Antigenic variation within the subunit protein of members of the colonization factor antigen I group of fimbrial proteins in human enterotoxigenic Escherichia coli

Colonization factor antigens (CFAs) of human enterotoxigenic Escherichia coli can be divided in groups based on the N-terminal amino acid sequence of their major subunit protein. One of the groups that has been distinguished in this way, is the CFA/I group of fimbriae. The sequence of the fimbrial subunit genes in the operons encoding the antigens CS4, CS14 and CS17, all members of this group, was determined. A duplication of the fimbrial subunit gene (csuA) was found in the CS14 operon, both genes encoding very similar proteins. Purified CS14 fimbriae consist of two proteins with different molecular masses (15.5 and 17.0 kDa) but identical N-terminal amino acid sequences, which strongly suggests that both csuA genes are transcribed. A phylogenetic tree derived from the amino acid sequences of the CFA/I, CS1, CS2, CS4, CS14, CS17 and CS19 subunit proteins shows that CS1, CS17 and CS19 belong to the same subgroup. CFA/I, CS4 and CS14 belong to a second subgroup, while CS2 is distinct within the CFA/I group of fimbriae. The genetic similarity between CS1, CS17 and CS19 is reflected in the substantial immunological cross-reactivity observed, both between their protein subunits and intact fimbriae.     

Gene encoding the major subunit of CS1 pili of human enterotoxigenic Escherichia coli.

Some enterotoxigenic strains of Escherichia coli (ETEC) utilize the CS1 pilus for colonization of human intestinal epithelium. We have cloned the gene which encodes the major CS1 subunit and called it cooA (for coli surface antigen one). Hybridization showed that the ETEC strain from which it was cloned carried cooA on a plasmid different from the one encoding its positive regulator, rns. Based on the cooA DNA sequence, cleavage with signal peptidase would be expected to produce a mature protein of 15.2 kDa; a 16-kDa polypeptide that reacted with CS1-specific antiserum was observed on electrophoresis. At the protein level, there was 92% similarity and 55% identity between cooA and cfaB, the major colonization factor antigen I (CFA/I) antigen. However, CS1-specific antisera did not react with CfaB. No hybridization was seen between either of two different cooA probes and total DNA from ETEC strains expressing AFA-1, CFA/I, CS2, CS3, CS4, CS5, or CS6.     

An F41-K88-related genetic determinant of bovine septicemic Escherichia coli mediates expression of CS31A fimbriae and adherence to epithelial cells.

A genetic determinant related to that encoding the F41 fimbrial adhesin was cloned from a bovine septicemic isolate of Escherichia coli. This determinant was found to mediate expression of morphologically distinct fimbriae in E. coli HB101. The gene encoding the fimbrial subunit protein was identified, and the nucleotide sequence was determined. Homology with the amino-terminal amino acid sequence of CS31A (J. Girardeau, M. Der Vartanian, J. Ollier, and M. Contrepois, Infect. Immun. 56:2180-2188, 1988) was observed, suggesting that this determinant encodes expression of the CS31A fimbrial antigen. The CS31A subunit gene was found to share extensive homology in its signal sequence to the subunit genes encoding the F41 and K88 adhesins. No apparent homology between the mature F41 and CS31A subunits was identified. However, substantial relatedness to the K88 fimbrial subunit was observed. Analysis of the protein products encoded by the CS31A, F41, and K88 determinants in maxicells established that despite extensive genetic similarities between the determinants, each encodes a distinct profile of proteins. E. coli HB101 harboring the cloned CS31A determinant was found to adhere to epithelial cells in a tissue culture assay, suggesting a role for CS31A in adherence. A CS31A-specific DNA hybridization probe detected homologous sequences among enterotoxigenic as well as septicemic E. coli isolates from calves.     

Monoclonal antibodies against enterotoxigenic Escherichia coli colonization factor antigen I (CFA/I) that cross-react immunologically with heterologous CFAs.

