Construction and expression of immunogenic hybrid enterotoxigenic Escherichia coli CFA/I and CS2 colonization fimbriae for use in vaccines

Enterotoxigenic Escherichia coli (ETEC) are an important cause of diarrheal morbidity in developing countries, especially in children and also of traveler's diarrhea. Colonization factors (CFs) of ETEC, like CFA/I and CS2 which are genetically and structurally related, play a substantial role in pathogenicity, and since intestinal–mucosal immune responses against CFs appear to be protective, much effort has focused on the development of a CF-based ETEC vaccine. We have constructed hybrid operons in which the major CS2 subunit-encoding cotA gene was inserted into the CFA/I operon, either replacing (hybrid I) or being added to the major CFA/I subunit-encoding cfaB gene (hybrid II). Using specific monoclonal antibodies against the major subunits of CFA/I and CS2, high levels of surface expression of both fimbrial subunits were shown in E. coli carrying the hybrid II operon. Oral immunization of mice with formalin-killed bacteria expressing hybrid II fimbriae induced strong CFA/I- and CS2-specific serum IgG + IgM and fecal IgA antibody responses, which were higher than those achieved by similar immunization with the reference strains. Bacteria expressing hybrid fimbriae are potential candidate strains in an oral-killed CF-ETEC vaccine, and the approach represents an attractive and novel means of producing a broad-spectrum ETEC vaccine.     

The fimbriae of human enterotoxigenic Escherichia coli strain 334 are related to CS5 fimbriae

Escherichia coli strain 334 is a human enterotoxigenic strain of serotype O15:H11 which had previously been shown to produce 'attachment pili'. These fimbriae were compared with other colonization factors. From strain 334 a mannose-resistant haemagglutination positive colony 334A and a mannose-resistant haemagglutination negative variant 334C were isolated. By electron microscopy the fimbriae of strain 334A were shown to have a helical structure resembling coli-surface-associated antigen (CS5) fimbriae. An antiserum was raised to strain 334A and absorbed with a fimbriae-negative variant of that strain, 334C. By immuno-electron microscopy this antiserum was shown to coat fimbriae of strain 334A but not CS5 fimbriae produced by strain E17018A. Conversely, CS5 antiserum did not coat the fimbriae produced by strain 334A. No antigenic cross-reaction was detected between these intact fimbriae when anti-strain 334A serum and CS5 antiserum were used in immunodiffusion tests. By enzyme-linked immunosorbent assays (ELISAs) the fimbriae of strain 334A were shown to be antigenically unrelated to most other human ETEC adhesins, namely colonization factor antigens (CFA/I, CFA/III and CFA/IV), coli-surface-associated antigens (CS1, CS2, CS3, CS4, CS6 and CS17) and putative colonization factors (PCFO159:H4 and PCFO166). However, a heated suspension of strain 334A reacted weakly with CS5 antiserum in an ELISA. By SDS-PAGE the fimbriae of strain 334A were shown to consist of subunits of similar size to CS5 subunits, that is about 21.5 kDa. Western immunoblotting revealed that the subunits of 334A and CS5 fimbriae shared common epitopes.(ABSTRACT TRUNCATED AT 250 WORDS)     

产肠毒素性大肠杆菌K88菌毛装配

K88菌毛介导产肠毒索性大肠杆菌在小肠上皮细胞的粘附,是引起新生仔猪腹泻的主要致病因子之一.菌毛的合成与装配是由fae操纵子调控的,fae操纵子包含10个基,faeA-fae J,其中有些基因表达菌毛装配所需的各种结构蛋白、分子伴侣和调控因子.菌毛的装配过程是由fae操纵子调控,通过分子伴侣,锚定蛋白的相互协同作用完成,组装成结构蛋白的多聚体.继阐明K88菌毛装配调控机理之后,K88菌毛在非毒素源性大肠杆菌及其它原核生物中装配也取得成功,同时菌毛结构蛋白在真核生物中组装也取得了很大进展.     

