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)     

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.     

The nucleotide sequence of a regulatory gene present on a plasmid in an enterotoxigenic Escherichia coli strain of serotype O167:H5

A DNA fragment that can functionally substitute for cfaD, the positive regulatory gene involved in expression of CFA/I fimbriae, has recently been cloned from an Escherichia coli strain of serotype O167:H5 that produces CS5 fimbriae. Nucleotide sequence determination showed that the fragment contained a gene, csvR (Coli Surface Virulence factor Regulator) homologous to the cfaD gene, which encoded for a protein of 301 amino acid residues. The csvR gene was found to be located between two different insertion sequences. Comparison of the amino acid sequence of the CsvR and CfaD proteins showed that CsvR is 34 amino acid residues longer at the C-terminus and, in the sequence, it also contains an insertion of two amino acid residues. The similarity between CfaD and Rns, the positive regulator of CS1 and CS2 expression, is much higher (97%) than between CsvR and CfaD (87%). This is reflected by the fact that the level of expression of CFA/I fimbriae induced by CsvR is not as high as when expression is induced by CfaD or Rns.     

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.     

Analysis of the first two genes of the CS1 fimbrial operon in human enterotoxigenic Escherichia coli of serotype 0139: H28

An oligonucleotide, derived from the N-terminal amino acid sequence of the CS1 fimbrial subunit protein was used to identify the subunit gene on recombinant plasmid pDEP23 containing the structural genes of the CS1 fimbrial operon. The nucleotide sequence of the subunit gene (csoA), encoding a protein of 171 amino acids, was determined. Flanking it upstream, a gene (csoB) encoding a protein of 238 amino acids was found. The CsoB and CsoA proteins are homologous to the CfaA and CfaB proteins in the CFA/I fimbrial operon. For all the CS1 producing strains investigated the structural genes are located on plasmids. Like CFA/I fimbriae, CS1 fimbriae are only expressed in the presence of a positive regulator, CfaD for CFA/I and Rns for CS1, respectively. The promoter region upstream of the csoB gene was cloned in front of the promoterless alkaline phosphatase (phoA) gene of the promoter-probe vector pCB267. PhoA activity was enhanced approximately two-fold by the introduction of compatible plasmids containing either rns or cfaD.     

CS3纤毛抗原表达调控机理的研究

ＣＳ３是某些肠毒素大肠杆菌菌体表面上的多聚物,它能使病原菌粘附于宿主的小肠上皮细胞上,是致病的重要因素．为了探索ＣＳ３菌毛抗原基因的表达调控机制,根据ＣＳ３亚基结构基因的核苷酸序列分析表明,在其翻译起始位点的上游存在着ｒｂｓ位点及原核启动子的－１０区和－３５区ＤＮＡ序列．采用基因重组技术将ＣＳ３结构基因上游１２０ｂｐ的ＤＮＡ片段亚克隆进缺乏启动子而只含报告基因ｌａｃＺ的质粒ｐＣＢ２６７中．凝胶滞留和启动报告基因表达的实验证明了ＣＳ３亚基结构基因具有自身的启动子（Ｐｓ）．将该启动子上游区域不同长度的核苷酸片段克隆进ｐＣＢ２６７中,报告基因表达结果表明ＣＳ３结构基因的表达受其上游区域的抑制．核苷酸序列分析发现,在Ｐｓ－３５区上游５５０ｂｐ和８４０ｂｐ处各存在一个富Ａ－Ｔ簇．结合原核启动子的一般作用规律推知,ＣＳ３的表达可能受ＤＮＡ结合蛋白型的正向调节因子的作用．用ＣＦＡ／１菌毛抗原基因的正向调节基因ｃｆａＤ对ＣＳ３基因进行的互补表达试验表明ｃｆａＤ基因不仅可消除上游区对Ｐｓ的抑制,而且可大幅度地提高Ｐｓ的启动能力．在分析表达调控的基础上获得ＣＳ３重组高效表达．同时提出了其表达调控模型．     

Polyphosphate and orthophosphate content during the life cycle of Myxococcus coralloides D

