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.     

Cloning of regulator genes controlling fimbrial production by enterotoxigenic Escherichia coli

Sequences regulating production of fimbriae were cloned from two enterotoxigenic Escherichia coli strains. One cloned region, from E. coli 0.25.H42, controlled expression of coli surface-associated (CS) antigen 4, whereas the function of the other, from E. coli 0167.H5, was unclear. Both regulators were related to the cfaD gene that controls expression of colonization factor antigen I (CFA/I) although low stringency conditions were required to show significant hybridization between cfaD and the regulatory fragment from E. coli 0167. The cloned regulatory genes promoted expression of CFA/I, CS1, CS2 and CS4 antigens but the levels of production in the presence of the 0167 regulator were lower than those promoted by the CS4 regulator or cfaD.     

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.     

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)     

Colonization factor antigen II (CFA/II) of enterotoxigenic Escherichia coli: molecular cloning of the CS3 determinant

The genes for the cell surface associated antigen CS3, produced by CFA/II type enterotoxigenic Escherichia coli, have been cloned in the plasmid vector pBR322 to produce a family of recombinant plasmids. These plasmids contain a series of HindIII fragments of which a fragment of 4.6 kb is common to all those expressing CS3. One of these plasmids, pPM474, has been subjected to mutagenesis with Tn1725 and deletions generated using Bal31. This has defined a minimum region of 3.75 kb necessary for the production of CS3 on the cell surface and implying genetic complexity as has been observed with other fimbrial antigens. Analysis of the plasmid encoded proteins in E. coli K-12 minicells has confirmed this complexity.     

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.     

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     

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.     

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.     

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.     

The possession of coli surface antigen CS6 by enterotoxigenicEscherichia coli of serogroups O25, O27, O148, and O159: a possible colonization factor?

A total of 134 enterotoxigenicEscherichia coli (ETEC) of serogroups O25, O27, O148, and O159 were tested in the enzyme-linked immunosorbent assays for the colonization factor antigens I (CFA/I), CFA/II (coli surface antigens CS1, 2 and 3) and putative colonization factor (PCF) 8775 (CS4, 5 and 6). CS6 was detected without CS4 or CS5 in 94% of the strains of serogroup O25, 86% of strains of serogroup O27, 87% of strains of serogroup O148, and 29% of strains of serogroup, O159. The frequency with which CS6 occurs in ETEC of common serotypes without the antigens CS4 or CS5 suggests that it might be a colonization factor.     

毒素源性大肠杆菌定居因子抗原CS6基因的克隆

通过构建人源毒素源性大肠杆菌野生株E519/66A大质粒的基因文库,成功地筛选出能表达定居因子抗原CS6菌毛的阳性克隆,初步确定了克隆DNA片段的限制性内切酶图谱。CS6抗原的编码和调控基因集中在一大小为4.6kb的DNA区段中,该片段能产生两种分子量大小不同、但抗原反应交叉的菌毛蛋白。本研究获得的CS6抗原阳性的重组菌株可用于人源ETEC多价疫苗的研制,克隆的基因片段亦可作为研究CS6菌毛蛋白基因表达及调控的基础。     

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.     

Morphological and immunocytochemical analysis of Escherichia coli-specific surface antigens in wildtype strains and in recombinant Vibrio cholerae

Adhesion is the first step in the pathogenesis of enterotoxigenic Escherichia coli infections. The genes encoding the most prevalent adhesion factors CFA/I, CS3 and CS6 were cloned into Vibrio cholerae strain CVD 103–HgR and expression of fimbriae was investigated in wildtype and recombinant strains by transmission electron microscopy in conjunction with immunolabelling and negative staining. Negative staining was effective in revealing CFA/I and CS3, but not CS6. Although morphology of fimbriae differed between wildtype and recombinant strains, corresponding surface antigens were recognized by specific antibodies. The present study provides evidence that ETEC-specific fimbriae can adequately be expressed in an attenuated V. cholerae vaccine strain and that immunoelectron microscopy is a critical tool to validate the surface expression of antigens in view of their possible suitability for recombinant vaccines.     

Properties of wild-type strains of enterotoxigenic Escherichia coli which produce colonization factor antigen II, and belong to serogroups other than O6

Enterotoxigenic strains of Escherichia coli, which belonged to serogroups other than O6 and produced colonization factor antigen II, usually produced only coli surface antigen 3 (CS3) and gave weak mannose-resistant haemagglutination of bovine erythrocytes. A non-autotransferring plasmid, NTP165, from a strain of E. coli O168. H16 coded for heat-stable enterotoxin, heat-labile enterotoxin and CS antigens. The CS antigens expressed after acquisition of plasmid NTP165 depended on the recipient strain: a biotype A strain of serotype O6. H16 expressed CS1 and CS3; a biotype C strain of serotype O6. H16 expressed CS2 and CS3; strain K12 and strain E19446 of serotype O139. H28 expressed only CS3. An exceptional wild-type strain, E24377, of serotype O139. H28 produced CS1 and CS3 when isolated; a variant of E24377 which had lost the plasmid coding for CS antigens produced both CS1 and CS3 after the introduction of NTP165.     