Erythrocyte and porcine intestinal glycosphingolipids recognized by F4 fimbriae of enterotoxigenic Escherichia coli

Enterotoxigenic F4-fimbriated Escherichia coli is associated with diarrheal disease in neonatal and postweaning pigs. The F4 fimbriae mediate attachment of the bacteria to the pig intestinal epithelium, enabling an efficient delivery of diarrhea-inducing enterotoxins to the target epithelial cells. There are three variants of F4 fimbriae designated F4ab, F4ac and F4ad, respectively, having different antigenic and adhesive properties. In the present study, the binding of isolated F4ab, F4ac and F4ad fimbriae, and F4ab/ac/ad-fimbriated E. coli, to glycosphingolipids from erythrocytes and from porcine small intestinal epithelium was examined, in order to get a comprehensive view of the F4-binding glycosphingolipids involved in F4-mediated hemagglutination and adhesion to the epithelial cells of porcine intestine. Specific interactions between the F4ab, F4ac and F4ad fimbriae and both acid and non-acid glycosphingolipids were obtained, and after isolation of binding-active glycosphingolipids and characterization by mass spectrometry and proton NMR, distinct carbohydrate binding patterns were defined for each fimbrial subtype. Two novel glycosphingolipids were isolated from chicken erythrocytes, and characterized as GalNAcα3GalNAcß3Galß4Glcß1Cer and GalNAcα3GalNAcß3Galß4GlcNAcß3Galß4Glcß1Cer. These two compounds, and lactosylceramide (Galß4Glcß1Cer) with phytosphingosine and hydroxy fatty acid, were recognized by all three variants of F4 fimbriae. No binding of the F4ad fimbriae or F4ad-fimbriated E. coli to the porcine intestinal glycosphingolipids occurred. However, for F4ab and F4ac two distinct binding patterns were observed. The F4ac fimbriae and the F4ac-expressing E. coli selectively bound to galactosylceramide (Galß1Cer) with sphingosine and hydroxy 24:0 fatty acid, while the porcine intestinal glycosphingolipids recognized by F4ab fimbriae and the F4ab-fimbriated bacteria were characterized as galactosylceramide, sulfatide (SO₃-3Galß1Cer), sulf-lactosylceramide (SO₃-3Galß4Glcß1Cer), and globotriaosylceramide (Galα4Galß4Glcß1Cer) with phytosphingosine and hydroxy 24:0 fatty acid. Finally, the F4ad fimbriae and the F4ad-fimbriated E. coli, but not the F4ab or F4ac subtypes, bound to reference gangliotriaosylceramide (GalNAcß4Galß4Glcß1Cer), gangliotetraosylceramide (Galß3GalNAcß4Galß4Glcß1Cer), isoglobotriaosylceramide (Galα3Galß4Glcß1Cer), and neolactotetraosylceramide (Galß4GlcNAcß3Galß4Glcß1Cer).     

A possible association between CFA (I) fimbriae and K99 fimbriae on Escherichia coli and bacterial adherence to human lymphocytes

We have explored a possible association between Escherichia coli binding to human lymphocytes and plasmid coded fimbriae on the bacterial surface. E. coli with or without the plasmid coded membrane CFA(I), K99 and K88 were mixed with freshly-drawn human peripheral blood lymphocytes. When the lymphocytes were mixed with E. coli possessing the CFA(I) fimbriae, 59% of the lymphocytes bound bacteria onto the surface, whereas only 22% of the lymphocytes bound the CFA(I)- derivative. The lymphocytes bound 53% and 56% of two K9+ strains, whereas 22% and 8% of the lymphocytes adhered the same strains without the K99 fimbriae. Twelve per cent and 7% of lymphocytes bound bacteria when the strain was K88+ or K88-, respectively. Likewise a low (8%) adherence to lymphocytes was found when the E. coli did not possess fimbriae or flagella.     

Screening for complex carbohydrates inhibiting hemagglutinations by CFA/I- and CFA/II-expressing enterotoxigenic Escherichia coli strains

Abstract Numerous structural families of naturally occurring glycopeptides and oligosaccharides have been evaluated as potential inhibitors of hemagglutinations mediated by CFA/I- and CFA/II-positive enterotoxigenic Escherichia coli strains. Among the preparations tested were glycopeptides with short O-linked (mucin-type) chains, various mixtures containing N-linked glycans (either oligomannoside-, hybrid- or complex-type), three fractions of human milk oligosaccharides, and glycopeptides derived from either pooled new-born meconiums or pooled human red blood cell membranes. In almost all cases, the same inhibitory preparations were active toward all E. coli strains. This emphasizes the close analogy between the carbohydrate specificities of the colonization factors concerned. Such inhibitors always contained lactosamine units in their oligosaccharide backbones, but this structural requirement alone was not sufficient for activity. The glycopeptide mixture derived from human erythrocyte membranes (known to contain blood group-related carbohydrate antigens carried by a lactosaminoglycan backbone) behaved as a potent hemagglutination inhibitor, especially towards CFA/II-expressing strains. This last result clearly indicates the structural family in which complex carbohydrates should be selected to establish precisely the specificity of these CFA/II adhesins.     

