Strain RDEC-1 Research Articles

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.

Towards a vaccine for attaching/effacing Escherichia coli: a LEE encoded regulator ( ler) mutant of rabbit enteropathogenic Escherichia coli is attenuated, immunogenic, and protects rabbits from lethal challenge with the wild-type virulent strain

The ler (LEE encoded regulator) gene product is a central regulator for the genes encoded on the locus of enterocyte effacement (LEE) pathogenicity island of attaching/effacing (A/E) pathogens, including human enteropathogenic E. coli (EPEC) and enterohemorrhagic E. coli (EHEC) as well as animal isolates. Although an in vivo role for Ler in bacterial virulence has not been documented, we hypothesized that a Ler deletion mutant should be attenuated for virulence but might retain immunogenicity. The goals of this study were to genetically characterize ler of a rabbit EPEC (rEPEC) strain (O103:H2), to examine the effect of ler on in vivo virulence, and to determine if intragastric inoculation of an attenuated rEPEC ler mutant was immunogenic and could protect rabbits against subsequent challenge with the wild-type virulent parent strain. The predicted ler gene product of rEPEC strain O103:H2 shares high homology (over 95% amino acid identity) with the Lers of another rEPEC strain RDEC-1 (O15:H-) and human EPEC and EHEC. A defined internal ler deletion mutant of rEPEC O103:H2 showed reduced production of secreted proteins. Although orogastric inoculation of rabbits with the virulent parent O103:H2 strain induced severe diarrhea, significant weight loss and early mortality with adherent mucosal bacteria found at sacrifice, the isogeneic ler mutant strain was well tolerated. Animals gained weight and showed no clinical signs of disease. Examination of histological sections of intestinal segments revealed the absence of mucosal bacterial adherence. This result demonstrates an essential role for Ler in in vivo pathogenicity of A/E E. coli. Single dose orogastric immunization with the rEPEC ler mutant induced serum IgG antibody to whole bacteria (but not to intimin). Immunized animals were protected against enteric infection with the WT virulent parent strain exhibiting normal weight gain, absence of diarrhea and absence of mucosally adherent bacteria at sacrifice. Such attenuated ler mutant strains may have potential for use as oral vaccines, or as vaccine vectors for delivery of foreign antigens. It remains to be determined whether such regulatory mutants can protect against infection with A/E bacteria of differing serotypes affecting different hosts.

Enteropathogenic Escherichia coli Strain RDEC-1 Produces a Novel Electrogenic Factor Active on Rabbit Ileum In Vitro

Attaching and effacing Escherichia coli demonstrate marked species specificity in inducing diarrhea, although its mechanism remains largely unclear. The purpose of this study was to investigate the existence of a soluble, species-specific factor that induces diarrhea in an in vitro model. Stripped rabbit ileum was mounted in Ussing chambers, and changes in potential difference and short-circuit current were monitored after the addition of bacterial culture supernatant. The culture supernatant from rabbit-specific strain RDEC-1, but not from human-specific enteropathogenic Escherichia coli strain E2348/69, induced an increase in potential difference and short-circuit current in rabbit ileum mounted in Ussing chambers. This electrical signal was related to chloride ion secretion, was absent in colonic tissue, and was retained in the 30 to 100-KDa fraction of the supernatant. Preliminary experiments failed to show an involvement of calcium or cyclic nucleotides as intracellular messengers. RDEC-1 cured of a 42-MDa plasmid lost the enterotoxicity whereas conjugation of the plasmid into the negative E. coli recipient HB101 resulted in the expression of toxicity. The authors describe a novel, species-specific factor that helps to explain RDEC-1 diarrhea, which may be relevant to the pathogenesis of enteropathogenic Escherichia coli infection.

Escherichia coli strain RDEC-1 AF/R1 endogenous fimbrial glycoconjugate receptor molecules in rabbit small intestine.

