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.     

Attachment pili from enterotoxigenic Escherichia coli pathogenic for humans.

Pili from enterotoxigenic Escherichia coli pathogenic for humans have been isolated by adsorption to the surface of erythrocytes followed by thermal elution. The pili are composed of two protein subunits with molecular weights of 13,100 and 12,500 as determined by sodium dodecyl sulfate-gel electrophoresis. These pili also bind to human buccal cells under temperature conditions (37 degrees C) which prevent the binding of these pili to the erythrocytes. Analogous temperature effects on binding have previously been observed with whole bacterial cells. This binding can be inhibited by antiserum prepared against the isolated pili.     

肠毒素性大肠杆菌CS3菌毛呈现载体的构建

目的 构建大肠杆菌ＣＳ３菌毛呈现载体,实现外源表位在细菌表面的呈现。方法 通过对ＣＳ３亚基蛋白二级结构、抗原表位、亲水性及柔韧性的预测分析,确定外源表位的插入位点,重叠延伸ＰＣＲ方法进行定点突变,将口蹄疫病毒ＶＰＩ插入到ＣＳ３中以验证表现呈现能力;用重组菌腹腔注射免疫小鼠以探讨其抗原性。结果 在大肠杆菌ＣＳ３的１３６位氨基酸残在后突变插入ＢａｍＨⅠ酶切点构建成呈现载体,全细胞ＥＬＩＳＡ、电镜和免疫     

Serotypes of attachment pili of enterotoxigenic Escherichia coli isolated from humans.

Pili from enterotoxigenic Escherichia coli isolated from humans have been partially purified, and antisera have been prepared. These pili were initially attached to erythrocytes and then removed by thermal elution for purification. Three distinct antigenic types of pili have been identified. Antisera against these three pili types reacted with 60 of 106 (56%) enterotoxigenic E. coli isolated from humans but not with nontoxigenic, normal human fecal isolates of E. coli nor with enterotoxigenic E. coli strains isolated from animals. There was no correlation between pili serogroup and any of the following toxin production (heat labile, heat stable, or both), O antigenic type, geographical source of isolation, or mannose-resistant hemagglutination patterns of various erythrocyte types.     

Characterization of the CsfC and CsfD proteins involved in the biogenesis of CS5 pili from enterotoxigenic Escherichia coli

The region required for biosynthesis of CS5 pili consists of six csf genes, with csfA encoding the major subunit. In this study, we describe the characterization of two of the genes constituting the region, csfC and csfD, but also identify the true morphology of the CS5 pilus by high resolution electron microscopy. CsfD was shown to be essential in the initiation of CS5 pilus biogenesis, did not possess any chaperone-like activity for the major subunit, and was an integral minor component of the pilus structure. Studies on CsfD translocation across the outer membrane in Escherichia coli K-12 using a csfA mutant also showed that CsfD is likely to be the first pilin subunit assembled. A specific in-frame deletion in the csfC gene resulted in the complete absence of cell surface CS5 pili and prevented the translocation of CsfA and CsfD pilins across the outer membrane. Specific cell localization studies showed an accumulation of CsfC in the outer membranes of E. coli K-12, while complementation experiments with homologous outer membrane assembly genes from CS1 and CFA/I pili systems were unable to restore assembly of CS5 pili. The CS5 pilus was shown to be a 2 nm flexible fibrillar structure, which adopted a predominantly open helical conformation under the electron microscope.     

Monoclonal antibodies against colonization factor antigen I pili from enterotoxigenic Escherichia coli.

Hybridomas secreting monoclonal antibodies directed against intact colonization factor antigen I pili have been produced by the fusion of spleen cells from immunized BALB/c mice with NS1/SP2 myeloma cells. The four monoclones with the highest antibody titer, as detected by enzyme-linked immunosorbant assay (ELISA), were chosen for antibody amplification by production of mouse ascitic fluid. These four were examined for antibody specificity by ELISA and immunoblot assays, using six different pilus types. Three of the four monoclonal isolates were specific for only colonization factor antigen I pili in both assays, whereas the remaining isolate showed a distinct cross-reactivity with K99 pili in the ELISA assay but not in immunoblot analysis. These results indicate that this monoclone may be recognizing a common structural element between the two adhesive pilus types.     