Binding of K99 fimbriae of enterotoxigenic Escherichia coli to pig small intestinal mucin glycopeptides

Binding of purified K99 fimbriae to cryostat sections of pig small intestine was detected. Binding sites were located in the mucus layer, but not in the submucosal connective tissue. High-Mr mucin glycopeptides from pig small intestine were found to bind to K99-fimbriated enterotoxigenic Escherichia coli, in contrast to non-fimbriated cells. Sialic acid specificity of K99 fimbriae was demonstrated by the significant reduction in binding upon desialylation of mucin glycopeptides. The binding was saturable and the dissociation constant was estimated to be 6 x 10(-7) M. Fimbriated bacteria were calculated to possess 2.3 x 10(3) binding sites per cell.     

Primary structure of the CFA1 fimbrial protein from human enterotoxigenic Escherichia coli strains

The complete amino acid sequence of the structural protein that constitutes the subunit of the CFA1 fimbria has been elucidated. The protein was fragmented by cyanogen bromide cleavage, and by enzymatic cleavage with trypsin. Secondary cleavage of the resulting peptides was performed with chymotrypsin, Staphylococcus aureus protease, and thermolysin. Sequential Edman degradation was performed manually. The CFA1 protein comprises 147 amino acid residues, with a molecular weight of 15058.     

Stimulation of mucosal immune response following oral administration of enterotoxigenic Escherichia coli fimbriae (CFA/I) entrapped in liposomes in conjunction with inactivated whole-cell Vibrio cholerae vaccine

In this study, we have searched for an effective mucosal vaccine. An oral enterotoxigenic E. coli vaccine containing colonization factor antigen (CFA/I) associated with inactivated whole-cell V. cholerae vaccine (WCV) has been tested for safety and immunogenicity in animals. Five groups of animals were used. The results showed the following: (a) vaccine containing CFA/I antigen entrapped in liposomes and associated with WCV (batch C) had increased titers of specific antibodies to CFA/I antigen in 15 to 18 (83.3%) animals; (b) specific Peyer's patches (PP), lymph nodes (LN) and spleen (SPL) lymphocytes proliferation was detected following in vitro restimulation with CFA/I antigen or WCV. This response gradually increased to the highest value by the 35th postimmunization day. Moreover, lower PP, LN and spleen (SPL) proliferation was observed in rabbits receiving soluble CFA/I antigen (S-CFA/I) or free liposomes (F-L) alone; (c) adhesion of E. coli H10407 strain labelled with 3H-leucine in immunized and control animals revealed the following local effects: (i) protection of rabbit intestinal mucosa against virulent E. coli cells; (ii) inhibition of adhesion of ETEC bacteria to intestinal mucosa and (iii) significantly faster release of E. coli H 10407 strain labelled with 3H-leucine from the intestinal tract of immunized animals. The histopathological and electron microscope findings confirmed the above results. The experimental results point out an efficient protection against infection with E. coli strains (ETEC), after mucosal vaccination with CFA/I antigen entrapped in liposomes associated with inactivated whole-cell Vibrio cholerae as immunological adjuvant.     

Off‐pathway assembly of fimbria subunits is prevented by chaperone CfaA of CFA/I fimbriae from enterotoxigenic E. coli

The assembly of the class 5 colonization factor antigen I (CFA/I) fimbriae of enterotoxigenic E. coli was proposed to proceed via the alternate chaperone‐usher pathway. Here, we show that in the absence of the chaperone CfaA, CfaB, the major pilin subunit of CFA/I fimbriae, is able to spontaneously refold and polymerize into cyclic trimers. CfaA kinetically traps CfaB to form a metastable complex that can be stabilized by mutations. Crystal structure of the stabilized complex reveals distinctive interactions provided by CfaA to trap CfaB in an assembly competent state through donor‐strand complementation (DSC) and cleft‐mediated anchorage. Mutagenesis indicated that DSC controls the stability of the chaperone‐subunit complex and the cleft‐mediated anchorage of the subunit C‐terminus additionally assist in subunit refolding. Surprisingly, over‐stabilization of the chaperone‐subunit complex led to delayed fimbria assembly, whereas destabilizing the complex resulted in no fimbriation. Thus, CfaA acts predominantly as a kinetic trap by stabilizing subunit to avoid its off‐pathway self‐polymerization that results in energetically favorable trimers and could serve as a driving force for CFA/I pilus assembly, representing an energetic landscape unique to class 5 fimbria assembly.     

