Positive regulation of colonization factor antigen I (CFA/I) production by enterotoxigenic Escherichia coli producing the colonization factors CS5, CS6, CS7, CS17, PCFO9, PCFO159:H4 and PCFO166

Entertoxigenic Escherichia coli (ETEC) strains of nineteen serogroups which produced colonization factors (coli-surface-associated antigens CS5, CS6, CS7 and CS17, colonization factor antigen CFA/III and putative colonization factors PCFO159:H4, PCFO166 and PCFO9) were tested for hybridization with a DNA probe containing the cfaD sequence that regulates expression of CFA/I. Strong colony hybridization, similar to that with the CFA/I-positive control strain H10407, occurred with ETEC strains of serogroups O27, O159 and O169 which produced CS6 antigen, and with all the strains which produced PCFO166 fimbriae. Weak colony hybridization, compared to the control strain, was found with ETEC producing CS5 fimbriae with CS6 antigen, CFA/III fimbriae with CS6 antigen, CS7 fimbriae or PCFO159:H4 fimbriae. CS6-antigen-positive strains of serogroups O79, O89 and O148 and all the CS17-antigen-positive and PCFO9-fimbriae-positive strains were negative in colony hybridization tests with the cfaD probe. Plasmid DNA of nine ETEC strains and their colonization-factor-negative derivatives was tested for hybridization with the cfaD probe and with ST and LT oligonucleotide probes. The sequences that hybridized with the cfaD probe were on the plasmids which coded for enterotoxin production. Fifteen strains were transformed with NTP513, a recombinant plasmid which contains the CFA/I region 1 fimbrial subunit operon but lacks a functional cfaD sequence, in order to determine whether DNA in any of these strains could substitute for the cfaD sequence in the regulation of production of CFA/I fimbriae.(ABSTRACT TRUNCATED AT 250 WORDS)     

Expression of plasmids coding for colonization factor antigen II (CFA/II) and enterotoxin production in Escherichia coli

Two plasmids transferred from enterotoxigenic Escherichia coli (ETEC) of serotype O6. H16 and biotypes A and C coded for mannose-resistant haemagglutination (MRHA) and production of heat-stable enterotoxin (ST) and heat-labile enterotoxin (LT). Both plasmids were nonautotransferring being mobilized most efficiently by the R plasmid R100-1. They were similar in their genetic properties being incompatible with each other and plasmids of the Inc group FI. The wild-type strains produced the colonization factor antigen II (CFA/II) which was made up of different coli surface antigens (CS). The biotype A strains produced CS1 and CS3 while the biotype C strains produced CS2 and CS3. These three antigens have the ability to cause MRHA. When plasmids coding for MRHA were transferred to K12 strains, the degree of haemagglutination was markedly reduced and only CS3 was produced. When both plasmids were transferred back into biotype A strains, good MRHA was restored and the strains produced CS1 and CS3. In a biotype C strain CS2 and CS3 were formed. The production of the antigens was compared by enzyme-linked immunosorbent assay (ELISA). The strains were also examined by electron microscopy where it was found that CS1 and CS2 were fimbrial antigens while CS3 was not.     

Structural and regulatory genes for coli surface associated antigen 4 (CS4) are encoded by separate plasmids in enterotoxigenic Escherichia coli strains of serotype 025.H42

Two enterotoxigenic Escherichia coli strains of serotype 0.25.H42 that produced coli surface associated antigens CS4 and CS6 hybridized with a probe containing the cfaD sequence that regulates expression of colonization factor antigen CFA/I. Transformation of a cloned cfaD gene into some derivatives of the strains that were negative for CS4 and CS6 resulted in expression of CS4 but not CS6. By hybridization the sequence that regulated CS4 production in the wild type 025 strains was located on a plasmid that also encoded the CS6 antigen. The structural genes for the CS4 antigen were on a separate plasmid. The 025 strains carried a third plasmid encoding enterotoxin production which was therefore unlinked to regulation sequences or genes encoding CS antigens.     

