New fimbrial antigen F165 from Escherichia coli serogroup O115 strains isolated from piglets with diarrhea

Sixteen strains of Escherichia coli serogroup O115 isolated from piglets with diarrhea were examined for mannose-sensitive or mannose-resistant hemagglutination (MSHA or MRHA, respectively) for the presence of fimbriae by electron microscopy and for enterotoxigenicity by the ligated gut loop technique in 10-day-old piglets. Four strains demonstrated MRHA of sheep, goat, pig, dog, cat, chicken, and human erythrocytes but no MRHA of calf, horse, guinea pig, and rabbit erythrocytes. They were divided into pattern I (MSHA negative) and pattern II (MSHA positive). The remaining 12 strains were classified as pattern III (MRHA negative, MSHA positive) and pattern IV (hemagglutination negative). An antiserum produced against the MRHA-positive, MSHA-negative strain 4787 and absorbed by the same strain grown at 15 degrees C agglutinated all of the MRHA-positive strains but none of the MRHA-negative strains and completely inhibited the MRHA of these strains. The surface antigen against which this absorbed antiserum was directed was designated "F165." Fimbriae (pili) purified from strain 4787 hemagglutinated erythrocytes in the same mannose-resistant pattern as the strain itself and reacted with the anti-F165 antiserum in an enzyme-linked immunosorbent assay, thus demonstrating the fimbrial nature of the hemagglutinating F165 antigen. The F165 antigen showed no serological relationship with the fimbrial antigens F4, F5, F6, and "F41". A positive correlation between the presence of F165 and the lack of enterotoxigenicity was demonstrated. Thus, we found a new mannose-resistant, hemagglutinating fimbrial antigen, F165, which is produced only by nonenterotoxigenic strains of E. coli serogroup O115. The possible role of F165 as a virulence attribute of E. coli strains causing extraintestinal disease is discussed.     

Identification of a new fimbrial structure in enterotoxigenic Escherichia coli (ETEC) serotype O148:H28 which adheres to human intestinal mucosa: a potentially new human ETEC colonization factor.

Three important fimbrial colonization factor antigens (CFAs) designated CFA/I, CFA/II, and E8775 were identified originally in some human enterotoxigenic Escherichia coli (ETEC) strains because of their mannose-resistant hemagglutination properties. To identify CFA, in strains lacking mannose-resistant hemagglutination properties we exploited the ability of human ETEC strains to adhere to human proximal small intestinal mucosa. ETEC strain B7A (O148:H28) was selected for study because it belongs to an epidemiologically important serotype and does not produce a known CFA, and yet it is known to be pathogenic and cause diarrheal disease in human volunteers. Results of an human enterocyte adhesion assay indicated that some bacteria in cultures of B7A produced adhesive factors. To select for such bacteria, cultured human duodenal mucosal biopsy samples were infected with B7A for up to 12 h, after which time a large percentage of the mucosal surface became colonized by bacteria. A new fimbrial structure morphologically distinct from CFA/I, CFA/II, and E8775 fimbriae and consisting of curly fibrils (approximately 3 nm in diameter) was readily identified when bacteria were subcultured from the mucosa and examined by electron microscopy. Identical fimbriae were produced by ETEC strain 1782-77 of the same serotype. Identification of these fimbriae only on bacteria subcultured from human intestinal mucosa strongly suggests that they promote mucosal adhesion of ETEC serotype O148:H28 and thus represent a potentially new human ETEC CFA.     

Hemagglutination by Escherichia coli in septicemia and urinary tract infections.

The agglutination of erythrocytes from various animal species by Escherichia coli was studied. The 405 strains of E. coli were isolated from urine in patients with urinary tract infections, from blood in septicemic patients, or from feces in persons without intestinal or urinary disorders. In urinary tract infections, d-mannose-resistant agglutination (MRHA) of human erythrocytes was the most common finding (23% of the strains). The highest frequency of mannose-sensitive hemagglutination (MSHA) attributed to type I (common type) pili occurred with guinea pig erythrocytes (11.5%). Of the 78 E. coli strains isolated from blood cultures, 11 (14%) produced MRHA of human erythrocytes and only one gave MSHA. In the stool cultures, only 1 of 170 E. coli strains was MSHA reacting, whereas 28 strains (16.5%) showed MRHA of human erythrocytes. No MRHA strain reacted with antiserum against colonization factor antigen (CFA)/I of pilus nature in enterotoxigenic human E. coli strains (O78:H12). MRHA of bovine erythrocytes, reputedly typical of enterotoxigenic E. coli of serogroups O6 and O8, was shown by only two strains, neither of which agglutinated with CFA/II antiserum. The most common hemagglutinating pattern of E. coli from urine and blood thus was MRHA for human erythrocytes. This agglutination may have been caused by pili or other surface properties of one or more serotypes. These may represent a new class of colonization-promoting antigens (adhesins).     

