Adhesion of enterotoxigenic Escherichia coli to the human colon carcinoma cell line Caco-2 in culture.

Enterotoxigenic Escherichia coli (ETEC) strains possessing colonization factor antigen I (CFA/I), CFA/II, CFA/III, and antigen 2230 were tested for their ability to adhere to the following cell lines: HeLa, HEp-2, HRT 18, Hutu 80, MDBK, MDCK, Vero, and Caco-2. ETEC strains adhered only to the Caco-2 cell line. Irrespective of the known adhesive factors, the ETEC strains that adhered to the brush border of human enterocytes also adhered to the Caco-2 cell line. The negative variants, which were cured of the plasmid encoding the adhesive factor, did not adhere. Adhesion of ETEC strains no longer occurred when the Caco-2 cells were pretreated with the homologous colonization factor antigen or when the bacterial cells were pretreated with homologous antibodies raised against the adhesive factors. This indicates that this adhesion is specific and that a different receptor exists for each type of adhesion factor. Electron micrographs of cross sections of the monolayer showed that the adhesion of ETEC strains to the brush border microvilli does not induce any lesion. Therefore, the Caco-2 cell line behaves in the same way as human enterocytes do.     

Gene cluster for assembly of pilus colonization factor antigen III of enterotoxigenic Escherichia coli

The assembly of pilus colonization factor antigen III (CFA/III) of enterotoxigenic Escherichia coli (ETEC) requires the processing of CFA/III major pilin (CofA) by a prepilin peptidase (CofP), similar to other type IV pilus formation systems. CofA is produced initially as a 26.5-kDa preform pilin (prepilin) and then processed to a 20.5-kDa mature pilin by CofP which is predicted to be localized in the inner membrane. In the present experiment, we determined the nucleotide sequence of the whole region for CFA/III formation and identified a cluster of 14 genes, including cofA and cofP. Several proteins encoded by cof genes were similar to previously described proteins, such as the toxin-coregulated pili of Vibrio cholerae and the bundle-forming pili of enteropathogenic E. coli. The G+C content of the cof gene cluster was 37%, which was significantly lower than the average for the E. coli genome (50%). The introduction of a recombinant plasmid containing the cof gene cluster into the E. coli K-12 strain conferred CFA/III biogenesis and the ability of adhesion to the human colon carcinoma cell line Caco-2. This is the first report of a complete nucleotide sequence of the type IV pili found in human ETEC, and our results provide a useful model for studying the molecular mechanism of CFA/III biogenesis and the role of CFA/III in ETEC infection.     

Detection of enterotoxigenic Escherichia coli colonization factor antigen I in stool specimens by an enzyme-linked immunosorbent assay.

An enzyme-linked immunosorbent assay (ELISA) was employed to detect and quantitate the fimbrial colonization factor antigen (CFA/I) of enterotoxigenic Escherichia coli in stool specimens obtained from adult cases of diarrhea in which CFA/I-positive E. coli was the known causative agent. The inhibition method, or blocking technique, was used. In this method, a standardized dilution of human anti-CFA/I serum was preincubated with dilutions of stool extract before transfer to CFA/I-coated microtiter plate wells, and then ELISA was performed with alkaline phosphatase-conjugated anti-human immunoglobulin. CFA/I purified from E. coli strain H-10407 (O78:H11) was used. Acute-phase diarrheal stool specimens were found to contain approximately 3.0 mg of antigen (mean value) per g stool, whereas control (CFA/I-negative) specimens contained insignificant amounts (less than 0.03 mg/g) of antigen. Also, CFA/I was detected in culture fluids of CFA/I positive enterotoxigenic E. coli belonging to a variety of serotypes and was undetectable in similar preparations from P-strains (spontaneous CFA/I-negative derivatives) of the same test cultures. Equivalent results were obtained in ELISA tests by using bacterial cells taken from isolated colonies grown on CFA agar. These results indicate that the ELISA technique will be useful for the diagnosis of diarrhea caused by CFA/I-positive enterotoxigenic E. coli.     

New fimbrial antigenic type (E8775) that may represent a colonization factor in enterotoxigenic Escherichia coli in humans.

