源性传染病,主要致病特征之一是能引起人肠上皮细胞特征性的A/E损伤,A/E损伤主要是由LEE致病岛所编码的毒力因子所引起,ler是LEE致病岛毒力基因群的中心调节基因,对LEE致病岛所编码的毒力因子有正调控作用。O157:H7另一个毒力因子是由整合到染色体上的原噬菌体编码的Stx毒素。以O157:H786-24为始发菌株,利用自杀性质粒pCVD442和同源重组的原理构建了O157:H7的ler基因缺失突变菌株(缺失了ler基因中第73-351位的碱基,共279bp),并利用噬菌体消除技术筛选到消除了编码Stx的原噬菌体DNA的菌株,构建出了O157:H7ler/stx基因缺失突变弱毒菌株,并对该菌株的Vero细胞毒性、小鼠模型的安全性以及乳鼠的被动免疫保护作用进行了研究。结果表明,O157:H7ler/stx基因缺失突变菌株丧失了对Vero细胞的毒性作用,并丧失了对实验小鼠的致病性,具有良好的安全性。乳鼠被动免疫保护性实验表明,用该菌株免疫母鼠后,乳鼠通过吸吮母乳可以获得良好的被动免疫保护作用。因此本研究所构建的O157:H7ler/stx基因缺失突变弱毒菌株可作为预防EHEC O157:H7感染的疫苗候选株,为最终研究制出O157的基因工程菌苗奠定基础。     

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.     

Optimizing the Protection of Cattle against Escherichia coli O157:H7 Colonization through Immunization with Different Combinations of H7 Flagellin,Tir, Intimin-531 or EspA

Enterohemorrhagic Escherichia coli (EHEC) are important human pathogens, causing hemorrhagic colitis and hemolytic uraemic syndrome in humans. E. coli O157:H7 is the most common serotype associated with EHEC infections worldwide, although other non-O157 serotypes cause life-threatening infections. Cattle are a main reservoir of EHEC and intervention strategies aimed at limiting EHEC excretion from cattle are predicted to lower the risk of human infection. We have previously shown that immunization of calves with recombinant versions of the type III secretion system (T3SS)-associated proteins EspA, intimin and Tir from EHEC O157:H7 significantly reduced shedding of EHEC O157 from experimentally-colonized calves, and that protection could be augmented by the addition of H7 flagellin to the vaccine formulation. The main aim of the present study was to optimize our current EHEC O157 subunit vaccine formulations by identifying the key combinations of these antigens required for protection. A secondary aim was to determine if vaccine-induced antibody responses exhibited cross-reactive potential with antigens from other EHEC serotypes. Immunization with EspA, intimin and Tir resulted in a reduction in mean EHEC O157 shedding following challenge, but not the mean proportion of calves colonized. Removal of Tir resulted in more prolonged shedding compared with all other groups, whereas replacement of Tir with H7 flagellin resulted in the highest levels of protection, both in terms of reducing both mean EHEC O157 shedding and the proportion of colonized calves. Immunization of calves with recombinant EHEC O157 EspA, intimin and Tir resulted in the generation of antibodies capable of cross-reacting with antigens from non-O157 EHEC serotypes, suggesting that immunization with these antigens may provide a degree of cross-protection against other EHEC serotypes. Further studies are now required to test the efficacy of these vaccines in the field, and to formally test the cross-protective potential of the vaccines against other non-O157 EHEC.     

Draft genome sequences of six Escherichia coli isolates from the stepwise model of emergence of Escherichia coli O157:H7

Enterohemorrhagic Escherichia coli (EHEC) of serotype O157:H7 has been implicated in food-borne illnesses worldwide. An evolutionary model was proposed in which the highly pathogenic EHEC O157:H7 serotype arose from its ancestor, enteropathogenic E. coli (EPEC) O55:H7 (sorbitol fermenting [SOR(+)] and β-glucuronidase positive [GUD(+)]), through sequential gain of virulence, phenotypic traits, and serotype change. Here we report six draft genomes of strains belonging to this evolutionary model: two EPEC O55:H7 (SOR(+) GUD(+)) strains, two nonmotile EHEC O157:H(-) strains (SOR(+) GUD(+)) containing plasmid pSFO157, one EHEC O157:H7 (SOR(-) GUD(+)) strain, and one O157:H7 strain containing plasmid pSFO157 (SOR(+) GUD(+)).     

