A sialoglycoprotein complex linked to the microvillus cytoskeleton acts as a receptor for pilus (AF/R1) mediated adhesion of enteropathogenic Escherichia coli (RDEC-1) in rabbit small intestine.

Escherichia coli strain RDEC-1 is an enteroadherent, diarrheagenic pathogen in rabbits that utilizes AF/R1 pili for initial (stage 1) adherence, but the host receptors for this adhesion are unknown. Here we demonstrate that RDEC-1 binds, via AF/R1 pili, to a specific rabbit ileal microvillus membrane glycoprotein receptor complex of subunits 130 and 140 kD. The binding involves sialic acid present on oligosaccharide moieties of the glycoprotein receptor. Furthermore, the microvillus membrane glycoprotein receptor complex appears to be associated with cytoskeletal components via brush border myosin 1. This newly described link between AF/R1 receptor and cytoskeletal components suggests that, in addition to this function in mucosal adherence, the pili may facilitate subsequent (second stage) close effacing attachment of RDEC-1 to the host epithelium by influencing cytoskeletal function.

Characterization of the eaeA gene from rabbit enteropathogenic Escherichia coli strain RDEC-1 and comparison to other eaeA genes from bacteria that cause attaching-effacing lesions.

A number of enteric pathogens, including enteropathogenic (EPEC) and enterohemorrhagic (EHEC) Escherichia coli, Hafnia alvei, a strain of Citrobacter freundii, and rabbit EPEC strain RDEC-1 cause attaching-effacing (AE) lesions in the gut mucosa. These bacteria have a pathogenicity cassette (locus of enterocyte effacement or LEE) containing the eaeA gene. This gene encodes intimin, an outer membrane protein required for production of AE lesions. RDEC-1, a non-invasive enteropathogen in young rabbits, produces AE lesions morphologically indistinguishable from lesions caused by human AE bacterial strains. The RDEC-1 example of E. coli diarrhea in rabbits is an important model for studying the pathogenesis of AE bacteria in a natural infection and for analyzing specific roles of the components of LEE. In order to better understand the role of intimin in the development of AE lesions, a portion of DNA within RDEC-1 LEE, containing the eaeA gene and an upstream open reading frame (ORF), was sequenced. The RDEC-1 eaeA gene shared 87%, 92%, and 93% DNA sequence identity and > 80% amino acid sequence identity with the eaeA genes of C. freundii biotype 4280, EHEC O157:H7, and EPEC O127:116, respectively. The carboxy-terminal 280 amino acid residues of intimin has 80%, 56%, and 54% identity with C. freundii, EHEC O157:H7, and EPEC O127:H6 intimins, respectively. The predicted protein encoded by the upstream ORF (156 amino acids) shares 95%, 97%, and 99% amino acid identity with predicted proteins from C. freundii. EHEC O157:H7, and EPEC O127:H6, respectively. The high degree of sequence homology of the ORF and the eueA gene of RDEC-1 with those of other AE bacteria suggests an evolutionary relationship of LEE and supports and facilitates the use of the RDEC-1 model for studying the role of LEE in pathogenesis.

The CS6 colonization factor of human enterotoxigenic Escherichia coli contains two heterologous major subunits.

The genes encoding the CS6 colonization factor were cloned from two human enterotoxigenic Escherichia coli strains of different serotypes. The DNA sequences from both clones were nearly identical and contained four open reading frames. Two of them have homology to genes encoding molecular chaperones and ushers found in many other operons encoding colonization factors. The two remaining open reading frames encode two heterologous major subunit proteins which makes CS6 unique because other colonization factors have only one major subunit. Upstream and downstream of the CS6 operon the DNA sequences of the clones diverged abruptly.

Assessment of attenuated Salmonella typhimurium strains designed as potential carriers of foreign antigens for mucosal localization and immuno-genicity in a rabbit model.

Neither wild type nor attenuated S. typhimurium strains induced diarrheal illness in rabbits. All strains localized to the Peyer's patch at higher concentrations than in lumenal contents or adjacent ileum. Wild type S. typhimurium C5 induced a typhoid-like illness in rabbits with severe weight loss, bacteremia, persistent splenic colonization, and serum IgG response. Both attenuated strains were disseminated to spleen (day 3) but produced minimal systemic illness. They induced biliary IgA responses greater than the wild type (day 7), but minimal serum IgG responses. Both mutants of S. typhimurium are suitable for further development as live enteric vaccines to carry foreign antigens since they localize to Peyer's patch after oral inoculation, induce biliary antibody, and produce minimal systemic disease. The attenuated strains tested are systemically disseminated. It remains to be determined whether dissemination (determined by a large virulence plasmid) is necessary for the desired mucosal immune response or acceptable for an oral vaccine strain.