An enterotoxigenic strain of Escherichia coli O25:H42 (strain E8775), isolated from a patient in Bangladesh with diarrhea, caused mannose-resistant hemagglutination (MRHA) of human and bovine erythrocytes. The strain did not show slide agglutination or immunodiffusion precipitin lines with antiserum specific for the colonization factor antigen CFA/I or CFA/II. A variant E. coli strain, E8775-B, did not cause MRHA or produce enterotoxin. Electron microscopy revealed the presence of fimbriae on the surface of strain E8775 but not strain E8775-B. When strain E8775 was grown at 22 degrees C, it became MRHA negative and fimbriae were absent. An antiserum prepared against strain E8775 was absorbed with strain E8775-B to make an antiserum specific for the fimbrial antigen. Using this absorbed antiserum, we found the fimbrial antigen in 48 of 742 enterotoxigenic E. coli strains. The 48 strains belonged to serogroups O25, O115, and O167. It is suggested by analogy to the properties of previously described colonization factors that these fimbriae may play a part in the colonization of the intestinal epithelium.     

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 and molecular studies of plasmids coding for colonization factor antigen I and heat-stable enterotoxin in several escherichia coli serotypes.

Plasmids coding for colonization factor antigen I (CFA/I) and heat-stable enterotoxin (ST) were identified in 10 strains of human enterotoxigenic Escherichia coli. The strains, which belonged to serogroups O63, O114, O128, and O153, were isolated in Bangladesh, Latin America, Spain, and South Africa. Two strains produced heat-labile enterotoxin in addition to ST. CFA/I-ST plasmids were mobilized from two O128 strains into E. coli K-12 with the R factor R1-19K-. Like the prototype CFA/I-ST plasmid NTP113, mobilized previously from an E. coli O78 strain into K-12, these two plasmids were non-autotransferring. All 10 CFA/I-ST plasmids were incompatible with NTP113 and had molecular weights ranging from 59 X 10(6) to 72 X 10(6). The molecular properties of seven of these plasmids were compared with those of six CFA/I-ST plasmids previously mobilized from O78 strains from Ethiopia, South Africa, and Bangladesh and with those of one plasmid coding for CFA/I, ST and heat-labile enterotoxin from a South African strain of serogroup O63. Digestion with the restriction endonuclease HindIII showed that several plasmids had very similar fragment patterns and two were identical. Generally, a larger proportion of HindIII fragments were of common size in digests of plasmids identified in strains from related geographical areas, regardless of serogroup. However, all except one plasmid shared five or six HindIII fragments of the same size, one of which had been shown previously to be involved in CFA/I production. There was at least 90% DNA homology between CFA/I-ST plasmids with a molecular weight of about 58 X 10(6) from O78 strains from different sources. Most of the DNA sequences of these plasmids were present in a larger CFA/I-ST plasmid (72 X 10(6) from an O128 strain. The results of genetic and molecular studies suggest that CFA/I and ST production is determined by very similar plasmids in different serogroups of human enterotoxigenic E. coli from several sources.     

Detection of colonization factor antigen I-positive enterotoxigenic Escherichia coli with a cloned polynucleotide probe.

We compared a new colony hybridization assay with an established enzyme-linked immunosorbent assay for detection of enterotoxigenic Escherichia coli (ETEC) expressing colonization factor antigen I (CFA/I). The tests were applied to 135 human ETEC strains. Of these isolates, 30 had previously been characterized for CFAs. A strain harboring the plasmid vector of the polynucleotide gene probe, nine non-ETEC strains from healthy infants, and eight ETEC strains of animal origin were included for further evaluation of probe specificity. The two assays showed a high level of concordance in the specific detection of ETEC strains expressing CFA/I. A total of 24 strains tested positive in the CFA/I hybridization assay, while 23 of those strains were positive in the CFA/I enzyme-linked immunosorbent assay. The single discrepant result could be explained by the loss of a regulatory gene. The strain harboring the plasmid vector of the probe, the non-ETEC E. coli strains, and the ETEC strains of animal origin were all negative in the CFA/I probe assay.     

Purification and characterization of the CFA/I antigen of enterotoxigenic Escherichia coli.