Mobilization of CS fimbriae-associated plasmids of enterotoxigenic Escherichia coli of serotype O6: K15: H16 or H- into various wild-type hosts

Abstract CS fimbriae-associated plasmids of two enterotoxigenic Escherichia coli strains of serotype O6: K15: H16 or H- (biotypes A and F) with M _r values of 51 × 10⁶ and 72 × 10⁶, respectively, were mobilized into various alternative host bacteria. Expression of CS1 or CS2 fimbriae was obtained when either of the CS fimbriae-associated plasmids was introduced into CS Fim⁻, O6: K15: H16 or H- recipients with rhamnose-negative and rhamnose-positive fermentation phenotypes, respectively, whereas CS3 fimbriae were expressed irrespective of the biotype of the recipient. On transfer into a CS Fim⁻ variant of an enterotoxigenic O8: H9 strain and into two K-12 strains, a CS3-fimbriae-only phenotype was conferred by the presence of either of the plasmids. When a CS Fim⁻ variant of a Rha⁺ CS2-fimbriae-only strain of serotype O6: K15: H16 harboured either of the plasmids, both CS2 and CS3 fimbriae were expressed, indicating that the rare CS2-fimbriae-only wild-type phenotype is probably due to the presence of a defective plasmid in such strains. Mobilization of the 51 MDa CS fimbriae-associated plasmid into five non-enterotoxigenic Rha⁺ porcine isolates of E. coli with O6 serotypes other than O6: K15: H16 or H- yielded CS3-fimbriae-only transconjugants. Thus the correlation between a Rha⁺ fermentation phenotype and expression of CS2 fimbriae does not hold in general for O-group 6 strains.     

Long-term systemic and mucosal antibody responses measured in BALB/c mice following intranasal challenge with viable enterotoxigenic Escherichia coli

The immunogenicity induced in BALB/c mice following intranasal challenge with a viable nonlethal dose (1.2 x 10(8) CFU) of enterotoxigenic Escherichia coli (ETEC) strain E23477A (O139:H28:CS1:CS3:LT+:ST+) was studied over a 140-day period. Serum IgG and IgM antibodies against coli surface antigen 3 (CS3), O139 lipopolysaccharide and heat-labile enterotoxin were measured by day 14 and remained at elevated levels out to day 140. The serum IgG response to the somatic antigens (CS3 and O139 lipopolysaccharide) was significantly greater (P < 0.05) than the IgG response to heat-labile enterotoxin, and the serum IgG response to CS3 was significantly greater (P < 0.05) than the IgG response to O139 lipopolysaccharide. The predominant serum IgG subclasses to CS3 were IgG1 and IgG2a, and they were significantly greater (P < 0.05) than IgG2b and IgG3. The predominant serum IgG subclass response to O139 lipopolysaccharide was initially IgG3 until day 56, after which IgG1 was predominant. The serum subclass response to CS3 indicated a mixed T helper 1/2 (Th1/Th2) profile, whereas the response to O139 lipopolysaccharide was primarily that of a Th2-type, at least over time. Fecal IgG and IgA responses to CS3 and O139 lipopolysaccharide were detected by day 14 and were measured out to day 140, with the CS3 fecal antibody responses being significantly greater (P < 0.05) than the O139 lipopolysaccharide and heat-labile enterotoxin fecal antibody responses. The aim of this study is the development of the intranasal mouse model that can aid in better understanding the immunopathology of ETEC infection and in screening of vaccine candidates prior to volunteer trials.     

Serologically distinct fimbriae on enterotoxigenic Escherichia coli of serotype O6:K15:H16 or H-

Abstract Serologically distinct, surface-associated, mannose-resistant haemagglutinins have been reported on enterotoxigenic Escherichia coli of serotype O6:K15:H16 or H- of human origin. Using immune electron microscopy, these haemagglutinins, termed the CS1 and CS2 antigens, have been identified as serologically distinct fimbriae, the presence of which correlates with the rhamnose fermentation phenotype of strains. The CS1 and CS2 fimbriae are morphologically indistinguishable from common type fimbriae. In contrast, using the same technique, no labelling of fimbriae was obtained with specific antibodies to another protein surface antigen termed the CS3 antigen, which is common to most strains of this serotype and also found on certain enterotoxigenic E. coli of serotype O8:H9. The morphological nature of the CS3 antigen was not disclosed.     

Plasmids coding for heat-labile enterotoxin production isolated from Escherichia coli O78: comparison of properties.

Nineteen enterotoxigenic Escherichia coli strains of serogroup O78, isolated in different geographical areas from humans with diarrheal diseases, were tested for their ability to transfer enterotoxin production. All of the strains originally produced heat-labile enterotoxin, and 16 also produced heat-stable enterotoxin and colonization factor antigen I. Plasmids coding for the production of heat-labile enterotoxin only were transferred from 13 strains. Some properties of these plasmids were compared. All were fi+, but they could be divided into three groups on the basis of their incompatibility reactions, ability to restrict E. coli K-12 phages, and size. The three heat-labile enterotoxin plasmids isolated from African strains all belonged to one enterotoxin plasmid group. The heat-labile enterotoxin plasmids from the Asian strains were divided into two groups, those from serotype O78.H11 differing from those from serotype O78.H12.     