Cloning of the RDEC-1 locus of enterocyte effacement (LEE) and functional analysis of the phenotype on HEp-2 cells.

EPEC Escherichia coli are an important cause of epidemic diarrhea in infants. The disease is characterized by attaching and effacing (A/E) lesions where the bacteria attach intimately to the enterocyte surface resulting in localized destruction of microvilli. A 35-kb chromosomal locus termed LEE (locus of enterocyte effacement) in an EPEC strain (E2348/69) has recently been found and is thought to contain all the necessary genes for A/E lesions. RDEC-1 is a strain of E. coli that causes diarrhea in rabbits by a similar mechanism and serves as a model for human EPEC disease. We report 1) the cloning of the RDEC-1 LEE, 2) show that the RDEC-1 LEE is similar in size to the LEE in E2348/69, 3) the RDEC-1 LEE possesses all four regions of the E2348/69 LEE, 4) there are restriction site polymorphisms between the RDEC-1 LEE and that of E2348/69, and 5) the RDEC-1 LEE clone is functionally similar to E2348/69 in its fluorescent actin staining test and suggests that the LEE may be sufficient for the production of A/E lesions by these strains.

Protection against Shiga toxin-producing Escherichia coli infection by transcutaneous immunization with Shiga toxin subunit B.

Enterohemorrhagic Escherichia coli (EHEC) strains are important human food-borne pathogens. EHEC strains elaborate potent Shiga toxins (Stx1, and/or Stx2) implicated in the development of hemorrhagic colitis (HC) or hemolytic-uremic syndrome (HUS). In this report, we evaluated the immunogenicity and protective efficacy of Stx1 subunit B (StxB1) administered by transcutaneous immunization (TCI). Three groups of Dutch Belted rabbits received patches containing StxB1, StxB1 in combination with Escherichia coli heat-labile enterotoxin (LT), or LT alone. An additional group of naïve rabbits served as controls. The protective efficacy following TCI with StxB1 was assessed by challenging rabbits with a virulent Stx1-producing strain, RDEC-H19A, capable of inducing HC and HUS in rabbits. Antibodies specific to StxB1 from serum and bile samples were determined by enzyme-linked immunosorbent assay and toxin neutralization test. Rabbits immunized with StxB1 demonstrated improved weight gain and reduced Stx-induced histopathology. Rabbits receiving StxB or StxB1/LT showed a significant increase in serum immunoglobulin G titers specific to StxB1 as well as toxin neutralization titers. These data demonstrated that the StxB delivered by TCI could induce significant systemic immune responses. Thus, Stx subunit B vaccine delivered by a patch for a high-risk population may be a practical approach to prevent (and/or reduce) Stx-induced pathology.

LEE-encoded regulator (Ler) mutants elicit serotype-specific protection, but not cross protection, against attaching and effacing E. coli strains.

We previously showed that single dose orogastric immunization with an attenuated regulatory Lee-encoded regulator (ler) mutant of the rabbit enteropathogenic Escherichia coli (REPEC) strain E22 (O103:H2) protected rabbits from fatal infection with the highly virulent parent strain. In the current study we assessed the degree of homologous (serotype-specific) and heterologous (cross-serotype) protection induced by immunization with REPEC ler mutant strains of differing serotypes, or with a prototype strain RDEC-1 (O15:H-) which expresses a full array of ler up-regulated proteins. We constructed an additional ler mutant using RDEC-1 thus, permitting immunization with a ler mutant of either serotype, O15 or O103, followed by challenge with a virulent REPEC strain of the same or different serotypes. Consistent with our previous data, the current study demonstrated that rabbits immunized with a RDEC-1 ler mutant were protected from challenge with virulent RDEC-H19A (RDEC-1 transduced with Shiga toxin-producing phage H19A) of the same serotype. Rabbits immunized with RDEC-1 or E22 derivative ler mutants demonstrated significant increase in serum antibody titers to the respective whole bacterial cells expressing O antigen but not to the LEE-encoded proteins. However, immunization with the ler mutants of either E22 or RDEC-1 failed to protect rabbits from infections with virulent organisms belonging to different serotypes. In contrast, rabbits immunized with the prototype RDEC-1 were cross protected against challenge with the heterologous E22 strain as shown by normal weight gain, and the absence of clinical signs of disease or characteristic attaching and effacing (A/E) lesions. Immunization with RDEC-1 induced significantly elevated serum IgG titers to LEE-encoded proteins. We thus, demonstrated homologous protection induced by the REPEC ler mutants and heterologous protection by RDEC-1. The observed correlation between elevated immune responses to the LEE-encoded proteins and the protection against challenge with heterologous virulent REPEC strain suggests that serotype-non-specific cross protection requires the expression of, and induction of antibody to, LEE-encoded virulence factors.