The fimbral colonization factor antigen CFA/I of enterotoxigenic Escherichia coli was purified and characterized. The initial purification step was release of these fimbriae from the bacterial cells by homogenization with a Waring blender. Common fimbriae and flagellar antigen were avoided by careful control of growth conditions and the use of a nonmotile (H-) mutant of the prototype strain H-10407 (O78:H11). The essential purification steps were membrane filtration (Millipore Corp.), ammonium sulfate fractionation, and negative diethylaminoethyl-Sephadex column chromatography. Yields were approximately 4.0 mg of CFA/I protein per g (wet weight) of bacteria. Purified CFA/I is a fimbrial molecule 7.0 nm in diameter and has an average molecular weight of 1.6 X 10(6), as determined by sedimentation equilibrium. CFA/I is a polymer of identical subunits of molecular weight 23,800 with an N-terminal valine, 37% hydrophobic amino acid residues, and 11 residues of proline per mol. The purified antigen retains its morphology, antigenicity, and biological activity. Purified antigen retains its morphology, antigenicity, and biological activity. Purified CFA/I exhibits mannose-resistant hemagglutination of human group A, bovine, and chicken erythrocytes, as do CFA/I-positive bacteria. This was demonstrated by sensitizing latex microbeads with the purified antigen since cell-free CFA/I fimbriae do not hemagglutinate erythrocytes. Thus, CFA/I detached from the bacteria are monovalent; however, purified CFA/I antigen retains an affinity for the epithelial cells of rabbit small intestine and blocks adhesion of CFA/I-positive bacteria. These results demonstrate that purified CFA/I is a good candidate for use in an oral vaccine for immunoprotection against diarrhea caused by CFA/I-positive enterotoxigenic E. coli.     

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.     

Glucose up-regulates expression of the differentiation-associated brush border binding site for enterotoxigenic Escherichia coli colonization factor antigen I in cultured human enterocyte-like cells.

The association of enterotoxigenic Escherichia coli expressing colonization factor antigen I (CFA/I) with the cultured human colon adenocarcinoma cell, a model of the mature enterocyte of the small intestine, is dependent on the binding of CFA/I to a brush border-associated component. Binding of the purified radiolabeled [125I]CFA/I- and 14C-labeled CFA/I-positive bacteria could be displaced by an increasing concentration of unlabeled CFA/I. Moreover, we showed that expression of the specific CFA/I binding developed as a function of cell differentiation in Caco-2 cells, whereas expression of the nonspecific binding did not. Expression of the brush border differentiation-associated component acting as a binding site for CFA/I was up-regulated by glucose. Indeed, the enterocyte-like HT-29 glc- cell subpopulation not expressing the CFA/I binding site when cultured in dialyzed serum and hexose-free medium regained the ability to bind CFA/I when the cells were returned to culture medium containing glucose. Furthermore, expression of the brush border-associated CFA/I binding site in the enterocyte-like Caco-2 cells was repressed when the cells were cultured in hexose-free conditions.     

Immune responses to novel Escherichia coli and Salmonella typhimurium vectors that express colonization factor antigen I (CFA/I) of enterotoxigenic E. coli in the absence of the CFA/I positive regulator cfaR.

An asd-stabilized plasmid carrying enterotoxigenic Escherichia coli cfaABCE genes was constructed and called pJGX15C-asd+. Expression of colonization factor antigen I (CFA/I) by this plasmid occurs independently of the cfaABCE positive regulator cfaR in attenuated Salmonella delta aro delta asd strain H683 and nonpathogenic laboratory E. coli asd strain chi 6212. Oral immunization of mice with nonpathogenic E. coli chi 6212 (pJGX15C-asd+) does not elicit significant serum or mucosal responses against CFA/I. In contrast, oral immunization with a single dose of attenuated S. typhimurium H683(pJGX15C-asd+) elicits a 10(5)-fold increase in CFA/I-specific serum immunoglobulin G and significant elevation of CFA/I-specific immunoglobulin A-secreting B cells in the lamina propria, mesenteric lymph nodes, and spleen. Thus, only the Salmonella-CFA/I construct effectively delivered CFA/I to the inductive sites of the gut-associated and systemic lymphoid tissues.     

Adhesion of enterotoxigenic Escherichia coli to human small intestinal enterocytes.

An improved enterocyte adhesion assay has been used to examine a collection of 44 strains of enterotoxigenic Escherichia coli (ETEC) for their ability to adhere to the brush border of isolated human duodenal enterocytes. Fourteen strains showed good adhesion; in each case the ability to adhere correlated with the production of colonization factor antigen I or II (CFA/I or CFA/II) fimbriae. CFA/II-positive producing coli surface antigens 1 and 3 (CS1 and CS3), coli surface antigens 2 and 3 (CS2 and CS3), and only coli surface antigen 3 (CS3) each showed good adhesion. CS3-mediated brush border attachment of CFA/II-positive ETEC was demonstrated by electron microscopy with monospecific antibody and an immunogold labeling technique. One CFA/I-positive ETEC strain was nonadherent in the assay, as were ETEC producing type 1 somatic fimbriae. Five animal ETEC strains producing K88, K99, F41, and 987P fimbriae were slightly more adhesive than control strains, but adhesion was significantly less than that of CFA-positive ETEC. Twenty five human ETEC strains that lacked CFA/I and CFA/II were nonadherent, suggesting either that the surface antigens responsible for adhesion to human intestinal mucosa in these strains were not being produced or that mucosal receptors for these strains are present in regions of the small intestine other than the duodenum.     