Escherichia coli strain RDEC-1 causes a diarrheagenic infection in rabbits with AF/R1 fimbriae, which have been identified as an important colonization factor in RDEC-1 adherence leading to disease. The AF/R1-mediated RDEC-1 adherence model has been used as a model systems for E. coli diarrheal diseases. In this study, RDEC-1 adhered specifically to small intestinal brush borders, with both sialic acid and beta-galactosyl residues apparently involved. The AF/R1-mediated adherence activity of [(14)C]-labeled RDEC-1 was analyzed quantitatively by using 24-well plates coated with purified brush borders and purified microvilli. Two microvillus membrane proteins (130 and 140 kDa) were individually isolated, and chicken antibody raised to each protein inhibited bacterial adherence. These same two proteins, previously shown to be recognized by AF/R1, were individually digested with trypsin, and the amino acid sequences of peptides were determined by reversed-phase capillary liquid chromatography-mass spectrometry tandem mass spectrometry (LC-MS). This LC-MS analysis indicated that these proteins are subunits of the rabbit sucrase-isomaltase protein (SI) complex. Guinea pig serum raised to purified rabbit SI complex inhibited bacterial adherence to microvilli. Additionally, as determined by high-performance thin-layer chromatography and autoradiography, RDEC-1 adhered selectively, via AF/R1 fimbriae, to a glycolipid tentatively identified as galactosylceramide (Gal beta 1-1Cer) in the lipid extract of rabbit small intestinal brush borders. RDEC-1 adherence to Gal beta 1-1Cer was partially inhibited in the presence of galactose. These combined results indicate that the endogenous receptor molecule for AF/R1 fimbriae of RDEC-1 is each individual component of the SI complex, although binding to glycolipid may be responsible for an additional adherence mechanism.

Proliferative enterocolitis associated with dual infection with enteropathogenic Escherichia coli and Lawsonia intracellularis in rabbits.

Both enteropathogenic Escherichia coli (EPEC) and an obligate intracellular bacterium, previously referred to as an intracellular Campylobacter-like organism and now designated Lawsonia intracellularis, have been reported as causes of enterocolitis in rabbits. An outbreak of enterocolitis in a group of rabbits, characterized by an unusually high rate of mortality, was found to be associated with dual infection with EPEC and L. intracellularis. The EPEC strain was found to have eaeA gene homology but was negative for afrA homology. The absence of the afrA gene, which encodes the structural subunit for the AF/R1 pilus, indicates that this rabbit EPEC strain is distinct from the prototypic RDEC-1 strain. This finding suggests that rabbit EPEC strains widely reported in Western Europe, which lack AF/R1 pili, are also present in rabbits in the United States. Dual infection with these two pathogens in rabbits has not been previously reported and may have contributed to the unusually high mortality observed in this outbreak.

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:H6, 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 eaeA 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.

137 ATTACHING AND EFFACING BACTERIA INDUCE AN INCREASE IN TISSUE PERMEABILITY THAT IS SPECIES-SPECIFIC

Introduction. A three-steps of Enteropathogenie Escherichia coli (EPEC) pathogenicity include a fimbrial-mediated loose adherence to the enterocyte apical membrane (step. 1), followed by intimate adherence with formation of characteristic attaching and affacing (A/E) lesions (step 2). The genetic elements necessary for step 2 are clustered in a 35kb region called Locus for Enterocyte Effacement (LEE). Finally, step 3 involves an intracellular signalling cascade that seems to mediate ion secretion. Other data shows that EPEC decrease tissue resistance and increase transepithelial flux of mannitol, which may be a complementary mechanism of diarrhea. Aim. We investigated whether the decrease in tissue resistance is species-specific and whether intimate adherence is necessary for the resistance drop to occur. Methods. Stripped rabbit ileal mucosa was mounted in Ussing chambers, batheds with Ringer's solution, gassed with 95%O2-5%CO2 and potential difference (PD) and short-cirtcuit current (Isc) were monitored after challenging the tissue with PBS-resuspended cultures of wild type A/E forming bacteria and genetically derived strains. Tissue resistance (Rt) was calculated from PD and Isc according to Ohm's law and its variation (ΔRt) was expressed as percentage of the basal Rt value obtained ca 10 min after mounting the tissue. Results and conclusions. Rabbit specific A/E strain RDEC-1 induced a ΔRt of -17% (±3) while negative control (i.e. no fimbria, no A/E formation) strain DH5α induced a decrease of -7% (±2). Wild type, human specific EPEC strain E2348/69 did not induce a significant Δrt while transforming it with the plasmid (pM5) containing RDEC-1 fimbria gene evokes a ΔRt of -27% (±4). No significant ΔRt were noted transforming pM5 into a negative control strain. Finally, cloning RDEC-1 LEE and pM5, but not the LEE alone, into DH5α induced a ΔRt comparable to wild type RDEC-1. In summary, our data show that the ability of decreasing Rt is dependent on species-specific step 1 adherence. However, fimbrial adherence per se is not sufficient to give this effect, since elements contained in the LEE, most likely involved in A/E lesion formation, are required. Using optical and electron microscopy we are currently looking for a morphologic correlation between A/E lesions and decreased Rt. We are also engineering more selective genetic constructs to find out which gene(s) in the LEE is (are) involved in the process.