Cloning of the genes for AF/R1 pili from rabbit enteroadherent Escherichia coli RDEC-1 and DNA sequence of the major structural subunit.

AF/R1 pili on the surface of Escherichia coli RDEC-1 promote attachment of the bacteria to rabbit intestinal brush borders. In order to characterize AF/R1 pili and manipulate their expression, we cloned the genes necessary for AF/R1 expression; determined the size of proteins produced in minicells; located the gene encoding the major structural subunit, named AfrA; and determined the DNA sequence of afrA as well as the sequence of 700 additional nucleotides upstream of afrA. Two contiguous EcoRI fragments spanning 7.9 kilobases were cloned from the 86-megadalton plasmid of RDEC-1 into vector pUC19 to make plasmid pW1. Bacteria carrying pW1 produced AF/R1 pili that were recognized by AF/R1-specific antiserum and promoted adherence of bacteria to brush borders prepared from rabbit intestine. Proteins with a molecular weight of 17,000 (17K proteins), which was the size of AfrA, as well as 15K, 15.5K, 26K, 28K, and 80K proteins were detected in minicells carrying pW1. The gene afrA was located by using an oligonucleotide probe, and its DNA sequence was determined. The DNA sequence of 700 additional nucleotides upstream was determined because this sequence may be important in the regulation of AF/R1 expression.

Rabbit mucosal receptors for an enteropathogenic Escherichia coli strain: appearance of bacterial receptor activity at weaning.

Adherence studies using the rabbit enteropathogenic Escherichia coli strain RDEC-1 indicate that this strain attaches in a species-specific manner to receptors located on its host intestinal epithelial cells. Because intestinal epithelial cells undergo marked developmental changes within the first few weeks of life, we designed a study to determine whether the presence of bacterial receptors on rabbit brush borders changed during this time. The adherence of RDEC-1 to rabbit brush borders isolated from rabbits aged 2-35 days and from adult rabbits was examined. No adherence of RDEC-1 was detected to rabbit brush borders isolated from rabbits 15 days or younger. Receptors for RDEC-1 were first detected on rabbit brush borders from 21-day-old rabbits and by 35 days, RDEC-1 receptor activity on rabbit brush borders had reached adult levels. Piliated, enteroadherent, human Escherichia coli pathogens did not adhere to rabbit brush borders of any age group indicating that nonspecific adherence did not occur.

Mucosal immune responses to intestinal bacterial pathogens.

Current advances in the study of gut mucosal immunology and molecular biology have enhanced our ability to understand the pathogenesis of enteric bacterial infections as well as the role of the immune system in mediating both tissue injury and protection. In this article, we review the immunopathogenesis and the protective immune response to three enteric pathogens, Vibrio cholerae, Shigella, and Salmonella. Each of these pathogens has a distinctive mechanism by which it causes disease, ie, epithelial attachment, epithelial invasion, and epithelial invasion with systemic dissemination. Pathogenicity and immune response can be conceptualized in terms of the interaction of these enteric pathogens with the gut epithelial compartment, immune inductive sites (Peyer's patch of the small intestine and lymphoid follicles of the colon), and a common immune effector compartment in the laimina propria where protective antibody is secreted. V cholerae, the representative noninvasive pathogen, has fimbrial adhesins that mediate attachment and colonization of the luminal surface of epithelial cells where organisms secrete cholera toxin (CT), a potent enterotoxin that induces a voluminous diarrhea via adenylate cyclase-dependent chloride secretion. Protective immunity is based on secretory (s) immunoglobulin A directed against whole-cell components that prevent attachment to gut epithelial cells and is enhanced by CT, an immunogen with potent adjuvant activity. Shigella, an enteric pathogen that locally invades gut epithelium, subverts the usual mechanism of immune sampling by initially invading via M cells overlying inductive sites. Subsequent macrophage invasion induces apoptosis and the release of interleukin-1, a proinflammatory cytokine. This seems to be a critical initiating event in immune-mediated tissue injury. Protective immunity is serotype specific. Infection caused by Salmonella is characterized by mucosal invasion and systemic spread mediated by the organisms ability to survive within macrophages. Both antibody and cell-mediated immunity are important for protection against Salmonella.