Enterotoxigenic Escherichia coli binds to enterocytes in the small intestine by means of antigenically distinct colonization factors (CFs), usually termed colonization factor antigens (CFAs), coli surface antigens (CS), or putative colonization factor antigens (PCFs). To explore the immunological relationship between different CFs, we dissociated CFA/I fimbriae into subunits and produced monoclonal antibodies (MAbs) against these subunits. We selected three MAbs that cross-reacted immunologically with a number of different, whole purified CFs in a dot blot test and with the corresponding subunits in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. One of the MAbs, i.e., subunit CFA/I 17:8 (S-CFA/I 17:8), reacted more strongly with subunits of CFA/I than with whole purified fimbriae. This MAb cross-reacted with whole purified fimbriae and subunits of CS4, PCFO166, CS1, and CS2. Moreover, it bound strongly to a peptide of 25 amino acids corresponding to the N-terminal end of CFA/I. The other two MAbs, i.e., S-CFA/I 5:6 and S-CFA/I 8:11, cross-reacted with CS1, CS2, CS4, PCFO166, and CS17 fimbriae but reacted only slightly or not at all with the CFA/I peptide. MAbs S-CFA/I 17:8 and S-CFA/I 5:6 were shown to inhibit hemagglutination by bacterial strains that express either CFA/I, CS1, or CS4. In addition, the binding of enterotoxigenic E. coli strains expressing CFA/I, CS2, CS4, and PCFO166 to enterocyte-like cell-line Caco-2 was inhibited by both MAbs. These results show that several antigenically different CFs have common epitopes and that among these at least one is located in the N-terminal end of the subunit protein. Moreover, antibodies against the common epitopes seem to block binding of the bacterial strains that express different CFs to both erythrocytes and Caco-2 cells.     

Genetic analysis of the gene cluster encoding nonfimbrial adhesin I from an Escherichia coli uropathogen.

The chromosomally encoded nonfimbrial adhesion I (NFA-I) from Escherichia coli urinary tract isolate 827 (O83:K1:H4) mediates agglutination of human erythrocytes. Subclones were constructed from an NFA-I-expressing recombinant E. coli K-12 clone, derived from a genomic library of E. coli 827. Minicell analysis and nucleotide sequencing revealed that proteins of 30.5, 9, 80, 15, and 19 kDa encoded on a stretch of approximately 6 kb are involved in the expression of NFA-I. NFA-I exhibits a polymeric structure, which disintegrates with elevated temperature into a 19-kDa monomer but with some relatively stable dimers. By using gold-conjugated monoclonal antibodies directed against NFA-I in electron microscopy, the adhesin could be localized on the outer surface of the recombinant E. coli K-12 bacteria. The nucleotide sequence of the nfaA gene encoding the monomeric structural subunit of the adhesin was determined. An open reading frame of 184 amino acids encoding the NfaA precursor, which is processed to the mature protein, was found; it consisted of 156 amino acids with a calculated molecular weight of 16,000. Peptide sequencing of the NFA-I subunit protein confirmed that this open reading frame corresponds to the NfaA coding locus. Furthermore, the nucleotide sequence of the open reading frame termed NfaE, located at the proximal part of the DNA stretch responsible for NFA-I expression, was elaborated. NfaE consists of 247 amino acids, including a presumptive 29-amino-acid signal peptide, leading to a molecular weight of 24,000 for the mature protein. The nfaE sequence shares homology with the 27-kDa CS3 protein, which is involved in the assembly of CS3 fibrillae, and might encode the 30.5-kDa protein, detected in minicells.     

Nucleotide sequence of the gene encoding the major subunit of CS3 fimbriae of enterotoxigenic Escherichia coli.

The complete nucleotide sequence of a 612-base-pair DNA fragment containing the gene for the major fimbrial subunit of CS3 of enterotoxigenic Escherichia coli is presented. A possible promoter region, a ribosome-binding site, and two potential signal peptidase cleavage sites are indicated. Unlike the best-studied fimbrial proteins, the predicted CS3 sequence has no Cys residues.     

Identification and characterization of CS20, a new putative colonization factor of enterotoxigenic Escherichia coli.