Identification of eae sequences in enteropathogenic Escherichia coli strains from rabbits.

DNA sequences coding for attachment and for verotoxin production were investigated in a collection of enteropathogenic Escherichia coli strains from rabbits. All of the strains produced diarrhea after experimental infection, attached to the brush borders of the intestinal lining, and possessed homology to the eae probe, whereas strains isolated from healthy rabbits did not. Sequences homologous to the AF/R1 fimbriae of strain RDEC-1 were not found. One strain reacted with the probe for the Shiga-like toxin type I gene.

Pili in microspheres protect rabbits from diarrhoea induced by E. coli strain RDEC-1

We tested whether pilus proteins of rabbit diarrhoeagenic Escherichia coli (RDEC-1), incorporated into biodegradable microspheres, could function as safe and effective oral immunogens in the rabbit diarrhoea model. The RDEC-1 adhesin, AF/R1, incorporated into poly(D,L-lactide-co-glycolide) microspheres, was administered intraduodenally. Vaccinated and unvaccinated rabbits were challenged with RDEC-1 and killed 1 week later. Vaccination with AF/R1 in microspheres did not cause diarrhoea or weight loss. After challenge, rabbits given AF/R1 in microspheres, in contrast to unvaccinated animals, remained in good health. RDEC-1 attachment to caecal epithelium of vaccinated rabbits was reduced (p = 0.02), whereas numbers of RDEC-1 in intestinal fluids were little affected. Also, in vaccinated animals, biliary anti-AF/R1 IgA levels were increased, and AF/R1-induced blast-cell transformation was vigorous in spleen cell cultures. We conclude that vaccination with AF/R1 in microspheres was safe and protected rabbits against RDEC-1 disease, probably by interfering with adherence of the bacteria to the intestinal mucosa. The interference might have been due to the presence of specific antibodies secreted in bile.

Identification and molecular characterization of a Salmonella typhimurium gene involved in triggering the internalization of salmonellae into cultured epithelial cells.

Penetration of intestinal epithelial cells is an important step in the pathogenesis of Salmonella infections. We have characterized a gene, invE, that is necessary for Salmonella invasion of cultured epithelial cells. The predicted amino acid sequence of InvE showed significant homology to the Yersinia outer membrane protein YopN (LcrE). Strains of Salmonella carrying mutations in invE were unable to penetrate Henle-407 human intestinal cells and Madin-Darby canine kidney cells, although they were fully capable of attaching to the same cells. Unlike wild-type Salmonella typhimurium, invE mutants failed to change the intracellular free calcium levels or the distribution of polymerized actin in cultured epithelial cells; neither did they alter the normal architecture of the microvilli of polarized Madin-Darby canine kidney cells. Wild-type S. typhimurium was able to rescue the invasive phenotype of the invE mutants in simultaneous infections of cultured epithelial cells although it did not rescue the Escherichia coli strain RDEC-1. We hypothesize that invE mutants are deficient in triggering the intracellular events that lead to bacterial internalization.

Effect of colonic inflammation on mucin inhibition of Escherichia coli RDEC-1 binding in vitro