Combined vaccine regimen based on parenteral priming with a DNA vaccine and administration of an oral booster consisting of a recombinant Salmonella enterica serovar Typhimurium vaccine strain for immunization against infection with human-derived enterotoxigenic Escherichia coli strains

Repeated evidence has demonstrated that combined primer-booster immunization regimens can improve both secreted and humoral immune responses to antigens derived from viral, bacterial, and parasitic pathogens. For the present work, we evaluated the synergic serum immunoglobulin G (IgG) and fecal IgA antibody responses elicited in BALB/c mice who were intramuscularly primed with a DNA vaccine, pRECFA, followed by oral boosting with an attenuated Salmonella enterica serovar Typhimurium vaccine (HG3) strain, with both vaccines encoding the structural subunit (CfaB) of the CFA/I fimbriae produced by human-derived enterotoxigenic Escherichia coli (ETEC) strains. The immunological properties of the vaccine regimen were evaluated according to the order of the administered vaccines, the nature of the oral antigen carrier, the age of the vaccinated animals, the interval between the priming and boosting doses, and the amount of injected DNA. The production of gamma interferon and the IgG2a subclass in serum indicated that mice immunized with the primer-booster regimen developed prevailing type 1 T-cell-dependent immune responses. The synergic effect of the vaccine regimen on the induced antibody responses was also revealed by its ability to block the adhesive properties of CFA/I fimbriae expressed by live bacteria, as shown by the inhibition of Caco-2 cell and human erythrocyte binding. Moreover, DBA2 newborn mice were protected from lethal challenges with a CFA/I+ ETEC strain after the incubation of live bacteria with serum samples harvested from mice who were subjected to the primer-booster regimen. We propose, therefore, that the DNA primer-Salmonella booster regimen represents an alternative for the development of vaccines requiring both mucosal and systemic antibody responses for immunological protection.     

Detection and characterization of colonization factor of enterotoxigenic Escherichia coli isolated from adults with diarrhea.

The fimbriate colonization factor antigen (CEA) of Escherichia coli strain H-1047 was isolated and used to prepare anti-CFA antiserum. Enterotoxigenic E. coli (ETEC) isolated from 29 adults with diarrhea acquired in Mexico were examined for CFA by using this serum. Retrospectively, it was found that ETEC possessing the H-10407-type CFA were isolated from 25 (86%) of these diarrhea cases as compared with 2 of 11 (18%) from asymptomatic controls from whom ETEC had been isolated. CFA was found onE. coli of various serotypes, as demonstrated by bacterial agglutination by the anti-CFA serum. Heat treating the cells at 65 degress C for 1 h prevented the agglutination. CFA-positive strains did not react with anti-CFA serum when the cultures were grown at a low incubation temperature (18 degrees C). E. coli isolates identified serologically as CFA positive were shown to adhere to the intestinal villous surfaces of infant rabbits. By the indirect immunofluorescence technique, it was found that adhesion occurred preferentially in the upper 20 cm of the small intestine. Also, the ability or inability of various isolates to adhere to intestinal mucosa in vivo correlated with the presence or absence of fimbriae on the cells when grown in vitro. Agglutinability with anti-CFA serum, fimbriae, and adhesiveness were spontaneously lost by many isolates after laboratory passage in a manner previously described with E. coli H-10407. These observations suggest that the H-10407-type CFA plays a role in the virulence of ETEC possessing this antigen.     

CS22, a novel human enterotoxigenic Escherichia coli adhesin, is related to CS15

Enterotoxigenic Escherichia coli (ETEC) expresses a broad spectrum of O:H antigens. Serogroup O20 is one of the most prevalent among the ETEC strains lacking any of the defined colonization factors (CFs), in Argentina. An O20:H- strain, ARG-3, adhered to Caco-2 cells and exhibited a thermoregulated 15.7-kDa protein band upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). An antiserum against this protein inhibited ARG-3 adhesion to Caco-2 cells and bound to very thin fibrilla-like structures on the bacterial surface. A 15.7-kDa protein-defective mutant failed to adhere to Caco-2 cells and lacked immunogold-labeled surface structures. The N-terminal amino acid sequence of the structural subunit showed 95% homology to that of CS15 of ETEC (former antigen 8786) and 65% homology with fimbria SEF14 of Salmonella enterica serovar Enteritidis. Nevertheless, the molecular size of ARG-3 adhesin was different from that of CS15, as revealed by SDS-PAGE and mass spectrometry. Both proteins are immunologically related, yet not identical, since an antiserum against the 15.7-kDa protein reacted solely with ARG-3 after absorption with bacteria bearing CS15. Moreover, only under low stringency conditions could DNA from strain ARG-3 be amplified by PCR using primers derived from the nfaA sequence of CS15. Thus, from the DNA sequence obtained from the ARG-3 PCR product, it could be deduced that the subunit protein differed in 30 residues from that of CS15. ARG-3 adhesin was found in 60% of the O20:H- CF-negative ETEC strains from Argentina; however, it appeared restricted to this serotype. We propose the designation CS22 for the herein identified nonfimbrial adhesin of human ETEC.     