An enterotoxigenic Escherichia coli (ETEC) strain producing a previously undescribed putative colonization factor was isolated from a child with diarrhea in India. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of bacterial heat extracts revealed a polypeptide band of 20.8 kDa when the bacteria were grown at 37 degrees C which was absent after growth at 22 degrees C. A specific rabbit antiserum raised against the purified 20.8-kDa protein bound specifically to the fimbriae, as shown by immunoelectron microscopy, and inhibited bacterial adhesion to tissue-cultured Caco-2 cells. Transformation with a recombinant plasmid harboring the cfaD gene, which encodes a positive regulator for several ETEC fimbriae, induced hyperexpression of the 20.8-kDa fimbrial subunit and a substantial increase in the proportion of bacterial cells that were fimbriated. The N-terminal amino acid sequence of the polypeptide showed 65 and 60% identity to the PCFO20 and 987P fimbriae of human and porcine ETEC, respectively. We propose the term CS20 for this new putative colonization factor of human ETEC.     

Genetic relationship of putative colonization factor O166 to colonization factor antigen I and coli surface antigen 4 of enterotoxigenic Escherichia coli.

Plasmid DNA from two strains of enterotoxigenic Escherichia coli harboring genes encoding coli surface antigen 4 (CS4) and from seven Indian enterotoxigenic E. coli isolates cross-hybridized at low stringency but not at high stringency with two polynucleotide probes derived from the colonization factor antigen I (CFA/I) operon. Low-stringency Southern blot hybridization of PstI-digested plasmid DNA from the seven Indian isolates yielded characteristic restriction fragment patterns, distinct from those of CS4- and CFA/I-associated plasmid DNA. Two of the Indian strains were transformed with a recombinant plasmid harboring the cfaD gene, which encodes a positive regulator of CFA/I and CS4 genes. The cfaD transformants produced large amounts of putative colonization factor O166 (PCFO166) irrespective of whether the nutrient agar contained bile salts, a growth factor otherwise required for adequate PCFO166 expression. A considerable interstrain variation in the level of PCFO166 production could be explained by differences in the proportion of bacteria that were fimbriated, as visualized by electron microscopy. The N-terminal amino acid sequence of PCFO166 fimbrial protein showed a high degree of homology with the corresponding sequences of CFA/I and CS4.     

Characterization of CS4 and CS6 antigenic components of PCF8775, a putative colonization factor complex from enterotoxigenic Escherichia coli E8775.

PCF8775 is a putative colonization factor complex present on the surface of 10 to 20% of enterotoxigenic Escherichia coli strains and has been reported to be composed of antigen CS6 (morphology undefined) expressed alone or together with either of the rigid fimbrial antigens CS4 and CS5. To better define the individual components of this complex and the determinants of their expression, we prepared antiserum to the PCF8775 complex as it was expressed on prototype strain E8775 and then used the antiserum to identify the subunit structure of the antigens, to study their morphology, and to detect expression of individual components of the complex after transfer of plasmids into laboratory strain HB101. CS4 was purified from strain E8775, confirmed to be fimbrial by electron microscopy, and found to be composed of a 22-kilodalton protein subunit whose N-terminal amino acid sequence (1 to 20) was similar to that of colonization factor antigen I. Transconjugants that express CS6 but not CS4 were obtained by mating prototype strain E8775 with HB101. CS6 expression was mediated by a 61-megadalton plasmid. Expression of CS6 in the transconjugants correlates with expression of a 16-kilodalton cell surface protein. The CS6 antigen was confirmed to be present on the cell surface by immunogold labeling, but its morphology was beyond the limits of resolution by electron microscopy.     

Role of PCF8775 antigen and its coli surface subcomponents for colonization, disease, and protective immunogenicity of enterotoxigenic Escherichia coli in rabbits.

