Mulberry Leaf-Derived Morin Activates ß-Catenin by Binding to Frizzled7 to Promote Intestinal Stem Cell Expansion upon Heat-Stable Enterotoxin b Injury.

Intestinal stem cells (ISCs) sustain epithelial renewal by dynamically altering behaviors of proliferation and differentiation in response to various nutrition and stress inputs. However, how ISCs integrate bioactive substance morin cues to protect against heat-stable enterotoxin b (STb) produced by Escherichia coli remains an uncertain question with implications for treating bacterial diarrhea. Our recent work showed that oral mulberry leaf-derived morin improved the growth performance in STb-challenged mice. Furthermore, morin supplementation reinstated the impaired small-intestinal epithelial structure and barrier function by stimulating ISC proliferation and differentiation as well as supporting intestinal organoid expansion ex vivo. Importantly, the Wnt/ß-catenin pathway, an ISC fate commitment signal, was reactivated by morin to restore the jejunal crypt-villus architecture in response to STb stimulation. Mechanically, the extracellular morin-initiated ß-catenin axis is dependent or partially dependent on the Wnt membrane receptor Frizzled7 (FZD7). Our data reveal an unexpected role of leaf-derived morin, which represents molecular signaling targeting the FZD7 platform instrumental for controlling ISC regeneration upon STb injury.

Escherichia cryptic clade I is an emerging source of human intestinal pathogens.

BACKGROUND: Within the genus Escherichia, several monophyletic clades other than the traditionally defined species have been identified. Of these, cryptic clade I (C-I) appears to represent a subspecies of E. coli, but due to the difficulty in distinguishing it from E. coli sensu stricto, the population structure and virulence potential of C-I are unclear. RESULTS: We defined a set of true C-I strains (n = 465), including a Shiga toxin 2a (Stx2a)-producing isolate from a patient with bloody diarrhoea identified by the retrospective analyses using a C-I-specific detection system. Through genomic analysis of 804 isolates from the cryptic clades, including these C-I strains, we revealed their global population structures and the marked accumulation of virulence genes and antimicrobial resistance genes in C-I. In particular, half of the C-I strains contained hallmark virulence genes of Stx-producing E. coli (STEC) and/or enterotoxigenic E. coli (ETEC). We also found the host-specific distributions of virulence genes, which suggests bovines as the potential source of human infections caused by STEC- and STEC/ETEC hybrid-type C-I strains, as is known in STEC. CONCLUSIONS: Our findings demonstrate the emergence of human intestinal pathogens in C-I lineage. To better understand the features of C-I strains and their infections, extensive surveillance and larger population studies of C-I strains are needed. The C-I-specific detection system developed in this study will be a powerful tool for screening and identifying C-I strains.

Strong Association between Diarrhea and Concentration of Enterotoxigenic Escherichia coli Strain TW10722 in Stools of Experimentally Infected Volunteers.

Enterotoxigenic Escherichia coli (ETEC) strains are a major cause of diarrheal illness in children and travelers in low- and middle-income countries. When volunteers are infected with ETEC strains, as part of experimental infection studies, some do not develop diarrhea. To improve our understanding of how these volunteers are protected, we investigated the association between stool ETEC DNA concentration, as determined by quantitative PCR, and the development and severity of disease in 21 volunteers who had been experimentally infected with ETEC strain TW10722. We found a strong association between maximum stool ETEC DNA concentration and the development of diarrhea: all of the 11 volunteers who did not develop diarrhea had <0.99% TW10722-specific DNA in their stools throughout the follow-up period of up to 9 days, while all of the 10 volunteers who did develop diarrhea had maximum DNA concentrations of ≥0.99%. Most likely, these maximum stool TW10722 DNA concentrations reflect the level of intestinal colonization and the risk of experiencing diarrhea, thereby, seems to be directly dependent on the level of colonization. Thus, the development and availability of vaccines and other prophylactic measures, even if they only partially reduce colonization, could be important in the effort to reduce the burden of ETEC diarrhea.

Whole Genome Sequencing and CRISPR/Cas9 Gene Editing of Enterotoxigenic Escherichia coli BE311 for Fluorescence Labeling and Enterotoxin Analyses.

Some prevention strategies, including vaccines and antibiotic alternatives, have been developed to reduce enterotoxigenic Escherichia coli proliferation in animal production. In this study, a wild-type strain of BE311 with a virulent heat-stable enterotoxin gene identical to E. coli K99 was isolated for its high potential for gene expression ability. The whole genome of E. coli BE311 was sequenced for gene analyses and editing. Subsequently, the fluorescent gene mCherry was successfully knocked into the genome of E. coli BE311 by CRISPR/Cas9. The E. coli BE311−mCherry strain was precisely quantified through the fluorescence intensity and red colony counting. The inflammatory factors in different intestinal tissues all increased significantly after an E. coli BE311−mCherry challenge in Sprague−Dawley rats (p < 0.05). The heat-stable enterotoxin gene of E. coli BE311 was knocked out, and an attenuated vaccine host E. coli BE311-STKO was constructed. Flow cytometry showed apoptotic cell numbers were lower following a challenge of IPEC-J2 cells with E. coli BE311-STKO than with E. coli BE311. Therefore, the E. coli BE311−mCherry and E. coli BE311-STKO strains that were successfully constructed based on the gene knock-in and knock-out technology could be used as ideal candidates in ETEC challenge models and for the development of attenuated vaccines.