Colonization factor antigen CFA/IV (PCF8775) of human enterotoxigenic Escherichia coli: nucleotide sequence of the CS5 determinant.

Human enterotoxigenic Escherichia coli isolates expressing the colonization factor antigen CFA/IV (previously designated PCF8775) produce plasmid-encoded CS5 fimbriae. The nucleotide sequence of the region encoding the major CS5 fimbrial subunit was determined. The subunit is synthesized as a precursor of 203 amino acids (20.85 kDa) with a mature protein of 181 amino acids corresponding to a size of 18.6 kDa. The CS5 subunit shows homology to the corresponding component of porcine enterotoxigenic E. coli F41, particularly within the signal sequence and at the carboxy terminus.     

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.     

Prevalence of the enterotoxigenic Escherichia coli colonization factors CFA/I, CFA/II and E8775 isolated in the South Pacific (New Caledonia, Republic of Vanuatu and Wallis and Futuna)

We tested the expression of adherence properties of enterotoxigenic Escherichia coli (ETEC) strains isolated in New-Caledonia, Vanuatu and Wallis and Futuna by examining for the presence of colonization factor E8775 using an agglutination test and an immuno-diffusion technique with specific antisera. Approximately 19% of ETEC strains possessed CFA/I and 21% a CFA/II. The E8775 antigen was found on 1.8% of the strains. This last factor was found on strains of the serogroup 025 from Vanuatu. Two strains 078 usually CFA/I+ possessed a CFA/II and three strains of the serogroup 0126 possessed a CFA/I. The results of this study emphasis the need to continue the search for other mechanisms of adhesion used by ETEC strains without any of the three factors of colonization.     

Hemagglutination activity and colonization factor antigens I and II in enterotoxigenic and non-enterotoxigenic strains of Escherichia coli isolated from humans.

We examined 205 enterotoxigenic strains of Escherichia coli for colonization factor antigens (CFA) I and II, using an immunodiffusion technique with specific antisera. A total of 36 strains of serogroups O63, O78, O114, O128, and O153 and 1 rough strain possessed CFA/I and gave a single precipitin line; 47 strains of serogroups O6, O8, O80, and O115 possessed CFA/II. The latter strains gave a major precipitin line (component 3) when tested with specific antisera prepared against strain E1392 or PB-176 (both E. coli O6.H16; biotype A). However, all 16 strains of E. coli O6.H16 belonging to biotype A gave a second precipitin line (component 1) when tested with both antisera. When CFA/II-positive strains were tested with a specific antiserum prepared against E. coli O6.H16 strains of biotype B or C, all strains gave component 3, but 16 of 17 strains of E. coli O6.H16 belonging to biotype B, C, or F gave a second precipitin line (component 2) not given by strains of biotype A. CFA/II-positive strains of serogroups other than O6 gave only component 3 in tests with all specific antisera. Nine enterotoxigenic strains of serotypes O7, O15, O25, O115, and O128 gave mannose-resistant hemagglutination of human or calf erythrocytes but lacked CFA/I or CFA/II. Although mannose-resistant hemagglutination was common in non-enterotoxigenic strains of E. coli, none of the non-enterotoxigenic strains possessed CFA/I or CFA/II; these strains included fecal strains of serogroups O6, O8, O63, and O78, fecal strains of enteropathogenic serogroups, and strains from extraintestinal sources.     

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.     

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.     

Comparison of methods for detection of colonization factor antigens on enterotoxigenic Escherichia coli.

Fecal Escherichia coli isolates from 196 patients with watery diarrhea and 68 healthy individuals (controls) were analyzed in Bangladesh immediately after isolation for the presence of colonization factor antigen (CFA) I or II (CFA/I or CFA/II, respectively) by a mannose-resistant hemagglutination (MRHA) test with six species of erythrocytes and by a slide agglutination test with absorbed CFA/I or CFA/II antisera. The presence of CFAs was confirmed by immunodiffusion analyses done in Sweden. By these methods, it was found that 49 of 69 enterotoxin-producing E. coli strains isolated from patients carried CFA/I or CFA/II, whereas none of the nonenterotoxigenic E. coli isolates or the three toxin-positive strains isolated from healthy individuals carried these adhesins. All E. coli strains retained their MRHA ability after transportation to Sweden followed by one subculture and after storage at -70 degrees C (but not at room temperature) for 1 to 2 years without further subculturing. After 5 to 10 subcultures of the fresh isolates, however, 70% of the initially CFA/I- and 80% of the initially CFA/II-carrying strains analyzed did not hemagglutinate. The efficacy of different methods for detecting CFAs on the fresh isolates was compared with that of immunodiffusion. The sensitivity of MRHA with human blood group A erythrocytes for the detection of CFA/I was high (97%), but the specificity was only 69%. The sensitivity of MRHA with bovine erythrocytes for the detection of CFA/II in Bangladesh was very low but increased considerably when chicken erythrocytes were also used. Whereas both false-positive and false-negative reactions were obtained when absorbed CFA antisera were used for agglutination, antisera against purified CFAs were equally effective as immunodiffusion in identifying CFA/I and CFA/II-carrying strains.     