Expression of CS fimbriae in Escherichia coli strains of human and animal origin belonging to O-serovar 6, K-serovar 15 or H-serovar 16

Abstract A non-conjugative CS-fimbriae-associated plasmid pCS001 was mobilized into rifampicin-resistant mutants of strains of Escherichia coli of O-serovar 6 or K-serovar 15 or H-serovar 16. By a variety of serological procedures only the production of CS3 fimbriae was detected in transoconjugants. The finding extend previous observations that only strains of E. coli of serotype O6:K15:H16 or H− and of appropriate biotype are able to express either CS1 or CS2 fimbriae, as even a recipient of serotype O6:K15:H31 possessing a rhamnose-positive fermentation phenotype did not express either of these two fimbriae. The results indicate that expression of CS1 or CS2 fimbriae probably involves chromosomal determinants only found in strains of serotype O6:K15:H16 or H−.     

Cloning of genes encoding coli-surface (CS) antigens in enterotoxigenic Escherichia coli

Abstract Sequences encoding the CS6 antigen of colonisation factor antigen (CFA)IV were cloned on a 3kb Cla I fragment. The recombinant plasmid pDEP5 coded for surface expression of CS6 measured by ELISA and production of CS6 polypeptides was detected in E. coli minicells. The genes for the CS1, CS2 and CS3 components of colonisation factor antigen CFA/II were cloned together on a length of DNA corresponding to about 17kb. CS3 was always expressed but production of either CS1 or CS2 depended on the serotype and biotype of the host strain. Separate subclones were obtained that expressed CS3 or CS1 and CS2.     

Characterization of a putative colonization factor (PCFO166) of enterotoxigenic Escherichia coli of serogroup O166

Enterotoxigenic Escherichia coli (ETEC) of serogroup O166 gave mannose-resistant haemagglutination (MRHA) with bovine and human erythrocytes. The strains did not react with antisera prepared against the known colonization factors CFA/I, CFA/II, CFA/III, CFA/IV and PCFO159:H4. Strain E7476 of serotype O166:H27, which produced heat-stable enterotoxin (ST), was examined initially. It produced fimbriae about 7 nm in diameter. On SDS-PAGE two possible fimbrial polypeptides of molecular mass 15.5 and 17.0 kDa were seen. When variants of strain E7476 were isolated, loss of ST and MRHA together was associated with loss of a 98 MDa plasmid, while loss of ST alone correlated with plasmid deletion. An absorbed anti-strain E7476 antiserum reacted specifically with the 15.5 and 17.0 kDa polypeptides in Western immunoblotting and bound to the intact fimbriae by immuno-electron microscopy. When this antiserum was used in an ELISA to examine other strains of serogroup O166, a positive reaction was obtained with all the ST- and MRHA-positive strains. One strain of serotype O71:H27 and two strains of serotype O98:H- also reacted with the absorbed anti-strain E7476 antiserum. The antiserum did not react with ETEC carrying known colonization factors. E. coli K12 and a number of E. coli of different serotypes carrying a plasmid coding for ST transferred from strain E7476, all gave MRHA and reacted with the absorbed anti-strain E7476 antiserum. The term putative colonization factor O166 (PCFO166) is proposed to describe the adhesive factor(s) on ETEC of serogroup O166 because of the similarity of properties with those of known colonization factors.     

Plasmids of enterotoxigenic Escherichia coli H10407: evidence for two heat-stable enterotoxin genes and a conjugal transfer system

Three species of plasmids, associated with virulence and conjugal transfer, were identified in a clinically isolated enterotoxigenic Escherichia coli strain, H10407 (serotype O78:H11). pCS1, a non-self-transmissible plasmid species with a molecular weight of 62 X 10(6) and a 47 mol% guanine-plus-cytosine content, specified colonization factor antigen I and heat-stable enterotoxin (ST) production, as reported by others previously. A second non-self-transmissible plasmid species, designated pJY11, with a molecular weight of 42 X 10(6) and a 51 mol% guanine-plus-cytosine content, specified ST and heat-labile enterotoxin production and manifested T5/T6 phage restriction. The third plasmid species, pTRA1, also had a molecular weight of 42 X 10(6) and had a guanine-plus-cytosine content of 51 mol%; this species was self-transmissible and promoted transfer of both pCS1 and pJY11 to other bacterial cells. pCS1 may have originated from species of bacteria with a lower guanine-plus-cytosine content than E. coli. Finally, although demonstrating some heterogeneity with each other, both STs encoded by pCS1 and pJY11 belonged to the STa group.     