Intestinal mucus protects the underlying epithelium against adhesion and invasion by microbial pathogens and their products. In inflamed colonic mucosa there are both histochemical and biochemical changes in the major organic constituent of mucus, goblet cell-derived mucin. To determine if these changes result in differences in functional properties of mucin, inhibition of adherence of piliated Escherichia coli, strain RDEC-1 (serotype O15:H-), by mucin purified from distal colons of normal rabbits was compared with inhibition by mucin from colons of rabbits with dinitrochlorobenzene-induced colitis. Histology from inflamed colons revealed evidence of chronicity with a chronic inflammatory cell infiltrate, depletion of mucous glands, and branching of crypts. Mucin was purified from crude mucus of distal colonic mucosa by sequential ultracentrifugation and characterized by gel electrophoresis and amino acid analysis. The rabbit enteropathogen RDEC-1 was grown to promote expression of adherence pili. A nonpiliated mutant, strain M34, was used as a negative control. A concentration-dependent inhibition of piliated RDEC-1 binding was shown using mucins derived from both inflamed and noninflamed colons. However, equivalent dry weights of mucin purified from inflamed colons showed less inhibition of bacterial binding (10.3% inhibition ± 5.2%, mean ± SD) compared with mucin from normal colons (47.6% ± 10.8%, P < 0.05). Mucin purified from additional animals with hapten-induced colitis but treated with the oral leukotriene B4-receptor antagonist SC-41930 showed intermediate inhibition of RDEC-1 binding (35.6% ± 14.3%). It is concluded that mucin derived from inflamed distal colon of rabbits failed to inhibit in vitro binding of piliated RDEC-1, and by decreasing mucosal inflammation, this functional alteration of mucin was partially reversed.

Effect of colonic inflammation on mucin inhibition of Escherichia coli RDEC-1 binding in vitro

Intestinal mucus protects the underlying epithelium against adhesion and invasion by microbial pathogens and their products. In inflamed colonic mucosa there are both histochemical and biochemical changes in the major organic constituent of mucus, goblet cell-derived mucin. To determine if these changes result in differences in functional properties of mucin, inhibition of adherence of piliated Escherichia coli, strain RDEC-1 (serotype O15:H-), by mucin purified from distal colons of normal rabbits was compared with inhibition by mucin from colons of rabbits with dinitrochlorobenzene-induced colitis. Histology from inflamed colons revealed evidence of chronicity with a chronic inflammatory cell infiltrate, depletion of mucous glands, and branching of crypts. Mucin was purified from crude mucus of distal colonic mucosa by sequential ultracentrifugation and characterized by gel electrophoresis and amino acid analysis. The rabbit enteropathogen RDEC-1 was grown to promote expression of adherence pili. A nonpiliated mutant, strain M34, was used as a negative control. A concentration-dependent inhibition of piliated RDEC-1 binding was shown using mucins derived from both inflamed and noninflamed colons. However, equivalent dry weights of mucin purified from inflamed colons showed less inhibition of bacterial binding (10.3% inhibition +/- 5.2%; mean +/- SD) compared with mucin from normal colons (47.6% +/- 10.8%; P less than 0.05). Mucin purified from additional animals with hapten-induced colitis but treated with the oral leukotriene B4-receptor antagonist SC-41930 showed intermediate inhibition of RDEC-1 binding (35.6% +/- 14.3%). It is concluded that mucin derived from inflamed distal colon of rabbits failed to inhibit in vitro binding of piliated RDEC-1, and by decreasing mucosal inflammation, this functional alteration of mucin was partially reversed. (Gastroenterology 1992 Apr;102(4 Pt 1):1199-211)

Effect of cow's milk protein absorption on the anaphylactic and systemic immune responses of young rabbits during bacterial diarrhoea.

In vivo and in vitro studies recently showed that intestinal permeability to cow's milk proteins such as beta-lactoglobulin increased transiently in infant rabbits infected by the enteroadherent Escherichia coli strain RDEC-1 at weaning. The consequences of this enhanced permeability for local anaphylactic and/or systemic immune responses were studied in infected and age-matched control rabbits at weaning, given either water or cow's milk diluted in water in addition to their solid diet. The systemic immune response was determined by measuring IgG, IgA, IgM, and IgE antibodies to cow's milk proteins. A rise in IgG and IgM, but not IgA or IgE, antibody titres to milk protein was observed. Further, infected milk-drinking rabbits had significantly higher beta-lactoglobulin and casein IgG titres than milk-drinking controls. The anaphylactic response was tested by mounting ileal segments in Ussing chambers, adding the milk proteins to the serosal side of the tissue and recording the variation in short-circuit current in order to detect any electrogenic chloride secretion. No change in short-circuit current was observed in the presence of milk proteins in infected or age-matched control milk-drinking rabbits, indicating the absence of any immediate hypersensitivity reaction. These results indicate that at weaning, the rabbit is not prone to developing anaphylactic responses to milk proteins. The rise in milk protein IgG antibodies that followed infection with RDEC-1 might favour the elimination of milk protein-IgG immune complexes from the systemic circulation. The present results emphasize the evidence that genetic background and/or animal species greatly influence the immune response to an antigenic load.