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.     

Presence and characterization of a mosaic genomic island which distinguishes sorbitol-fermenting enterohemorrhagic Escherichia coli O157:H- from E. coli O157:H7

A mosaic genomic island comprising Shigella resistance locus (SRL) sequences flanked by segments of Escherichia coli O157:H7 strain EDL933 O islands 43, 81, and 82 was identified in sorbitol-fermenting (SF) enterohemorrhagic Escherichia coli (EHEC) O157:H(-) strain 493/89. This mosaic island is absent from strain EDL933. PCR targeting the SRL-related sequence is a useful tool to distinguish SF EHEC O157:H(-) from EHEC O157:H7.     

Use of immunoglobulin enriched bovine colostrum against oral challenge with enterohaemorrhagic Escherichia coli O157:H7 in mice

An immunoglobulin enriched bovine colostrum preparation, IMMULAC (New Zealand Dairy Group, Cambridge, New Zealand), contains antibodies against various bacterial antigens. In the present study, we investigated the protective effects of a commercial bovine colostrum preparation against infections with enterohaemorrhagic Escherichia coli (EHEC) O157:H7 in a murine model. Balb/c mice were given drinking water containing streptomycin for 3 days before and following oral challenge with streptomycin-resistant EHEC O157:H7 strain (O157-SM(R)). In mice pretreated with streptomycin, EHEC O157:H7 maintained stable levels of bacterial colonization in the intestines for the 3-week experimental time period. Oral administration of colostrum resulted in rapid decrease in the bacteria numbers compared with administration of skim-milk. Colostrum showed no direct in vitro bactericidal properties against either EHEC O157:H7. When sections prepared from cecum walls of streptomycin-pretreated mice were incubated in vitro with EHEC O157:H7, the colostrum significantly prevented the attachment of the organisms to the sections when compared with skim-milk. These results indicate that oral administration of bovine colostrum effectively protects mice against food-borne infections by inhibiting bacterial attachment to the intestinal mucous membrane, colonization and growth in the intestinal tract.     

肠出血性大肠杆菌O157∶H7 EspA和EspB蛋白的重组表达及其免疫效果

目的：通过DNA重组技术表达肠出血性大肠杆菌（EHEC）0157：H7的EspA和EspB蛋白,并分析它们的免疫保护性。方法：采用PCR技术从EHEC0157：H7基因组中扩增espA和espB基因,连接至pET-22b（4-）载体上,转化至宿主细胞大肠杆菌BL21（DE3）,经IPTG诱导表达,用亲和层析纯化目的蛋白,SDS-PAGE测定其相对分子质量,免疫小鼠分析其免疫保护性。结果：重组espA和espB基因片段的测序结果与GenBank中的相应基因序列完全一致,一致性均为100％;得到了纯度为95％以上的重组EspA和EspB蛋白,免疫小鼠所得到的抗体效价均为10^6。结论：重组EspA和EspB蛋白获得了可溶性表达,表达的蛋白具有良好的免疫保护性,为进一步制备疫苗奠定了基础。     

EHEC O157:H7 z3276基因缺失株构建及其生物学特性

【目的】肠出血性大肠杆菌O157:H7是世界范围内重要的动物源性致病菌之一,可感染人。I型菌毛是多种致病性大肠杆菌(如肾盂肾炎型大肠杆菌等)可表达的一种黏附结构,与细菌吸附黏膜表面密切相关。然而,O157:H7 fim操纵子上几个核苷酸的缺失却导致其不能表达I型菌毛。BLAST比对结果表明O157:H7独有的开放阅读框z3276编码的氨基酸序列与其他大肠杆菌I型菌毛高度同源,这可能是对O157:H7不能表达I型菌毛的补偿机制,但确切功能尚不清楚。本文探究z3276基因的生物学功能。【方法】利用O157:H7 86-24参考菌株构建z3276基因缺失株(?z3276),并构建其互补株(C?z3276),进而比较亲本株、?z3276与C?z3276的生物学特性及对小鼠致病性差异。【结果】与亲本株相比,?z3276进入对数生长期的时间延后,在半固体琼脂平板上的迁移直径明显缩小,生物被膜形成能力显著减弱。?z3276对HEp-2细胞的黏附和侵袭能力并无明显变化,但对IPEC-J2细胞的侵袭能力明显减弱。在小鼠攻毒试验中,?z3276组排菌数量减少、排菌持续时间缩短。C?z3276各项特性均能回复到与亲本株一致的水平。【结论】z3276基因可能是O157:H7重要的毒力相关因子。     