Enterohemorrhagic Escherichia coli: a family of emerging pathogens.

Enterohemorrhagic Escherichia coli (EHEC) have emerged over the last decade as important enteric pathogens because of their potential to induce both hemorrhagic colitis and fatal hemolytic uremic syndrome (HUS). HUS following EHEC colitis has become the leading cause of pediatric renal failure requiring kidney transplant in North America. The ability for EHEC to induce disease is dependent upon their ability to adhere to the intestinal mucosa in an intimate fashion, and to produce potent cytotoxins. These virulence factors (toxin production and enteroadherence) have been implicated in the pathogenesis of EHEC-induced disease. In this review we will discuss the symptomatology, epidemiology, laboratory diagnosis, pathogenesis, complications, treatment, and prevention of EHEC disease. We will review HUS with emphasis on treatment and prevention. Finally we will review animal models for EHEC infection in order to discuss their role in developing new strategies for the treatment and prevention of EHEC-associated diseases.

Adherence of an enterotoxigenic Escherichia coli strain, serotype O78:H11, to purified human intestinal brush borders.

The human enterotoxigenic Escherichia coli pathogen designated H10407 expresses two different types of surface pili, one designated type 1 pili and the other designated colonization factor antigen I (CFA/I), CFA/I pili are thought to promote the adherence of H10407 to the mucosa of the human small bowel. H10407 was grown under conditions which promoted the expression of either type 1 pili or CFA/I pili, and in each case, the adherence of H10407 to purified human intestinal brush borders was quantitated. The adherence assays revealed that H10407 adhered to human brush borders only when it expressed CFA/I pili. It appears that in vitro adherence of H10407 to human intestinal epithelial cells is dependent on the expression of CFA/I.

Pilus-mediated interactions of the Escherichia coli strain RDEC-1 with mucosal glycoproteins in the small intestine of rabbits.

Escherichia coli strain RDEC-1 (serotype 015:NM) is an effacing adherent enteropathogen that binds to the intestine of rabbits in a manner morphologically identical to the binding of human enteropathogenic E. coli strains to human intestine. The rabbit enteropathogen adheres to mucosal enterocytes in vivo and to microvillus membranes in vitro. Binding of RDEC-1 to ileal brush borders and to M cells overlying Peyer's patches is mediated by pili (fimbriae) expressed on the cell surface of bacteria. To examine whether similar binding occurs to glycoproteins present in the intestinal lumen, RDEC-1 was fed to rabbits and the intestinal luminal contents were examined for in vivo colonization by RDEC-1. In addition, preparations of rabbit luminal glycoproteins were tested for their ability to agglutinate RDEC-1 in vitro. After the infection of rabbits, RDEC-1 organisms were found in the intestinal lumen associated with glycoproteins, as defined by positive histochemical staining of luminal material by periodic acid-Schiff and Alcian blue. In vitro aggregation of RDEC-1 by luminal glycoproteins of rabbit intestine, a luminal glycoprotein fraction purified on column chromatography and rabbit ileal microvillus membranes, was determined using a microtiter plate assay and an aggregometer. Nonpiliated RDEC-1 did not interact with either of the intestinal glycoprotein preparations or microvillus membranes. RDEC-1 expressing mannose-resistant AF/R1 pili or mannose-sensitive type 1 pili coaggregated with both rabbit luminal glycoprotein preparations and rabbit ileal microvillus membranes at equivalent protein concentrations. These studies demonstrate that RDEC-1 are associated with luminal glycoproteins during in vivo infection of rabbits, and that piliated RDEC-1, but not nonpiliated, coaggregate with rabbit glycoprotein preparations in vitro. Luminal glycoproteins contained within the mucus layer may serve as a site for replication and colonization of bacteria before bacterial enteroadherence.

Regional differences in attachment of enteroadherent Escherichia coli strain RDEC-1 to rabbit intestine: luminal colonization but lack of mucosal adherence in jejunal self-filling blind loops.