肠毒素性大肠杆菌CS3纤毛抗原在痢疾菌中的表达及其免疫效果研究

首先通过体内外重组的方法,构建了福氏2a痢疾菌T32asd基因缺陷的突变体FaD,作为抗原载体菌;同时,构建包含asd基因的表达质粒pYX102,与FaD一起,构成宿主-载体平衡致死系统,用于在没有抗生素条件选择的情况下,稳定表达克隆在表达质粒上的外源抗原基因.将肠毒素性大肠杆菌的CS3菌毛抗原基因克隆至pYX102,构建成重组表达质粒pYX103,ELISA检测结果证实CS3在痢疾菌中可以很好地表达.免疫小鼠后可诱生相应的抗体,虽然口服免疫和注射免疫产生的CS3抗体效价有一定差别,但对痢疾菌的毒株攻击均可提供较好保护.该结果为细菌性腹泻疫苗的研制提供了候选株.     

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)     

Isolation and characterization of a new fimbrial antigen (CS1541) from a porcine enteroxigenic Escherichia coli O8: KX105 strain

Abstract A fimbrial antigen (CS1541) was extracted and purified from the porcine enterotoxigenic Escherichia coli strain 1541P (O8:K-:H9). CS1541 fimbriae appeared as long thin filaments 3–5 mm in diameter. CS1541 antigen consisted of two peptide bands of about 18 and 19 kDa as demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting. It was expressed both at 37°C and 15°C and did not demonstrate hemagglutinating properties. It was antigenically distinct from the fimbrial antigens K88, K99, F41, FY(Att25), F165, type 1, CFA-I, and CFA-II complex but demonstrated serological cross-reactions with the 987P fimbrial antigen.     

Prevalence of CS31A and F165 surface antigens in Escherichia coli isolates from animals in France, Canada and India

Bovine and porcine enterotoxigenic and non-enterotoxigenic Escherichia coli isolates from France, Canada, and India were characterized with respect to serogroup and production of fimbrial antigens CS31A and F165. Of 231 bovine isolates from the 3 countries, 20.5% produced CS31A alone, 17.7% produced F165 alone, and 17.3% produced both CS31A and F165. On the other hand, of 84 porcine isolates from Canada, 1.2% produced CS31A alone, 14.3% produced F165 alone, and no isolate produced both CS31A and F165. CS31A was found together with F5 (K99) in 7 of 16 bovine enterotoxigenic E. coli isolates of serogroups 08, 09, 020, and 023, but was not found in any of 20 F4 (K88)- or 5 F6 (987P)-positive porcine enterotoxigenic E. coli isolates. F165 was not found in enterotoxigenic E. coli. Among non-enterotoxigenic isolates, CS31A and F165 were mainly associated with serogroups 08, 09, 011, 015, 017, 023, 025, 078, 0101, 0115, 0117, 0141, and 0153.     

Further characterization of Escherichia coli O153:H45, an ETEC serotype disseminated in Chile

The newly described stable enterotoxin producing, enterotoxigenic Escherichia coli, serotype O153:H45, capable of expressing colonizing factor antigen I, is frequently isolated as a cause of diarrhea among Chilean children. Hybridization studies of five new strains confirmed previous results which indicated that the stable enterotoxin genes are contained in nonconjugative plasmids ranging in size from 81 to 87 kilobases. The strains expressed similar antibiotic resistance and metabolic properties but differed in their plasmid content.     