Location of binding sites on common type 1 fimbriae from Escherichia coli.

Common type 1 fimbriae were isolated from Escherichia coli and their length distribution profile was determined before and after treatment with ultrasound. As fimbriae were shortened, so their haemagglutinating capacity decreased, but their ability to bind to erythrocytes did not decrease to the same extent. Isolated fimbriae did not agglutinate inside-out vesicles prepared from horse erythrocytes or liposomes, suggesting that the binding mechanism was not based on non-specific hydrophobic interactions. The results support a lateral rather than a terminal location for the fimbrial binding site responsible for haemagglutination.     

Structural characterization of CFA/III and Longus type IVb pili from enterotoxigenic Escherichia coli

The type IV pili are helical filaments found on many Gram-negative pathogenic bacteria, with multiple diverse roles in pathogenesis, including microcolony formation, adhesion, and twitching motility. Many pathogenic enterotoxigenic Escherichia coli (ETEC) isolates express one of two type IV pili belonging to the type IVb subclass: CFA/III or Longus. Here we show a direct correlation between CFA/III expression and ETEC aggregation, suggesting that these pili, like the Vibrio cholerae toxin-coregulated pili (TCP), mediate microcolony formation. We report a 1.26-Å resolution crystal structure of CofA, the major pilin subunit from CFA/III. CofA is very similar in structure to V. cholerae TcpA but possesses a 10-amino-acid insertion that replaces part of the α2-helix with an irregular loop containing a 3₁₀-helix. Homology modeling suggests a very similar structure for the Longus LngA pilin. A model for the CFA/III pilus filament was generated using the TCP electron microscopy reconstruction as a template. The unique 3₁₀-helix insert fits perfectly within the gap between CofA globular domains. This insert, together with differences in surface-exposed residues, produces a filament that is smoother and more negatively charged than TCP. To explore the specificity of the type IV pilus assembly apparatus, CofA was expressed heterologously in V. cholerae by replacing the tcpA gene with that of cofA within the tcp operon. Although CofA was synthesized and processed by V. cholerae, no CFA/III filaments were detected, suggesting that the components of the type IVb pilus assembly system are highly specific to their pilin substrates.     

Characterization of colonization factor antigen CFA/I from an enterotoxigenic Escherichia coli strain (0128:H12)

CFA/I antigen was isolated and purified from E. coli, mutant 279 B-1-14, serotype 0128:H12, and had the following biochemical and biological features: a) amino-acid content was similar to that of purified antigen prepared from strain H10407; b) latex particles sensitization with purified CFA/I antigen produced bovine and human erythrocytes group A/II hemagglutination in carbohydrates presence; c) purified anti-CFA/I specific antibodies agglutinated CFA/I-positive enterotoxigenic E. coli strains; d) 3H-leucine-labelled CFA/I antigen adhered to rabbits intestinal mucosa at significant values; e) intestinal mucosa pretreating with purified CFA/I antigen, followed by 3H-leucine labelled enterotoxigenic bacteria infection, had a least 3 local effects: 1) intestinal mucosa protection against parental enterotoxigenic bacteria; 2) inhibition of CFA/I-positive bacteria adherence to intestinal mucosa; 3) release of approximately 96% intraluminally inoculated bacteria.     

Characterisation of the binding sites for Escherichia coli heat-labile toxin type I in intestinal brush borders

Intestinal brush borders from Wistar rats contained a total of 20-30-times more binding sites for Escherichia coli heat-labile enterotoxin (LT-1) than for cholera toxin (CT). The results suggest that LT-1 binds to sites in addition to ganglioside GM1, the binding site for CT. Brush border proteins were separated by SDS-PAGE, blotted to nitrocellulose and the filters incubated with 125I-labeled toxins. [125I]LT-1 was shown to bind to a series of brush border galactoproteins ranging in size from 130-140 kDa. Binding was inhibited by unlabeled LT-1 (but not CT), and by ricin and free galactose. A number of brush border enzymes are large glycoproteins which can be solubilised by papain. The papain-solubilised sucrase-isomaltase complex was purified by affinity chromatography and shown to bind LT-1, as did the proteins in fractions enriched in maltase activity. However, such brush border galactoproteins do not account for all of the additional LT-1 binding sites. Thus, brush borders prepared from 1-15-day-old rabbits contained many more binding sites for LT-1 than CT despite the absence of any sucrase-isomaltase activity, and no [125I]LT-1 binding proteins could be detected by blotting. There was a marked variation in the number of LT-1 binding sites in different strains of rat, and between different species.     