Genetic Fusions of a CFA/I/II/IV MEFA (Multiepitope Fusion Antigen) and a Toxoid Fusion of Heat-Stable Toxin (STa) and Heat-Labile Toxin (LT) of Enterotoxigenic Escherichia coli (ETEC) Retain Broad Anti-CFA and Antitoxin Antigenicity

Immunological heterogeneity has long been the major challenge in developing broadly effective vaccines to protect humans and animals against bacterial and viral infections. Enterotoxigenic Escherichia coli (ETEC) strains, the leading bacterial cause of diarrhea in humans, express at least 23 immunologically different colonization factor antigens (CFAs) and two distinct enterotoxins [heat-labile toxin (LT) and heat-stable toxin type Ib (STa or hSTa)]. ETEC strains expressing any one or two CFAs and either toxin cause diarrhea, therefore vaccines inducing broad immunity against a majority of CFAs, if not all, and both toxins are expected to be effective against ETEC. In this study, we applied the multiepitope fusion antigen (MEFA) strategy to construct ETEC antigens and examined antigens for broad anti-CFA and antitoxin immunogenicity. CFA MEFA CFA/I/II/IV [CVI 2014, 21(2):243-9], which carried epitopes of seven CFAs [CFA/I, CFA/II (CS1, CS2, CS3), CFA/IV (CS4, CS5, CS6)] expressed by the most prevalent and virulent ETEC strains, was genetically fused to LT-STa toxoid fusion monomer 3xSTa_A14Q-dmLT or 3xSTa_N12S-dmLT [IAI 2014, 82(5):1823-32] for CFA/I/II/IV-STa_A14Q-dmLT and CFA/I/II/IV-STa_N12S-dmLT MEFAs. Mice intraperitoneally immunized with either CFA/I/II/IV-STa_-toxoid-dmLT MEFA developed antibodies specific to seven CFAs and both toxins, at levels equivalent or comparable to those induced from co-administration of the CFA/I/II/IV MEFA and toxoid fusion 3xSTa_N12S-dmLT. Moreover, induced antibodies showed in vitro adherence inhibition activities against ETEC or E. coli strains expressing these seven CFAs and neutralization activities against both toxins. These results indicated CFA/I/II/IV-STa_-toxoid-dmLT MEFA or CFA/I/II/IV MEFA combined with 3xSTa_N12S-dmLT induced broadly protective anti-CFA and antitoxin immunity, and suggested their potential application in broadly effective ETEC vaccine development. This MEFA strategy may be generally used in multivalent vaccine development.     

Antigen-Specific Memory B-cell Responses to Enterotoxigenic Escherichia coli Infection in Bangladeshi Adults

Background

Multiple infections with diverse enterotoxigenic E. coli (ETEC) strains lead to broad spectrum protection against ETEC diarrhea. However, the precise mechanism of protection against ETEC infection is still unknown. Therefore, memory B cell responses and affinity maturation of antibodies to the specific ETEC antigens might be important to understand the mechanism of protection.

Methodology

In this study, we investigated the heat labile toxin B subunit (LTB) and colonization factor antigens (CFA/I and CS6) specific IgA and IgG memory B cell responses in Bangladeshi adults (n = 52) who were infected with ETEC. We also investigated the avidity of IgA and IgG antibodies that developed after infection to these antigens.

Principal Findings

Patients infected with ETEC expressing LT or LT+heat stable toxin (ST) and CFA/I group or CS6 colonization factors developed LTB, CFA/I or CS6 specific memory B cell responses at day 30 after infection. Similarly, these patients developed high avidity IgA and IgG antibodies to LTB, CFA/I or CS6 at day 7 that remained significantly elevated at day 30 when compared to the avidity of these specific antibodies at the acute stage of infection (day 2). The memory B cell responses, antibody avidity and other immune responses to CFA/I not only developed in patients infected with ETEC expressing CFA/I but also in those infected with ETEC expressing CFA/I cross-reacting epitopes. We also detected a significant positive correlation of LTB, CFA/I and CS6 specific memory B cell responses with the corresponding increase in antibody avidity.

Conclusion

This study demonstrates that natural infection with ETEC induces memory B cells and high avidity antibodies to LTB and colonization factor CFA/I and CS6 antigens that could mediate anamnestic responses on re-exposure to ETEC and may help in understanding the requirements to design an effective vaccination strategies.     

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)     

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.     

Immune Response against Hamster Erythrocytes in the Low-Responder Mouse Strains

C57BL/6 and AKR mice were treated with hamster erythrocytes (HRBC) in complete Freund's adjuvant (CFA) or incomplete Freund's adjuvant (IFA) and the development of delayed hypersensitivity and antibody production were examined. 1) Delayed hypersensitivity against HRBC antigen, as determined by the peritoneal macrophage disappearance test, was detected in mice sensitized with HRBC in CFA but not in those sensitized with HRBC in IFA. 2) Antibody production against HRBC or hapten TNP after a booster injection of HRBC or trinitrophenylated HRBC (TNP-HRBC) in saline was enhanced by pretreatment with HRBC in CFA or IFA. 3) Delayed hypersensitivity was not detectable after a booster sensitization with HRBC in CFA in mice which had been pretreated with HRBC in IFA 2 weeks earlier. In the mice treated with both HRBC in IFA (day ?21) and in CFA (day ?7), however, an enhanced antibody production against HRBC or TNP was detected after an intravenous injection with HRBC or TNP-HRBC in saline (day 0). These results suggest that sensitized effector lymphocytes in delayed hypersensitivity and helper cells in antibody production may be derived from the same pool of unprimed T cells. The pool of unprimed T cells with a capacity to differentiate into either type of primed T cells may be exhausted after pretreatment with the antigen in IFA, and the primed helper T cells may not be able to differentiate into sensitized lymphocytes even after sensitization with the antigen in CFA, which favors development of delayed hypersensitivity in normal controls.