Adhesion of piliated Escherichia coli strains to phagocytes: differences between bacteria with mannose-sensitive pili and those with mannose-resistant pili.

Escherichia coli with mannose-resistant (MR) pili, in contrast to those with mannose-sensitive (MS) pili, did not adhere to rat peritoneal macrophages and human polymorphonuclear granulocytes, as measured by use of radioactive bacteria and by the chemiluminescence response induced by the cell contact. With some MS-piliated E. coli strains, unpiliated bacteria, obtained by growth at a pilus-restrictive temperature, did show MS adherence to phagocytes, presumably by virtue of bacterial cell wall adhesins which, like MS pili, recognize alpha-mannose-containing structures of the phagocyte membrane. Possible roles of MR pili, MS pili, and MS cell wall adhesins in the unspecific cellular host defense are discussed.     

Immunization of suckling pigs against enteric enterotoxigenic Escherichia coli infection by vaccinating dams with purified pili.

Pregnant swine (gilts) were vaccinated parenterally with a suspension of purified pili from the porcine enterotoxigenic Escherichia coli strain 987 (09:K103::NM). Gilts injected with placebo served as controls. Suckling pigs born to gilts in both groups were challenged intragastrically with virulent strain 987. The percentage of deaths, incidence and duration of diarrhea, numbers of E. coli in the ilea, and E. coli attachment to the villous epithelia were significantly less in suckling pigs of vaccinated gilts than in those of controls. These results are consistent with the hypothesis that pili of some enterotoxigenic E. coli facilitate adhesion to intestinal epithelia. Vaccination of dams with pili appears to be a means of immunizing against diarrheal disease caused by enterotoxigenic E. coli in suckling neonates. This work confirms the role of somatic pili as colonization and virulence factors and provides another example of safe and effective purified pilus vaccines.     

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.     

Colonization factor antigens I and II and type 1 somatic pili in enterotoxigenic Escherichia coli: relation to enterotoxin type.

Enterotoxigenic Escherichia coli (ETEC) isolates from 36 persons with acute traveler's diarrhea from whom no other pathogens were recovered were tested (after no more than three subcultures) for the presence of colonization factor antigens I and II (CFA/I and CFA/II) and type 1 somatic pili. CFA/I or CFA/II was identified in 7 of 10 strains with heat-labile and heat-stable enterotoxins (LT+/ST+), but in only 2 of 12 LT-/ST+ (P less than 0.05) and 0 of 14 LT+/ST- (P less than 0.02) strains. CFA pili were not found among 74 non-enterotoxigenic E. coli strains. Type 1 somatic pili were demonstrable in 42% of the 36 ETEC and in 49% of the 74 non-enterotoxigenic E. coli isolates. The nine ETEC isolates bearing a CFA were serially subcultured on 10 consecutive days and retested for CFA and toxin. After five subcultures only one strain had lost a CFA, but after 10 passages three strains were negative: two lost CFA/I and one lost CFA/II. The strain that lost CFA/II became negative for both LT and ST as well and was found to lack a 48- and a 60-megadalton plasmid. The two strains that lost CFA/I also became negative for ST, but plasmid analysis revealed no plasmid loss. Disappearance of the CFA/I phenotype without loss of a plasmid can be explained by phase variation, as exhibited by type 1 somatic pili, or by rearrangement of base sequences in the CFA/I plasmid genome. If purified pili vaccines are to provide broad-spectrum protection against ETEC diarrhea, the search must be intensified to identify the antigens responsible for adhesion to intestinal mucosa in the many ETEC strains that lack CFA/I and CFA/II.     

Characterization of new hydrophobic pili of human enterotoxigenic Escherichia coli: a possible new colonization factor.