Non-replicating oral whole cell vaccine protective against enterotoxigenic Escherichia coli (ETEC) diarrhea: stimulation of anti-CFA (CFA/I) and anti-enterotoxin (anti-LT) intestinal IgA and protection against challenge with ETEC belonging to heterologous serotypes

An oral killed (non-replicating) whole-cell anti-ETEC vaccine was prepared by treating enterotoxigenic Escherichia coli strain H-10407 (ST + LT +; 078: H11: CFA/I) with a 100%-lethal amount of colicin E2. Colicin E2 is a potent DNA endonuclease which enters the target bacterial cells without disrupting cellular integrity. Thus the vaccine consists of intact cells lacking chromosomal and plasmid DNA but possessing a normal complement of antigens, including CFA/I and enterotoxin(s), unaltered by chemical- or heat-treatment. Young healthy volunteers were administered two oral doses, one month apart, of approximately 3 x 10(10) vaccine cells. Of 22 vaccinees, 17 (77.3%) showed an intestinal anti-CFA/I IgA response and 19 (86.4%) showed an increase in intestinal anti-LT IgA. Twenty of 22 (90.9%) vaccinees had antibody responses to either CFA/I, LT, or both antigens, demonstrating that colicin E2-treated CFA-positive E. coli cells are an efficient vehicle in terms of delivery of antigens to the gut immune system. We previously demonstrated protection of vaccinees against challenge with the living homologous ETEC (strain H-10407). In this study, two groups of 8 vaccinees were challenged with a diarrheagenic dose of virulent ST + LT + ETEC of heterologous serotype; one group was challenged with a CFA/I-positive 063: H- strain and the other group was challenged with a CFA/II-positive 06: H16 strain. Approximately 75% efficacy was achieved in both challenge groups. None of the 16 vaccinees who had responded to both CFA/I and LT became ill upon challenge while both of the vaccinees who had not responded to either antigen did.(ABSTRACT TRUNCATED AT 250 WORDS)     

Transposition of ampicillin resistance to an enterotoxin plasmid in an Escherichia coli strain of human origin.

We examined a strain of Escherichia coli, serotype O159.H34, of human origin which produced heat-stable and heat-labile enterotoxins, was resistant to ampicillin, and produced colicin. By conjugation and transformation experiments plasmids coding for enterotoxin production (Ent), enterotoxin production and ampicillin resistance (Ap-Ent), ampicillin resistance (Ap), and colicin production were isolated. Both the Ent and Ap-Ent plasmids were autotransferring and belonged to the F-incompatibility complex. However, the Apr Ent+ transconjugants showed differences in their levels of resistance and in their abilities to propagate F-specific phages and to transfer resistance. The results suggested there was transposition from the small Ap plasmid to the Ent plasmid. The Ap-Ent plasmids were larger than the enterotoxin factor and when treated with restriction endonuclease BamHI showed an additional fragment not present in the enterotoxin plasmid. The insertion of ampicillin resistance probably occurred at different sites on the enterotoxin plasmid, resulting in the observed variation in phenotype.     

Characterization of glucosyltransferase expressed from a Streptococcus sobrinus gene cloned in Escherichia coli

The gene encoding a glucosyltransferase which synthesized water-insoluble glucan, gtfI, previously cloned from Streptococcus sobrinus strain MFe28 (mutans serotype h) into a bacteriophage lambda vector, was subcloned into the plasmid pBR322. The recombinant plasmid was stable in Escherichia coli and gtfI was efficiently expressed. The GTF-I expressed in E. coli was compared to the corresponding enzymes in S. sobrinus strains MFe28 (serotype h), B13 (serotype d) and 6715 (serotype g) and shown to resemble them closely in molecular mass and isoelectric point. The insoluble glucan produced by GTF-I from recombinant E. coli consisted of 1,3-alpha-D-glycosyl residues (approximately 90%). An internal fragment of the gtfI gene was used as a probe in hybridization experiments to demonstrate the presence of homologous sequences in chromosomal DNA of other streptococci of the mutans group.     

肠毒素大肠杆菌定居因子CFA/Ⅰ和CS6在减毒福氏志贺氏菌中的共表达

定居因子CFA/I和CS6是肠毒素大肠杆菌 (ETEC)中重要的两种优势抗原 ,是ETEC疫苗研制的首选组分。采用基因重组技术将二者构建在以asd基因为选择标记的重组质粒上 ,与asd基因缺失突变型减毒福氏志贺氏菌FWL0 1构成宿主 载体平衡致死系统。实验结果表明 ,重组疫苗候选株能够稳定表达CFA/I和CS6抗原 ,并可在菌体表面形成相应菌毛。重组菌口服免疫BALB/c小鼠后 ,可诱生相应的抗CFA/I和CS6的特异性血清抗体IgG和分泌型抗体sIgA ,说明以志贺氏菌为载体 ,可以构建同时表达多个定居因子抗原的ETEC多价菌苗