The role of the PCF8775 antigen and its antigenic subcomponents, in particular, the coli surface (CS) antigen CS6, as colonization factors and protective antigens was studied in the reversible intestinal tie adult rabbit diarrhea model. This was done by testing the abilities of different mutants which carried one or two of the CS components to colonize the intestine and to induce protective immunity against reinfection with PCF8775-positive enterotoxin-producing Escherichia coli. Infection with enterotoxigenic E. coli carrying CS4-CS6, CS5-CS6, or CS6 alone induced diarrhea in 75% or more of the rabbits, whereas the corresponding nonenterotoxigenic mutants, as well as enterotoxigenic but CS-negative strains, induced diarrhea in only a few cases. Mutants carrying CS6 alone colonized the intestine equally as well as strains carrying CS4-CS6 or CS5-CS6 did, whereas CS-negative mutants were excreted in the stool for a significantly shorter period. Rabbits previously infected with mutants carrying CS6 alone or CS6 in combination with CS4 or CS5 developed diarrhea with a significantly lower frequency after reinfection with a normally highly diarrheagenic dose of enterotoxigenic CS4-CS6-positive E. coli bacteria than did animals immunized with corresponding CS-negative mutants. Fecal excretion of the rechallenge strain was also of considerably shorter duration than that observed after initial infection with corresponding strains in 27 of the 30 animals (90%) immunized with strains carrying CS6 alone or in combination with CS4 or CS5. Such reduced shedding of the challenge strain was only seen in a few rabbits (3 of 12) initially infected with CS-negative bacteria. These results suggest that the CS6 component of PCF8775 is a colonization factor in rabbits and that it is also capable of inducing protective immunity.     

Self-transmissible R plasmids encoding CS31A among human Escherichia coli strains isolated from diarrheal stools.

The CS31A antigen was first described for septicemic and enterotoxigenic bovine E. coli strains. In our study, of 597 human Escherichia coli strains isolated from diarrheagenic stools of hospitalized patients, 30 (5%) hybridized with the CS31A DNA probe. These CS31A-positive E. coli strains diffusely adhered to Caco-2 and/or HEp-2 cells and produced a major surface protein of either 30 or 30.5 kDa according to the strain. These proteins were antigenically related to the two forms of the CS31A antigen, namely, CS31A-L and CS31A-H. Genes encoding CS31A were located on 140-kb conjugative R plasmids. E. coli transconjugants expressed major surface proteins similar to those of the wild-type strains and adhered to Caco-2 and/or HEp-2 cells. An association of CS31A and another adhesive factor of the Dr family was found in 70% of wild-type strains, since 21 strains hybridized with the diffuse adhesion DNA probe corresponding to the accessory gene (daaC) of the F1845 adhesin. Comparison of the restriction patterns of the 140-kb R plasmids of the CS31A-positive E. coli strains showed these plasmids to be similar. Hybridization experiments indicated that the genes encoding CS31A and resistance to penicillin were located together on either of two 20- or 27-kb EcoRI restriction fragments in four E. coli strains. We reported a similar linkage between these genes in Klebsiella pneumoniae strains which produced CF29K, a CS31A-like antigen. These results suggest a horizontal transfer between E. coli and K. pneumoniae strains.     

Genetic control and properties of coli surface antigens of colonization factor antigen IV (PCF8775) of enterotoxigenic Escherichia coli.

Enterotoxigenic Escherichia coli producing coli surface antigen 4 (CS4), CS5, and CS6 of colonization factor antigen IV were examined. This factor was originally called putative colonization factor 8775 (PCF8775). All of the coli surface antigens were plasmid coded and were usually carried on the same plasmid as the genes coding for heat-stable toxin (ST) or heat-labile toxin (LT); thus, CS5-CS6-ST, CS6-ST, and CS6-LT plasmids were found. In strains of serotype O25:H42, the genes coding for CS4 and CS6 were on a plasmid separate from that containing the genes coding for ST and LT. CS4 and CS5 were fimbrial antigens with a subunit molecular mass of about 17.0 and 21.0 kilodaltons (kDa), respectively. CS6 was found as a single polypeptide with a molecular mass of about 14.5 kDa in strains of serotypes O25:H42, O27:H7, and O27:H20 when heated extracts were run on sodium dodecyl sulfate-polyacrylamide gels. CS6-positive extracts of strains of serogroups O148, O159, and O167 showed two bands with molecular masses between 14.5 and 16.0 kDa.     