Immunoglobulins, prepared from polyclonal rabbit antisera raised against Escherichia coli fimbrial antigens, colonization factor antigen (CFA)/I, and coli-surface-associated antigens (CS)1, CS2 and CS4, were used to assess antigenic cross-reactions between these four fimbrial types by Western immunoblotting. Antibodies in a serum, prepared against CS4, cross-reacted strongly with the fimbrial subunits of CFA/I, CS1 and CS2. Antibodies in sera prepared against CFA/I and CS1 gave weak reactions with CS1 or CFA/I respectively and also with CS2 and CS4, while the antiserum prepared against CS2 did not react. CS4 antiserum also reacted with the CS17 fimbrial subunit, but not with the subunits of fimbrial antigens: CFA/III, CS5, putative colonization factor (PCF) 0159:H4 or PCF0166.     

Expression of cloned plasmid regions encoding colonization factor antigen I (CFA/I) in Escherichia coli

Molecular cloning from a plasmid encoding colonization factor antigen I (CFA/I) and heat-stable enterotoxin isolated two regions, 1 and 2, that are required for the production of CFA/I fimbriae. The level of CFA/I synthesis measured by ELISA was similar in an Escherichia coli K12 strain carrying regions 1 and 2 cloned separately on compatible plasmid vectors to that in the same strain containing the parental plasmid. The structural gene for the CFA/I fimbrial subunit was within region 1. This region directed production in E. coli minicells of at least six independent polypeptides, of which the fimbrial subunit and at least three others appeared to be synthesized as precursor molecules that underwent processing. Cloned DNA containing CFA/I region 2 specified three polypeptides in minicells. Attempts to reduce the size of the cloned region 1 resulted in a derivative plasmid that carried the CFA/I structural gene but did not complement a region-2 recombinant plasmid to restore production of CFA/I fimbriae.     

CFA/I-ST plasmids: comparison of enterotoxigenic Escherichia coli (ETEC) of serogroups O25, O63, O78, and O128 and mobilization from an R factor-containing epidemic ETEC isolate

Colonization factor antigen I (CFA/I) plays an important role in the pathogenesis of diarrhea due to enterotoxigenic Escherichia coli. In this study, we examined 11 CFA/I+ enterotoxigenic E. coli from serogroups O25, O63, O78, and O128 and found that with all strains, spontaneous loss of CFA/I was associated with the loss of heat-stable toxin (ST) and with the loss of a single plasmid ranging in size from 54 to 60 megadaltons; when heat-labile toxin was lost, this was associated with the loss of another plasmid. The R factor of one strain, TX432 (O78:H12:CFA/I+; ST+), was found to mobilize the CFA/I-ST plasmid into E. coli K-12 at a frequency of 20%. These studies provide further evidence that CFA/I production is plasmid mediated in enterotoxigenic E. coli belonging to serogroups O25, O63, O78, and O128.     

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.     

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.     

Prevalence of Colonization Factor Antigens (CFAs) and Adherence to HeLa Cells in Enterotoxigenic Escherichia coli Isolated from Feces of Children in São Paulo

Fifty-eight enterotoxigenic Escherichia coli (ETEC) strains, isolated from children with and without diarrhea in Sao Paulo, were examined for the presence of colonization factor antigens (CFAs) and their ability to adhere to HeLa cells. Antisera to CFA/I, the coli surface (CS) antigens CS1CS3, CS2CS3, and CS2 of CFA/II, CFA/III, and CS5CS6 and CS6 of CFA/IV were used. CFAs were identified in 43% of the ETEC strains: 40% of the strains with CFAs harbored CFA/I, 24% carried CFA/II (CS1CS3), 24% carried CFA/IV (CS6), and 12% carried CFA/IV (CS5CS6). CFAs occurred mainly among ETEC strains producing only heat-stable (ST-I) enterotoxin and in strains also producing heat-labile toxin (LT-I). No ETEC strains tested expressed CFA/III. A marked change in serotypes of ST-I-producing strains was found in Sao Paulo between 1979 and 1990. Adherence to HeLa cells was detected in 14% of the ETEC strains. All of them had a diffuse adherence pattern and produced only ST-I, and 88% carried CS6 antigen.     