Enterotoxigenic Escherichia coli strains were divided into five groups on the basis of their bacterial surface hydrophobicity (Honda et al., FEMS Microbiol. Lett. 17:273-276, 1983). Strains in group III showed heat-stable high hydrophobicity, although they did not show mannose-resistant hemagglutination with either human or bovine erythrocytes. E. coli strain 260-1 in group III was characterized. Electron microscopic examination revealed the presence of pili on the surface of this strain, but not on that of strain 260-1a, which is a mutant of 260-1 showing low hydrophobicity. When strain 260-1 was grown at 18 degrees C, it did not produce pili or show high hydrophobicity. On homogenization of strain 260-1 grown at 37 degrees C the high hydrophobicity and the pili on its surface were lost. These results indicate that the pili of strain 260-1 are associated with the hydrophobicity. Strain 260-1 pili were purified to homogeneity by successive column chromatographies on Sepharose 4B and phenyl-Sepharose CL-4B. Their molecular weight was estimated to be about 18,000. An antigenic difference between purified pili of strain 260-1 and colonization factor antigens I and II was demonstrated. The colonization ability of E. coli 260-1 was shown by animal experiments on suckling mice and infant rabbits. From these results it is concluded that the pili of strains in group III of human enterotoxigenic E. coli, which may play a role in colonization, are of a new type.     

Rapid screening method for enterotoxigenic Escherichia coli.

A rapid screening method for detection of Escherichia coli producing heat-labile enterotoxin is described. Single colonies are transferred directly from primary culture plates into 96-well microculture plates containing 0.3 ml of brain heart infusion broth in each well. After 24 h at 37 degrees C, each brain heart infusion broth culture is assayed by the miniculture method in the corresponding well of a microculture plate in which Y-1 mouse adrenal tumor cells have been grown. All enterotoxigenic isolates detected by this method were confirmed in the assay but with culture supernatants.     

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.     

Quantitative method for enumeration of enterotoxigenic Escherichia coli.

A rapid method was developed to quantify toxigenic Escherichia coli, using a membrane filter procedure. After filtration of samples, the membrane filter was first incubated on a medium selective for E. coli (24 h, 44 degrees C) and then transferred to tryptic soy agar (3%; 6 h, 37 degrees C). To assay for labile toxin-producing colonies, the filter was then transferred to a monolayer of Y-1 cells, the E. coli colonies were marked on the bottom of the petri dish, and the filter was removed after 15 min. The monolayer was observed for a positive rounding effect after a 15- to 24-h incubation. The method has an upper limit of detecting 30 toxigenic colonies per plate and can detect as few as one toxigenic colony per plate. A preliminary screening for these enterotoxigenic strains in polluted waters and known positive fecal samples was performed, and positive results were obtained with fecal samples only.     

Type 1 pili (F1) of porcine enterotoxigenic Escherichia coli: vaccine trial and tests for production in the small intestine during disease.

This study was performed to determine whether the F1 (type 1) pili of a porcine strain of enterotoxigenic Escherichia coli are protective antigens and whether they are produced in the pig small intestine during disease caused by an enterotoxigenic E. coli. Reciprocal cross-absorption experiments with antisera prepared against F1 pili purified from enterotoxigenic E. coli 431 (O101:K30,99:H-:F1) and P14 (O149:K91,88ac:8H+:F1) demonstrated that the F1 antigens of the two strains were closely related or identical. Pregnant swine vaccinated with a vaccine prepared from strain P14 (F1+) responded with a significant increase in antibody against F1 in their serum and colostrum. However, the vaccinated dams did not significantly protect their suckling pigs against fatal challenge with strain 431. There was no evidence of F1 pilus production in the strain 431-infected pigs, as determined by immunofluorescent staining of ileal sections, direct electron microscopic examination of bacteria from ilea, and titration of serum agglutinins in convalescent pigs. It was concluded that strain 431 did not produce F1 in the small intestine during disease and that F1 was not a protective antigen in this system.     

Comparison of receptors for 987P pili of enterotoxigenic Escherichia coli in the small intestines of neonatal and older pig.