RDEC-1 (serotype 015:NM) is an enteropathogenic Escherichia coli that adheres to mucosal epithelial cells in the ileum, cecum, and colon, but not in the jejunum, of rabbits during diarrheal illness. As RDEC-1 can adhere to jejunal brush borders in vitro, rapid distal clearing of the enteropathogen from the upper gastrointestinal tract may explain the absence of in vivo adherence of RDEC-1 to rabbit jejunum. To test this hypothesis, self-filling blind loops (SFBL) were created in the jejunum of rabbits that were then inoculated with RDEC-1 to determine if stasis would promote increased luminal colonization and increased mucosal attachment of RDEC-1 in the proximal small intestine. Eight rabbits with SFBL and seven nonoperated littermates were inoculated with RDEC-1 4 weeks after surgery. Semiquantitative estimation of fecal shedding of RDEC-1 and the presence and severity of diarrhea were monitored for 7 days. Rabbits were then killed, and colonization of jejunum and ileum by anaerobic bacteria and luminal colonization of jejunum, ileum, and cecum by RDEC-1 was quantitated. Mucosal adherence of RDEC-1 to jejunum, ileum, and cecum was determined by Giemsa staining of intestinal sections obtained from rabbits with and without SFBL. In addition, brush border membranes (BBM) were prepared from blind loop jejunum, nonoperated jejunum, and ileum. In vitro adherence of RDEC-1 to brush borders was quantitated under phase contrast microscopy. Maximal fecal shedding of RDEC-1 began 3-4 days after infection in all rabbits, but diarrhea was more severe in rabbits with SFBL.(ABSTRACT TRUNCATED AT 250 WORDS)

Functional heterogeneity of intestinal Escherichia coli strains expressing type 1 somatic pili (fimbriae): assessment of bacterial adherence to intestinal membranes and surface hydrophobicity.

Although the role of host-specific, nonmannose-sensitive pilus adhesins in the intestinal adherence of pathogenic Escherichia coli is well established, a similar role for mannose-sensitive type 1 or common pili is less clear, since these structures can be expressed by most E. coli, even nonpathogens. We first examined whether type 1 pili, expressed by the rabbit-effacing, adherent, enteropathogenic E. coli strain RDEC-1, mediated interactions with intestinal membranes of several species and compared these interactions with those mediated by the nonmannose-sensitive adhesin of RDEC-1. We next grew a series of E. coli intestinal strains in static broth to promote type 1 pilus expression and determined whether E. coli expressing type 1 pili differed in their affinity for intestinal membranes (as measured by phase-contrast microscopy and aggregometry), hydrophobic surface properties, net negative surface charge (as measured by hydrophobic interaction chromatography and salt aggregation), and hemagglutination patterns. In contrast to the species-specific attachment to rabbit brush borders of RDEC-1 expressing its nonmannose-sensitive adhesin, type 1 pili on RDEC-1 mediated mannose-sensitive attachment to intestinal membranes of all four species tested. Expression of type 1 pili on other E. coli strains resulted in varying degrees of nonspecies-specific, mannose-sensitive attachment to intestinal membranes. This attachment correlated with increasing surface hydrophobicity rather than with hemagglutination patterns. These results indicate that various E. coli strains expressing type 1 pili are functionally heterogeneous and suggest that some type 1 pili might contribute to in vivo enteroadherence.

Quantitation of the adherence of an enteropathogenic Escherichia coli to isolated rabbit intestinal brush borders.

Two assays were developed to quantitate the adherence of an Escherichia coli strain (RDEC-1) known to colonize the mucosal surface of the small intestine of rabbits to brush borders isolated from rabbit intestinal epithelial cells. In the first assay, the mean adherence per rabbit brush border was determined by counting the number of organisms adhering to each of 40 brush borders under phase microscopy. The mean adherence of RDEC-1 (11.5 +/- 0.7 per rabbit brush border) was significantly greater than adherence of two nonpathogenic strains: HS (2.7 +/- 0.4 per rabbit brush border) and 640 (0.8 +/- 0.1 per rabbit brush border). A similar distinction between the adherence of RDEC-1 and the control (nonadherent) organisms could be made more rapidly by determining the percentage of the total number of brush borders which had 10 or more adherent organisms; this second assay was used to define the optimum conditions for adherence. Maximum adherence was seen within 15 min. Adherence was temperature dependent, with adherence after 1 min at 37 degrees C being fourfold greater than that at 4 degrees C. The pH optimum for adherence was between 6.5 and 7.0, and adherence was abolished below pH 5.0. With the first, more sensitive assay, the effect of electrolytes and a number of hexoses and hexosamines on adherence was analyzed. RDEC-1 adherence was inhibited at high ionic strengths; however, adherence was not influenced at moderately high concentrations (20 mg/ml) by either d-mannose or l-fucose, in contrast to the case for other reported enteric pathogens. These two quantitative in vitro assays for adherence produce consistent results and have been used to partially characterize the adherence of RDEC-1 to rabbit brush borders.