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)     

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     

Assessing the expression of enterotoxigenic Escherichia coli-specific surface antigens in recombinant strains by transmission electron microscopy and immunolabeling.

Infections with enterotoxigenic Escherichia coli (ETEC) are a major cause of travelers' diarrhea worldwide. Colonization of the small intestine mucosa is dependent on specific colonization factor antigens (CFA) and coli surface (CS) antigens. CFA/1, CS3, and CS6 are the most prevalent fimbrial antigens found in clinical isolates. The goal of our study was to visualize the morphology of CS3 and CS6 fimbriae in wild-type and recombinant E. coli strains by means of transmission electron microscopy in conjunction with negative staining and immunolabeling. Corresponding ETEC genes were cloned into E. coli K12 strain DH10B. Expression of fimbriae was dependent on culture conditions and sample handling. Specific immunolabeling of fimbriae unequivocally demonstrated the presence of all types of surface antigens investigated. Negative staining was effective in revealing CS3 but not CS6. In addition, this technique clearly demonstrated differences in the morphology of genetically and immunologically identical CS3 surface antigens in wild-type and recombinant strains. This paper provides a basis for the assessment of recombinant vaccines.     

Immune response to Escherichia coli antigens entrapped in liposomes. I. Immunopathological studies

In this study, we have searched for an effective mucosal delivery system for a purified E. coli antigen which elicits anticolonization and anti-toxic immunity. E. coli colonization factor antigen (CFA/I) and heat-labile enterotoxin (LT) were encapsulated in liposomes. To determine the efficacies of soluble and liposome-encapsulated E. coli antigens young rabbits were mucosally treated with three oral doses of E. coli antigens given 7 days apart. Ten days after the last booster, rabbits were orally challenged with 5 x 10(9) bacterial cells (O78:H11 serotype). The experimental results allow of making some remarks which can be correlated with the protection obtained in vaccinated animals: (a) immunization with E. coli antigens entrapped in liposomes ensured protection against ETEC strains; (b) lower protection against homologous and heterologous CFA/I +(LT- ST+) strains were noticed; (c) adhesion of labelled -3H-leucine-bacteria to the intestinal mucosa revealed a maximum distribution in duodenum-jejunum and minimum in the colonic mucosa; (d) it contributed to the release of inoculated virulent bacteria from intestinal tract; (e) humoral, cellular and histopathological findings confirm the afore mentioned observation. Summing up, these results suggest that liposomes are very good carriers for E. coli antigens and these findings highlight the potential use of LT and CFA/I antigens entrapped in liposomes as mucosal and humoral induction of immune response and make them a candidate for future use in prophylaxis of diarrhoea in man.     

Donor strand complementation governs intersubunit interaction of fimbriae of the alternate chaperone pathway

Fimbrial filaments assembled by distinct chaperone pathways share a common mechanism of intersubunit interaction, as elucidated for colonization factor antigen I (CFA/I), archetype of enterotoxigenic Escherichia coli (ETEC) Class 5 fimbriae. We postulated that a highly conserved beta-strand at the major subunit N-terminus represents the donor strand, analogous to interactions within Class I pili. We show here that CFA/I fimbriae utilize donor strand complementation to promote proper folding of and interactions between CFA/I subunits. We constructed a series of genetic variants of CfaE, the CFA/I adhesin, incorporating a C-terminal extension comprising a flexible linker and 10-19 of the N-terminal residues of CfaB, the major subunit. Variants with a donor strand complement (dsc) of >or= 12 residues were recoverable from periplasmic fractions. Genetic disruption of the donor beta-strand reduced CfaE recovery. A hexahistidine-tagged variant of dsc19CfaE formed soluble monomers, folded into beta-sheet conformation, displayed adhesion characteristic of CFA/I, and elicited antibodies that inhibited mannose-resistant haemagglutination by ETEC expressing CFA/I, CS4 and CS14 fimbriae. Immunoelectron microscopy indicated that CfaE was confined to the distal fimbrial tip. Our findings provide the basis to elucidate structure and function of this class of fimbrial adhesins and assess the feasibility of an adhesin-based vaccine.     

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)