Interaction of Escherichia coli with Different Fimbriae and Polymorphonuclear Leukocytes

The effects of Escherichia coli strains with various fimbriae on bacteria-polymorphonuclear leukocyte (PMN) interactions were studied. Strains of E. coli were cultivated at 37 degrees C to express and at 18 degrees C to suppress the formation of fimbriae. The presence of fimbriae was confirmed by electron microscopic studies and hemagglutination and salt aggregation tests. Fimbriated E. coli strains were more readily PMN associated than the nonfimbriated strains in the absence of opsonins, confirming the results of previous studies. However, the PMN chemiluminescence (CL) induced by the various strains in the absence of serum opsonins depended on the type of fimbriae they expressed. Strains with type 1 fimbriae expressing mannose-sensitive hemagglutination induced 5 to 15 times more CL than the same strains grown at 18 degrees C, i.e., not expressing this type of fimbriae. For strains showing mannose-resistant hemagglutination, the differences between fimbriated and nonfimbriated variants of the same strains grown at 37 and 18 degrees C, respectively, were less pronounced. Analysis of enterotoxigenic strains expressing colonization factor antigen I (CFA/I) fimbriae showed that these induced only 25 to 33% of the CL induced by the same E. coli strains not expressing CFA/I, whereas enterotoxigenic strains expressing CFA/II fimbriae induced 100 to 200% of the CL induced by the nonfimbriated variants. Although less CL was induced by bacteria with CFA/I fimbriae than by nonfimbriated variants, this situation was reversed when the microorganisms were opsonized. Thus, CFA/I fimbriae, while enhancing adhesion to cells, induce less activation of PMN-killing mechanisms in a serum-free environment. These findings may be relevant for the virulence in certain body sites, since CFA/I fimbriae, while facilitating adhesiveness, may protect the bacteria from PMN killing. Our findings indicate that PMN interactions with fimbriated E. coli in the host defense may be complex. Certain fimbriae may indeed be advantageous to the bacteria, enabling them to interact with PMNs without activating the bactericidal oxidative metabolism.     

Enterotoxigenic Escherichia coli strains belonging to a new serogroup, Escherichia coli O166.

Thirty-two strains of Escherichia coli belonging to a new O group, O166, were examined. Twenty-one strains had the flagella antigen H27, five had the H15 antigen, five had the H7 antigen, and one was nonmotile. All the H27 strains and the nonmotile strain produced heat-stable enterotoxin but not heat-labile enterotoxin. All the H7 strains produced heat-labile enterotoxin but not heat-stable enterotoxin. The remaining strains were nonenterotoxigenic. None of the strains possessed colonization factor antigens CFA/I, CFA/II, or PCF8775.     

Experimental enterotoxin-induced Escherichia coli diarrhea and protection induced by previous infection with bacteria of the same adhesin or enterotoxin type.

The diarrheal response to an initial and a second infection with Escherichia coli expressing various enterotoxins (the heat-stable toxin [ST] alone or in combination with the heat-labile toxin [LT]) and colonization factor antigens (CFA/I, CFA/II, or E8775-type) was studied in the reversible tie adult rabbit diarrhea model. An initial infection with high doses (1 X 10(10) to 5 X 10(11) bacteria) of the various strains regularly induced diarrhea which was usually self-limiting (only 7 of 85 animals died). The diarrheal response to equally effective doses of different strains producing both ST and LT (ST/LT) did not differ significantly with serotype or colonization factor antigen. ST/LT-producing strains appeared to induce severe disease more regularly than ST-producing strains carrying the same adhesin. Previous infection with CFA/I-carrying, ST/LT-producing E. coli protected all animals reinfected with an otherwise highly diarrheogenic dose of the same strain as well as against challenge with a CFA/I-carrying, ST/LT-producing strain with different O-, K-, and H-antigens. Fecal excretion of bacteria was also significantly reduced in the protected animals, although not completely eliminated. When only one of the two antigens, CFA/I and LT, was shared by the immunizing and rechallenge strains, partial protection was evident consistent with independent antibacterial (anti-CFA) and antitoxic (anti-LT) immune mechanisms. Oral immunization with purified CFA/I significantly reduced fluid secretion in intestinal loops infected with CFA/I-carrying enterotoxigenic bacteria.     