Genetic characterization and immunogenicity of coli surface antigen 4 from enterotoxigenic Escherichia coli when it is expressed in a Shigella live-vector strain

The genes that encode the enterotoxigenic Escherichia coli (ETEC) CS4 fimbriae, csaA, -B, -C, -E, and -D', were isolated from strain E11881A. The csa operon encodes a 17-kDa major fimbrial subunit (CsaB), a 40-kDa tip-associated protein (CsaE), a 27-kDa chaperone-like protein (CsaA), a 97-kDa usher-like protein (CsaC), and a deleted regulatory protein (CsaD'). The predicted amino acid sequences of the CS4 proteins are highly homologous to structural and assembly proteins of other ETEC fimbriae, including CS1 and CS2, and to CFA/I in particular. The csaA, -B, -C, -E operon was cloned on a stabilized plasmid downstream from an osomotically regulated ompC promoter. pGA2-CS4 directs production of CS4 fimbriae in both E. coli DH5alpha and Shigella flexneri 2a vaccine strain CVD 1204, as detected by Western blot analysis and bacterial agglutination with anti-CS4 immune sera. Electron-microscopic examination of Shigella expressing CS4 confirmed the presence of fimbriae on the bacterial surface. Guinea pigs immunized with CVD 1204(pGA2-CS4) showed serum and mucosal antibody responses to both the Shigella vector and the ETEC fimbria CS4. Among the seven most prevalent fimbrial antigens of human ETEC, CS4 is the last to be cloned and sequenced. These findings pave the way for CS4 to be included in multivalent ETEC vaccines, including an attenuated Shigella live-vector-based ETEC vaccine.     

Evolutionary and functional relationships of colonization factor antigen i and other class 5 adhesive fimbriae of enterotoxigenic Escherichia coli

Colonization factor antigen I (CFA/I) is the archetype of eight genetically related fimbriae of enterotoxigenic Escherichia coli (ETEC) designated class 5 fimbriae. Assembled by the alternate chaperone pathway, these organelles comprise a rigid stalk of polymerized major subunits and an apparently tip-localized minor adhesive subunit. We examined the evolutionary relationships of class 5-specific structural proteins and correlated these with functional properties. We sequenced the gene clusters encoding coli surface antigen 4 (CS4), CS14, CS17, CS19, and putative colonization factor antigen O71 (PCFO71) and analyzed the deduced proteins and the published homologs of CFA/I, CS1, and CS2. Multiple alignment and phylogenetic analysis of the proteins encoded by each operon define three subclasses, 5a (CFA/I, CS4, and CS14), 5b (CS1, CS17, CS19, and PCFO71), and 5c (CS2). These share distant evolutionary relatedness to fimbrial systems of three other genera. Subclass divisions generally correlate with distinguishing in vitro adherence phenotypes of strains bearing the ETEC fimbriae. Phylogenetic comparisons of the individual structural proteins demonstrated greater intrasubclass conservation among the minor subunits than the major subunits. To correlate this with functional attributes, we made antibodies against CFA/I and CS17 whole fimbriae and maltose-binding protein fusions with the amino-terminal half of the corresponding minor subunits. Anti-minor subunit Fab preparations showed hemagglutination inhibition (HAI) of ETEC expressing homologous and intrasubclass heterologous colonization factors while anti-fimbrial Fab fractions showed HAI activity limited to colonization factor-homologous ETEC. These results were corroborated with similar results from the Caco-2 cell adherence assay. Our findings suggest that the minor subunits of class 5 fimbriae may be superior to whole fimbriae in inducing antiadhesive immunity.     

Synthesis of hybrid molecules between heat-labile enterotoxin and cholera toxin B subunits: potential for use in a broad-spectrum vaccine.

Three variants of the cholera toxin B subunit (CTB) were generated by site-specific mutagenesis in which regions of the mature protein were altered to the composition found at the corresponding positions of the closely related B subunit of the heat-labile enterotoxin of enterotoxigenic Escherichia coli (LTB). The mutant proteins were expressed in Vibrio cholerae and purified from the growth medium. In the first of the mutant proteins, the first 25 amino acids corresponded to the sequence found in LTB, and in the second, changes were made at positions 94 and 95 of the mature protein. The third mutant protein combined the changes made in the first two. Analysis of the immunological properties of these novel proteins by using monoclonal antibodies and absorbed polyclonal antiserum demonstrated that they had acquired LTB-specific epitopes. Immunizations with the mutant proteins resulted in antisera containing LTB-specific as well as CTB-specific and cross-reactive antibodies. The sera were also found to be more strongly cross-reactive in the in vitro neutralization of both cholera toxin and heat-labile enterotoxin than were antisera raised against either CTB or LTB. The results suggest that such hybrid CTB-LTB proteins may be useful in a broad-spectrum vaccine against enterotoxin-induced diarrhea.     