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.

HeLa cell adherence, actin aggregation, and invasion by nonenteropathogenic Escherichia coli possessing the eae gene

Enteropathogenic Escherichia coli (EPEC) produce diarrhea in humans by a mechanism that involves close adherence to epithelial cells in the intestine and colon. Close adherence is associated with effacement of microvilli and condensation of actin beneath the bacteria, a process termed attaching/effacing adherence. Attaching/effacing adherence of EPEC occurs in vitro in tissue culture, simplifying the study of the molecular genetics of this process. An EPEC gene (eae) necessary for attaching/effacing adherence was recently characterized. Enterohemorrhagic E. coli and the rabbit-specific RDEC-1 strain adhere in a like fashion in vivo and hybridize with eae. However, these strains adhere poorly to tissue culture cells, complicating the in vitro study of attaching/effacing adherence. In order to develop an in vitro model for the study of attaching/effacing activity of non-EPEC bacteria, a plasmid encoding the F1845 adhesin of an E. coli strain (C1845) isolated from a patient with diarrhea was transformed into RDEC-1 and enterohemorrhagic E. coli. The transformed strains adhered in a diffuse pattern to HeLa cells, and they aggregated HeLa cell actin at points of adherence in the fluorescein-isothiocyanate-labeled phalloidin assay. They also invaded HeLa cells in a gentamicin invasion assay, although not to the extent seen with EPEC. The construction of adherent non-EPEC strains facilitates the molecular study of the attaching/effacing properties and invasiveness of these strains in tissue culture models.

Plasmid and chromosomal elements involved in the pathogenesis of attaching and effacing Escherichia coli

Attaching and effacing (A/E) intestinal lesions are produced by enteropathogenic Escherichia coli (EPEC), enterohemorrhagic E. coli (EHEC), and RDEC-1, a pathogen of weanling rabbits. We recently identified a chromosomal locus (eae[E. coli A/E]) which is required for A/E activity in a wild-type EPEC strain. Sequences homologous to those of an eae gene probe were detected in EPEC, RDEC-1, and EHEC isolates. We report here that the eae gene is chromosomally encoded in all EPEC and EHEC strains tested and in RDEC-1. In addition, the eae probe was found to be 100% sensitive and 98% specific in detecting E. coli of EPEC serogroups that demonstrate A/E activity. Ten percent of E. coli of EPEC serogroups that hybridized with the eae probe and produced A/E activity did not hybridize with the EAF (EPEC adherence factor) probe, a plasmid-associated diagnostic probe which is currently used to identify EPEC. In addition to A/E factors, plasmid-associated adhesins also contribute to the pathogenesis of EPEC and RDEC-1. To further investigate the role of plasmid-associated adherence, a hybrid RDEC-1-EPEC strain containing the adherence plasmid of an EPEC strain in the A/E background of RDEC-1 was constructed. This hybrid strain, unlike the parent RDEC-1 strain, produced A/E lesions on human tissue culture cells, which suggests that the EPEC adherence plasmid provides tissue specificity to the hybrid strain and that the A/E factors of RDEC-1 are not host restricted.

Antigen absorption in rabbit bacterial diarrhea (RDEC-1)

Intestinal absorption of antigenic (intact) and degraded beta-lactoglobulin and horseradish peroxidase was studied in rabbits experimentally infected at weaning with the rabbit-specific Escherichia coli strain RDEC-1 (015:NM). Transepithelial fluxes of both proteins were measured at four stages after infection: early, peak, late, and recovery, on segments of ileum and Peyer's patches mounted in Ussing chambers. During this period of observation, absorption of antigenic beta-lactoglobulin and intact horseradish peroxidase decreased significantly in the ileum and Peyer's patches of age-matched controls. No significant age-related decrease was observed in the controls for the transport of these proteins in degraded form. RDEC-1 infection caused, first, a rise in degraded protein fluxes across Peyer's patches only, during the early phase of the disease. Second, during the peak phase of infection, absorption of antigenic beta-lactoglobulin by the ileum rose to 74.0 ng/hr/cm2 (90% confidence interval: 63.1, 86.8) vs 15.8 (5.8, 42.8) in the controls (P = 0.07), and absorption of intact horseradish peroxidase rose to 21.8 (16.5, 26.7) ng/hr/cm2 vs 4.4 (0.7, 26.6) in control ileum, (P = 0.09). These rises, which were similar in Peyer's patches, delayed the physiologic decrease in protein absorption that occurs with age. These results indicate that increased antigen absorption is observed during bacterial diarrhea and that it might interfere with the immune responses of the host.