Vaccine Candidate Double Mutant Variants of Enterotoxigenic Escherichia coli Heat-Stable Toxin.

Heat-stable enterotoxin (ST) producing enterotoxigenic Escherichia coli (ETEC) strains are among the top four enteropathogens associated with moderate-to-severe diarrhea in children under five years in low-to-middle income countries, thus making ST a target for an ETEC vaccine. However, ST must be mutated to abolish its enterotoxicity and to prevent a potential immunological cross-reaction due to its structural resemblance to the human peptides uroguanylin and guanylin. To reduce the risk of eliciting cross-reacting antibodies with our lead STh-A14T toxoid, L9 was chosen as an additional mutational target. A double mutant vaccine candidate immunogen, STh-L9A/A14T, was constructed by conjugation to the synthetic virus-like mi3 nanoparticle using the SpyTag/SpyCatcher technology. This immunogen elicited STh neutralizing antibodies in mice, but with less consistency than STh-A14T peptide control immunogens. Moreover, individual sera from mice immunized with both single and double mutant variants displayed varying levels of unwanted cross-reacting antibodies. The lowest levels of cross-reacting antibodies were observed with STh-L9K/A14T control immunogens, suggesting that it is indeed possible to reduce the risk of eliciting cross-reacting antibodies by mutation. However, mutant-specific antibodies were observed for most double mutant immunogens, demonstrating the delicate balancing act between disrupting cross-reacting epitopes, keeping protective ones, and avoiding the formation of neoepitopes.

A statistical approach to identify prevalent virulence factors responsible for post-weaning diarrhoeic piglets.

A statistical approach was carried out to identify the prevalent virulence factors responsible for post-weaning diarrhoea (PWD). Healthy piglets' faecal samples and diarrhoeic piglets' rectal swab specimens were secured. Twenty-six (26) and 100 independent enterotoxigenic Escherichia coli (ETEC) strains were subsequently isolated. These strains were assessed utilising polymerase chain reaction to identify the encoding genes of six virulence factors: heat-labile enterotoxin (LT; encoded by eltAB), heat-stable enterotoxin A (STa; encoded by estA), heat-stable enterotoxin B (STb; encoded by estB), enteroaggregative E. coli heat-stable enterotoxin 1 (EAST1; encoded by astA), Shiga toxin 2e (Stx2e; encoded by stx2e), and F18 fimbriae (encoded by fedA). The LT and ST secretions were investigated using enzyme-linked immunosorbent assays. From direct observation, no stx2e was evident in the 126 strains. Among the 26 strains retrieved from the healthy piglets, none harboured fedA or secreted LT; 23% (6/26) secreted ST, and 50% (13/26) carried astA. A statistical regression was applied on the 100 E. coli strains retrieved from the diarrhoeic piglets, where fedA was set as the dependent variable and the enterotoxin secretions were set as the independent variables. The results exhibit that the LT secretion was the only significant factor (P < 0.000 1) correlated to fedA in the diarrhoeic piglets; thus, it is concluded that the prevalent virulence factors for PWD were the ECET strain with F18 fimbriae adhesion and LT secretion, but not astA or stx2e.

In Silico Prediction, Molecular Docking and Dynamics Studies of Steroidal Alkaloids of Holarrhena pubescens Wall. ex G. Don to Guanylyl Cyclase C: Implications in Designing of Novel Antidiarrheal Therapeutic Strategies.

The binding of heat stable enterotoxin (STa) secreted by enterotoxigenic Escherichia coli (ETEC) to the extracellular domain of guanylyl cyclase c (ECDGC-C) causes activation of a signaling cascade, which ultimately results in watery diarrhea. We carried out this study with the objective of finding ligands that would interfere with the binding of STa on ECDGC-C. With this view in mind, we tested the biological activity of a alkaloid rich fraction of Holarrhena pubescens against ETEC under in vitro conditions. Since this fraction showed significant antibacterial activity against ETEC, we decided to test the screen binding affinity of nine compounds of steroidal alkaloid type from Holarrhena pubescens against extracellular domain (ECD) by molecular docking and identified three compounds with significant binding energy. Molecular dynamics simulations were performed for all the three lead compounds to establish the stability of their interaction with the target protein. Pharmacokinetics and toxicity profiling of these leads demonstrated that they possessed good drug-like properties. Furthermore, the ability of these leads to inhibit the binding of STa to ECD was evaluated. This was first done by identifying amino acid residues of ECDGC-C binding to STa by protein-protein docking. The results were matched with our molecular docking results. We report here that holadysenterine, one of the lead compounds that showed a strong affinity for the amino acid residues on ECDGC-C, also binds to STa. This suggests that holadysenterine has the potential to inhibit binding of STa on ECD and can be considered for future study, involving its validation through in vitro assays and animal model studies.