Intranasal HIV-1-gp160-DNA/gp41 peptide prime-boost immunization regimen in mice results in long-term HIV-1 neutralizing humoral mucosal and systemic immunity

An intranasal DNA vaccine prime followed by a gp41 peptide booster immunization was compared with gp41 peptide and control immunizations. Serum HIV-1-specific IgG and IgA as well as IgA in feces and vaginal and lung secretions were detected after immunizations. Long-term humoral immunity was studied for up to 12 mo after the booster immunization by testing the presence of HIV-1 gp41- and CCR5-specific Abs and IgG/IgA-secreting B lymphocytes in spleen and regional lymph nodes in immunized mice. A long-term IgA-specific response in the intestines, vagina, and lungs was obtained in addition to a systemic immune response. Mice immunized only with gp41 peptides and L3 adjuvant developed a long-term gp41-specific serum IgG response systemically, although over a shorter period (1-9 mo), and long-term mucosal gp41-specific IgA immunity. HIV-1-neutralizing serum Abs were induced that were still present 12 mo after booster immunization. HIV-1 SF2-neutralizing fecal and lung IgA was detectable only in the DNA-primed mouse groups. Intranasal DNA prime followed by one peptide/L3 adjuvant booster immunization, but not a peptide prime followed by a DNA booster, was able to induce B cell memory and HIV-1-neutralizing Abs for at least half of a mouse's life span.     

Enhanced immunoprotective potential of Mycobacterium tuberculosis Ag85 complex protein based vaccine against airway Mycobacterium tuberculosis challenge following intranasal administration

This study examined the role of intranasal vaccination with Mycobacterium tuberculosis antigen85 complex proteins formulated in dimethyldioctadecylammonium bromide against airway Mycobacterium tuberculosis challenge in mice. Intranasal vaccination with antigen85A and antigen85B induced a significantly higher level of interferon-gamma, interleukin-12 and interleukin-4 in cervical lymph nodes together with IgA and IgG, predominantly IgG2a isotype in nasal secretion over subcutaneous vaccination. Further, intranasal vaccination with antigen85A and antigen85B imparted protection comparable with that obtained from intranasal or subcutaneous Mycobacterium bovis bacillus Calmette-Guerin immunization. These results suggest that mucosal vaccination via the intranasal route is of importance in the development of vaccine for tuberculosis.     

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.     

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.     

Protective effects of lactoferrin chimera and bovine lactoferrin in a mouse model of enterohaemorrhagic Escherichia coli O157:H7 infection

Mice orally infected with enterohaemorrhagic Escherichia coli (EHEC) O157:H7 were used to evaluate the activity of bovine lactoferrin (bLF) and the synthetic peptide LFchimera. Groups of BALB/c mice inoculated intragastrically with EHEC O157:H7 showed chronic intestinal infection with the pathogen that persisted over 6 days and resulted in a high mortality rate (90%). LFchimera and kanamycin significantly decreased (40%) this mortality rate (P = 0.028). On the other hand, although mice administered with bLF showed an important reduction in mortality (50%), this was not statistically significant (P = 0.070). In infected and untreated mice, severe tubular necrosis, glomerular lesions, and moderate intratubular hyaline casts were found in the kidney. However, in the bLF and LFchimera groups we found a reduction in the damage and a substantial decrease in the bacterial concentration excreted in feces 48 h after infection. Furthermore, sepsis caused by EHEC was reduced by the treatments, evidenced by the fact that bacteria were not detected in the kidney or liver 72 h after infection. The results suggest the bLF and LFchimera could have potential as therapeutics in EHEC infections.