毒素源性大肠杆菌CFA/I、CS_3重组菌株的粘附力、免疫原性和保护性的研究

本研究采用可逆性肠结扎成兔腹泻(RITARD)动物模型检测了毒素源性大肠杆菌(ETEC)定居因子抗原Ⅰ(CFA/Ⅰ)和表面抗原3(CS_3)重组菌株的肠道粘附力、免疫原性和保护性。经肠道及口服接种时重组菌株对肠道均有较强的粘附力,与CFAs阴性宿主菌相比P<0.01。口服免疫家兔时,第四周血清抗体滴度达到高峰,分别为1∶9 000和1∶80 000;肠道免疫家兔后血清效价在第二周达到高峰,滴度分别为1∶8 000和1∶60 000。在抗体滴度高峰时进行ETEC野生株攻毒,结果显示口服组抗腹泻保护率为66.7％～75％,肠道组为50.1％～71.4％。攻毒后免疫家兔的排菌天数较对照组明显减少。     

A Chimeric protein of CFA/I,CS6 subunits and LTB/STa toxoid protects immunized mice against enterotoxigenic Escherichia coli

Enterotoxigenic Escherichia Coli (ETEC) strains are the commonest bacteria causing diarrhea in children in developing countries and travelers to these areas. Colonization factors (CFs) and enterotoxins are the main virulence determinants in ETEC pathogenesis. Heterogeneity of CFs is commonly considered the bottleneck to developing an effective vaccine. It is believed that broad spectrum protection against ETEC would be achieved by induced anti‐CF and anti‐enterotoxin immunity simultaneously. Here, a fusion antigen strategy was used to construct a quadrivalent recombinant protein called 3CL and composed of CfaB, a structural subunit of CFA/I, and CS6 structural subunits, LTB and STa toxoid of ETEC. Its anti‐CF and antitoxin immunogenicity was then assessed. To achieve high‐level expression, the 3CL gene was synthesized using E. coli codon bias. Female BALB/C mice were immunized with purified recombinant 3CL. Immunized mice developed antibodies that were capable of detecting each recombinant subunit in addition to native CS6 protein and also protected the mice against ETEC challenge. Moreover, sera from immunized mice also neutralized STa toxin in a suckling mouse assay. These results indicate that 3CL can induce anti‐CF and neutralizing antitoxin antibodies along with introducing CFA/I as a platform for epitope insertion.

     

The binding of colonization factor antigens of enterotoxigenic Escherichia coli to intestinal cell membrane proteins

We examined the binding of colonization factor antigens (CFAs) of enterotoxigenic Escherichia coli to electrophoretically separated membrane components of rabbit intestinal brush borders or human intestinal (and non-intestinal) cell lines using an immunoblotting technique. Both CFA/I and CFA/II bound to distinct membrane components which seemed to be identical in rabbit brush borders and in a human intestinal cell line; these binding structures were mainly missing in membranes from epithelial cell lines of non-intestinal origin. Both shared and specific binding components were identified for CFA/I and the different subcomponents of CFA/II (CS1, CS2 and CS3), respectively. Chloroform-methanol extraction of lipids from the cell membranes did not change the binding pattern for either CFA/I or CFA/II suggesting that the binding occurred to (glyco)proteins rather than to (glyco)lipids.     

Determination of colonization factor antigens CFA in diagnosis of enterotoxigenic strains of Escherichia coli]

This study was aimed at establishment whether preliminary determination of colonization factor antigens CFA may be useful in selection of potentially pathogenic strains of Escherichia coli with serological types belonging to ETEC and 750 isolates of E. coli from children with symptoms of diarrhoea. Enterotoxigenicity of strains was evaluated by suckling mice test and culture of Y1 cell tissue. Colonization factor antigens CFA were evaluated on the basis of slide agglutination and agar gel immunodiffusion with application diagnostic sera prepared for this study. Ability of enterotoxin production was found in 25% strains of E. coli with serological types belonging to ETEC. In 90% these strains were isolated from cases of epidemic diarrhoea. ETEC strains were found in 11% of hospitalized children and in 5% who were treated outside of hospital because of diarrhoea. MRHA adhesins occurred on 80% of ETEC strains were all diagnosed as CFA/I. CFA/II were not found and in only three strains non-fimbrial CFA/IV was present. Preliminary determination of CFA during selection of ETEC strains presents as a very sensitive method (97%) and is also highly specific (99%). Application of this method will result in significant increase of affectivity of biological tests directed toward determination of E. coli enterotoxigenicity.