Ultrastructural study of adhesion of enterotoxigenic Escherichia coli to erythrocytes and human intestinal epithelial cells

The adhesion to erythrocytes and human intestinal epithelial cells of enterotoxigenic Escherichia coli strains H10407, B2C, and H10407P, expressing colonization factor antigen I (CFA/I), CFA/II, and type 1 fimbriae, respectively, was examined by electron microscopy. CFA and type 1 fimbriae were visualized by negative staining in thin sections after en bloc staining with ruthenium red and by immune labeling with antisera raised against purified fimbriae. By negative and ruthenium red staining, CFA/I, CFA/II, and type 1 fimbriae were indistinguishable and appeared as approximately 7-nm-diameter hollow cylindrical structures up to 1.5 micron in length; strain B2C also produced 2- to 3-nm-diameter flexible fibrillar fimbriae. Bacteria producing CFA/I, CFA/II, and type 1 fimbriae adhered to and agglutinated human, bovine, and guinea pig erythrocytes, respectively; CFA/I and CFA/II also mediated attachment of bacteria to the brush border of isolated human duodenal enterocytes. Electron microscopy of agglutinated erythrocytes and enterocytes with adherent bacteria showed, in each case, that bacterial adhesion involved the formation of many interactions between the tips of fimbriae and receptors on the erythrocyte or enterocyte brush border membrane. Immune labeling allowed different fimbrial antigens mediating bacterial attachment to human enterocytes to be identified.     

Identification of a cross-reactive continuous B-cell epitope in enterotoxigenic Escherichia coli colonization factor antigen I.

Enterotoxigenic Escherichia coli (ETEC) colonizes the intestine by means of several antigenically distinct colonization factors (CFs). Several of these CFs have very significant amino acid sequence similarity or identity, particularly in the N-terminal end. We have previously shown that a monoclonal antibody (MAb) raised against the subunits of colonization factor antigen I (CFA/I) fimbriae, which reacts with a peptide corresponding to the 25 N-terminal amino acids of such subunits, can inhibit attachment to intestinal cells of ETEC expressing heterologous as well as homologous CFs, with related amino acid sequences. In this study we have, by means of Pepscan analysis, determined the sequence of the MAb-specific linear epitope to be 15IDLLQ19. Parenteral immunization of rabbits with an N-terminal 25-mer synthetic peptide of CFA/I fimbrial subunit, either covalently coupled to bovine serum albumin or uncoupled, induced high titers of specific antibodies against this peptide as well as against CFA/I fimbriae. Increased titers against several heterologous CF fimbriae with a related N-terminal sequence were also induced, whereas no increase was seen against fimbriae with an unrelated sequence. Neither antisera against the coupled peptide nor antisera against the uncoupled peptide inhibited binding of CF-expressing bacteria to the human intestinal cell line Caco-2 in spite of high titers. The difference in the inhibitory capabilities of the antipeptide sera and the MAb might be due to slightly different epitope specificities. Thus, whereas the antipeptide sera bound to several continuous epitopes in the N-terminal end, none of them reacted specifically with the epitope 15IDLLQ19.     

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.     

Loss of CFA/I expression by enteropathogenic E. coli following exposure to antibody.

H-10407 (078, H11) an enteropathogenic strain of Escherichia coli carries a fimbriate plasmid-mediated adherence antigen on the cell surface (CFA/I) which facilitates colonization of human small intestine. This shows strong similarities to the K88 antigen of porcine enteropathogenic E. coli. K88 expression may be suppressed by antibody both in vivo and in vitro. Expression of CFA/I, detected by agglutination of human erythrocytes was progressively lost during in vitro culture with antisera containing antibodies specific for CFA/I but, unlike K88, CFA/I was re-expressed during further culture in the absence of antibody. Antibody to CFA/I seemed to exert a switching effect on expression of the adherence antigen.