Adhesive fimbriae associated with porcine enterotoxigenic Escherichia coli of the O141 serotype.

Enterotoxigenic Escherichia coli of the O141 serotype, isolated from piglets with postweaning coliform enteritis but producing none of the characterized adhesive fimbriae, was examined for fimbrial production by transmission electron microscopy. Two strains that produced numerous fimbriae were chosen for further characterization. The fimbriae were isolated and purified and had a subunit molecular weight of 17,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Using antiserum raised against this protein, we have shown it to be specific for the 17,000-molecular-weight band by immunoblotting and to be directed against the fimbriae by immunoelectron microscopy. These fimbriae were not produced when the bacteria were grown at 18 degrees C and did not show any mannose-resistant hemagglutination of the erythrocytes tested. We propose that these are a new type of adhesive fimbriae associated with porcine enterotoxigenic E. coli of the O141 serotype.     

Enteroadhesion fimbriae and enterotoxin of Escherichia coli: genetic transfer to a streptomycin-resistant mutant of the galE oral-route live-vaccine Salmonella typhi Ty21a.

An enterotoxigenic Escherichia coli plasmid encoding colonization factor antigen (CFA) I fimbriae and heat-stable toxin was transferred into a streptomycin-resistant mutant of the Salmonella typhi galE strain Ty21a (a live attenuated oral typhoid vaccine). The virulence plasmid-carrying transconjugants produced CFA I fimbriae and heat-stable toxin. The marked production of CFA I fimbriae was observed even in a vaccine medium for Ty21a. The data lead to a new type of potential live oral vaccine, S. typhi Ty21a producing enteroadhesion fimbriae.     

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.     

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.     

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.     

Adhesion and ultrastructural properties of human enterotoxigenic Escherichia coli producing colonization factor antigens III and IV.

Human enterotoxigenic Escherichia coli (ETEC) producing colonization factor antigen III (CFA/III) and coli surface antigens 4, 5, and 6 (CS4, CS5, and CS6) of CFA/IV were examined ultrastructurally and for ability to adhere to human small intestinal enterocytes and to cultured human intestinal mucosa. Strains of serotypes O25:H-, O25:H42, and O167:H5 producing CFA/III plus CS6, CS4 plus CS6, and CS5 plus CS6, respectively, showed good adhesion to human enterocytes (1.8 to 4.2 bacteria per brush border) and cultured human intestinal mucosa, whereas variants lacking these antigens or producing only CS6 were nonadherent (0 to 0.03 bacterium per brush border). By electron microscopy, CFA/III, CS4, and CS5 appeared as morphologically distinct rodlike fimbriae: CFA/III was 7 to 8 nm in diameter, CS4 was 6 to 7 nm in diameter, and CS5 was 5 to 6 nm in diameter. CS5 was unusual in that it appeared to be composed of two fine fibrils arranged in a double-helical structure. CS6 was difficult to characterize morphologically but possibly has a very fine fibrillar structure. By specific fimbrial staining and immunoelectron microscopy. CS4 and CS5 were shown to promote mucosal adhesion of ETEC; a similar adhesion role for the CS6 antigen could not be confirmed. ETEC strains of serotypes O27:H7, O27:H20, O148:H28, and O159:H20 which produced CS6 showed good adhesion to human enterocytes (1.6 to 3.0 bacteria per brush border), whereas variants which lacked CS6 were nonadherent (0 to 0.01 bacterium per brush border). These strains, however, also produced fimbrial or fibrillar surface antigens, in addition to CS6, which probably represent additional coli surface antigens responsible for the observed adhesive properties of these ETEC serotypes.     

Iron represses the expression of CFA/I fimbriae of enterotoxigenic E. coli.

Experiments were designed to study the effect of iron on the expression of CFA/I fimbriae by enterotoxigenic E. coli (ETEC). Addition of 0.05 mM ferrous sulfate to growth media decreased CFA/I antigen and fimbrial production by the CFA/I-positive ETEC strain H-10407 as measured by quantitative ELISA and hemagglutination assay. The repressive effect was reversed by the addition of the iron chelators, sodium citrate or dipyridyl. With a CFA/I subunit gene promoter-lacZ fusion, it was found that the activity of the subunit gene promoter was significantly higher in the presence of iron chelators than in medium containing iron in the fur+ strain DHB24. This difference was not observed in the fur mutant strain SBC24, suggesting that the global E. coli metalloregulatory protein Fur (ferric uptake regulation) is involved in the repression. The repressor may bind to the promoter of the CFA/I subunit gene since several potential Fur-binding sites were identified in the promoter area.