Enterotoxigenic Escherichia coli isolates that express 987P pili colonize the small intestine and cause diarrhea in neonatal (less than 6-day-old) but not in older (greater than 3-week-old) pigs. However, 987P+ E. coli isolates adhere in vitro to small-intestinal epithelial cells from pigs of both ages. This indicates that older pigs as well as neonatal pigs contain receptors for 987P pili and that resistance in older pigs is not due to a lack of intestinal receptors for 987P pili. In this study, we demonstrated that 3-week-old gnotobiotic pigs, like neonatal pigs, were colonized and developed diarrhea when challenged with 987P+ E. coli. We compared 987P receptors in small-intestinal epithelial cell brush borders and in intestinal washes (luminal contents) from less than 1-day-old, 3-week-old gnotobiotic, and 3- to 4-week-old weaned pigs. Samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and blotted onto nitrocellulose filters, and 987P binding was demonstrated by immunoassay using purified 987P pili. Multiple 987P-binding components ranging from 33 to 40 kDa were found in brush borders from both 987P-susceptible (neonatal and gnotobiotic) and 987P-resistant (older) pigs: 987P binding to these receptors, which we called 987R, did not correlate with 987P susceptibility. A less than 17-kDa 987P receptor, 987M, was found in the mucus fraction of intestinal washes from 987P-resistant older pigs. Only trace amounts of 987M were detected in 987P-susceptible neonatal and gnotobiotic pigs. 987M comigrated with the 987P receptor previously isolated from adult rabbits. Receptors for 987P in the mucus of older pigs may inhibit 987P-mediated intestinal colonization by preventing the attachment of 987P+ enterotoxigenic E. coli to intestinal epithelial receptors for 987P.     

Mucosal and systemic immune responses in patients with diarrhea due to CS6-expressing enterotoxigenic Escherichia coli

Colonization factor CS6 expressed by enterotoxigenic Escherichia coli (ETEC) is a nonfimbrial polymeric protein. A substantial proportion of ETEC strains isolated from patients in endemic settings and in people who travel to regions where ETEC is endemic are ETEC strains expressing CS6, either alone or in combination with fimbrial colonization factor CS5 or CS4. However, relatively little is known about the natural immune responses elicited against CS6 expressed by ETEC strains causing disease. We studied patients who were hospitalized with diarrhea (n = 46) caused by CS6-expressing ETEC (ETEC expressing CS6 or CS5 plus CS6) and had a disease spectrum ranging from severe dehydration (27%) to moderate or mild dehydration (73%). Using recombinant CS6 antigen, we found that more than 90% of the patients had mucosal immune responses to CS6 expressed as immunoglobulin (IgA) antibody-secreting cells (ASC) or antibody in lymphocyte supernatant (ALS) and that about 57% responded with CS6-specific IgA antibodies in feces. More than 80% of the patients showed IgA seroconversion to CS6. Significant increases in the levels of anti-CS6 antibodies of the IgG isotype were also observed in assays for ASC (75%), ALS (100%), and serum (70%). These studies demonstrated that patients hospitalized with the noninvasive enteric pathogen CS6-expressing ETEC responded with both mucosal and systemic antibodies against CS6. Studies are needed to determine if the anti-CS6 responses protect against reinfection and if protective levels of CS6 immunity are induced by vaccination.     

Test for enterotoxigenic Escherichia coli using Y-1 adrenal cells in miniculture.

A rapid, potentially clinically useful test for detection of enterotoxigenic Escherichia coli is described. Whole bacterial cultures of enterotoxigenic E. coli, when briefly exposed to Y1 adrenal cells in tissue miniculture, effect a rounding response in the tissue culture that can be discerned at 18 to 24 h. The tissue culture technique agreed with the rabbit ileal loop in all 58 enterotoxigenic and 52 non-enterotoxigenic E. coli strains tested.     

Use of monoclonal antibodies specific for the a determinant of K88 pili for detection of enterotoxigenic Escherichia coli in pigs.

Monoclonal antibodies directed against the a determinant of K88 pili from porcine enterotoxigenic Escherichia coli which react with all three K88 variants have been produced. These antibodies have been used for diagnosis of porcine enterotoxigenic E. coli in a direct enzyme-linked immunosorbent assay with sensitivity to 50 ng of pilus protein per ml.