Hydrophobic properties of the rabbit enteric pathogen Escherichia coli strain RDEC-1

Escherichia coli strain RDEC-1 (serotype 0.15: H- ) is a rabbit pathogen which attaches to the intestinal mucosa in a manner that is morphologically identical to the binding of enteropathogenic E. coli in the intestine of human infants. The organisms express mannose-resistant bacterial lectins previously defined as attachment factor, rabbit-1 (AF/R1) pili. We examined the effect of AF/R1 pilus expression on the hydrophobicity of RDEC-1. Hydrophobicity was determined by four complementary methods: the salt aggregation test, hydrophobic interaction chromatography, adherence to polystyrene, and contact angle with water measurements. The organisms were more hydrophobic when AF/R1 pili were expressed compared with bacteria grown to suppress pilus expression. Binding of bacteria to polystyrene surfaces was dependent on AF/R1 pilus expression. Attachment of piliated RDEC-1 to more hydrophilic surfaces such as sulphonated polystyrene was minimal. Adherence of piliated E. coli to polystyrene was reduced by 80% when propanol was added to reduce the surface tension of the suspending buffer. Conversely, in the presence of propanol adherence of piliated organisms to sulphonated polystyrene increased 12-fold. The data indicate that AF/R1 pilus expression increased the cell surface hydrophobicity of E. coli RDEC-1. In addition, expression of the hydrophobic adhesins mediated bacterial attachment to hydrophobic surfaces.

Bacterial cell surface hydrophobicity properties in the mediation of in vitro adhesion by the rabbit enteric pathogen Escherichia coli strain RDEC-1.

The role of hydrophobicity in the attachment of enteropathogens to gastrointestinal mucosa is controversial. In vitro binding of Escherichia coli RDEC-1 to rabbit intestine is dependent on the expression of pili. We examined in vitro adherence of piliated RDEC-1 after altering either the hydrophobicity of the organisms, the hydrophobicity of the substrate for attachment, or the surface tension of the suspending liquid. Hydrophobicity of RDEC-1 was determined using four complementary methods. In each assay piliated RDEC-1 demonstrated relatively more hydrophobic properties compared with both organisms grown to suppress pilus expression and a mutant that cannot express mannose-resistant pili. When piliated RDEC-1 were pretreated with tetramethyl urea to disrupt hydrophobic bonds surface hydrophobicity decreased. Concurrently, bacterial adherence to rabbit ileal microvillus membranes, mucus and mucin was reduced. Binding of piliated organisms to hydrophobic surfaces was significantly higher compared to both nonpiliated bacteria and the adherence of piliated RDEC-1 to relatively hydrophilic surfaces. Addition of propanol reduced the surface tension of the suspending liquid, and decreased adhesion of piliated RDEC-1 to polystyrene by 80%. Conversely, adherence of piliated organisms to a hydrophilic surface increased 12-fold after lowering the surface tension of the suspending liquid. We conclude that hydrophobic properties have a role in mediating in vitro adherence of this E. coli enteric pathogen.

Effect of oligomannoside-type glycopeptides in diarrheal disease of rabbits induced byEscherichia colistrain RDEC-1

We examined the effects of a soluble receptor-like complex carbohydrate (an oligomannoside-type glycopeptide) on adherence of a type 1 fimbriated rabbit enteropathogen, Escherichia coli strain RDEC-1. Oligomannoside-type glycopeptide, but not a non-glycosylated peptide mixture used as control, inhibited adherence of type 1 fimbriated RDEC-1 to both rabbit ileal brush borders and guinea pig erythrocytes in vitro. In contrast, during RDEC-1 infection of rabbits the receptor-like oligomannoside did not affect the presence and severity of diarrhea, fecal shedding of organisms, luminal colonization by RDEC-1, or enteroadherence of organisms.