Vi抗原影响产肠毒素大肠杆菌菌毛 在人伤寒沙门菌表面的装配

目的 观察人伤寒沙门菌Vi抗原对大肠杆菌菌毛抗原装配的影响。方法 利用体内、外同源重组系统,构建了VipR基因缺失突变的人伤寒沙门菌菌株,导致其Vi抗原的表达较相应野生菌株偏低。用包含产肠毒素大肠杆菌菌毛抗原基因的表达质料分别转化Vi表达弱化菌株和相应野生菌株,对两者表达的菌毛抗原进行含量分析。结果 产肠毒素大肠杆菌CS3、CFA－I在VipR突变体菌株表面的含量,均比在相应野生菌株表面的含量高。结论 Vi抗原的表达弱化可能有利于菌毛抗原在人伤寒沙门菌表面的装配。本研究结果对于产肠毒素大肠杆菌基因工程疫苗的构建有指导意义。     

Revised amino acid sequence for a heat-stable enterotoxin produced by an Escherichia coli strain (18D) that is pathogenic for humans.

The amino acid sequence of heat-stable enterotoxin produced by enterotoxigenic Escherichia coli 18D has been revised. Amino acids originally assigned to positions 11 and 18, i.e., Tyr and Asn, respectively, were found to occupy positions 18 and 11, respectively. Thus all heat-stable enterotoxins composed of 18 amino acids sequenced to date from human, porcine, and bovine isolates of E. coli have identical primary structures, i.e., Asn-Thr-Phe-Tyr-Cys-Cys-Glu-Leu-Cys-Cys-Asn-Pro-Ala-Cys-Ala-Gly-Cys-Tyr. Furthermore, all 18- and 19-amino-acid heat-stable enterotoxins from E. coli share an almost identical core sequence, i.e., 14 of the 15 carboxy-terminal amino acid residues are identical.     

Proteus mirabilis flagella and MR/P fimbriae: isolation, purification, N-terminal analysis, and serum antibody response following experimental urinary tract infection.

Urinary tract infection with Proteus mirabilis may lead to serious complications, including cystitis, acute pyelonephritis, fever, bacteremia, and death. In addition to the production of hemolysin and the enzyme urease, fimbriae and flagellum-mediated motility have been postulated as virulence factors for this species. We purified mannose-resistant/proteuslike (MR/P) fimbriae and flagella from strains CFT322 and HU2450, respectively. Electron microscopy revealed highly concentrated preparations of fimbriae and flagella. Fimbrial and flagellar structural subunits were estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be 18.5 and 41 kDa, respectively. N-terminal sequencing revealed that 10 of the first 20 amino acids of the major MR/P subunit matched the sequence of the P. mirabilis uroepithelial cell adhesin N terminus and 11 of 20 amino acids matched the predicted amino acid sequence of the Escherichia coli P fimbriae structural subunit, PapA. In addition, 90 and 80% homologies were found between the first 20 amino acids of P. mirabilis flagellin and those of Salmonella typhimurium phase-1 flagellin and the E. coli hag gene product, respectively. An enzyme-linked immunosorbent assay using purified antigens showed a strong reaction between the MR/P fimbriae or flagella and sera of CBA mice challenged transurethrally with P. mirabilis. A possible role for MR/P fimbriae in the pathogenesis of urinary tract infection is supported by (i) a strong immune response to the antigen in experimentally infected animals, (ii) amino acid sequence similarity to other enteric surface structure, and (iii) our previously reported observation that MR/P fimbriae are expressed preferentially as the sole fimbrial type in human pyelonephritis isolates.