Re-evaluation of a Neonatal Mouse Model of Infection With Enterotoxigenic Escherichia coli.

Enterotoxigenic E. coli (ETEC) is a common cause of diarrhea in children in low- and middle-income countries, and in travelers to these countries. ETEC is also an important cause of morbidity and premature mortality in piglets, calves, goat kids and lambs. The major virulence determinants of ETEC are enterotoxins and colonization factors, which enable the pathogen to colonize the small intestine and deliver enterotoxins, such as the heat-stable enterotoxins, STp and STh, to epithelial cells. Because most ETEC strains are host-specific, there are few convenient animal models to investigate the pathogenesis of ETEC infections or to evaluate specific anti-ETEC interventions, such as drugs and vaccines. An exception is ETEC strains bearing F41 pili, which mediate intestinal colonization of various young animals, including neonatal mice, to cause disease and in some cases death. In this study, we used the archetypal F41-producing bovine ETEC strain, B41 (O101:NM; K99, F41, STp) to validate and further explore the contribution of F41 and STp to bacterial virulence. By using targeted gene deletion and trans-complementation studies, augmented by whole genome sequencing, and in vitro and animal studies of virulence, we established that F41 mediates colonization of the mouse intestine and is essential for bacterial virulence. In addition, we showed for the first time that STp is as important as F41 for virulence. Together, these findings validate the use of neonatal mice to study the pathogenesis of F41-bearing ETEC and to investigate possible specific anti-ETEC interventions including vaccines that target heat-stable enterotoxins.

AU richness within the 5' coding region of the Escherichia coli heat-stable enterotoxin b mRNA affects toxin secretion.

The majority of protein secretion in bacteria is mediated by the T2SS pathway. Substrates processed through this pathway are guided by the N-terminal signal sequence within the nascent polypeptide. Recent experimental evidence suggests that in similar secretory pathways, such as the T3SS, information in the 5' coding region of the mRNA affects secretion and may also participate in mRNA localization. The majority of studies on the effects of AU richness on translation have focussed on the 5' UTR in mRNAs. To look at the effects of AU richness within the coding region of mRNA on secretion, we have generated several silent mutations within the 5' coding region of the E. coli heat-stable enterotoxin b (STb). This toxin is a well studied T2SS substrate. The mutations were generated such that AU richness within the 5' coding region (corresponding to the N-terminal signal sequence) was gradually reduced. Reduction of AU richness within the first 15 codons resulted in reduced secretion of the toxin as the AU/GC ratio was reduced from 2.13 for the WT STb to 1.65 (S-I) and subsequently to 1.30 (S-II). This reduction did not correlate with mRNA accumulation and decreased stability of the transcripts could not account for the reduced secretion observed. Reduction of AU richness beyond the first 15 codons recovered secretion efficiency of the toxin (S-III). To validate the experimental approach, a positive control was used in which a mutation involving the insertion of a positive charge within the hydrophobic domain of the N-terminal signal sequence was constructed. As expected, this mutation abolished secretion of the toxin. In conclusion, reducing AU richness within the 5'coding region in the STb mRNA reduces toxin secretion but other factors, such as formation of hairpins, must also be taken into consideration. This will have implications for both homologous and heterologous expression of STb for biological studies and for toxin production.

Phosphodiesterase 5 (PDE5) restricts intracellular cGMP accumulation during enterotoxigenic Escherichia coli infection.

Diarrhea caused by enterotoxigenic Escherichia coli (ETEC) has a continuing impact on residents and travelers in underdeveloped countries. Both heat-labile (LT) and heat-stable (ST) enterotoxins contribute to pathophysiology via induction of cyclic nucleotide synthesis, and previous investigations focused on intracellular signal transduction rather than possible intercellular second messenger signaling. We modeled ETEC infection in human jejunal enteroid/organoid monolayers (HEM) and evaluated cyclic nucleotide pools, finding that intracellular cAMP was significantly increased but also underwent apical export, whereas cGMP was minimally retained intracellularly and predominantly effluxed into the basolateral space. LT and virulence factors including EatA, EtpA, and CfaE promoted ST release and enhanced ST-stimulated cGMP production. Intracellular cGMP was regulated by MK-571-sensitive export in addition to degradation by phosphodiesterase 5. HEMs had limited ST-induced intracellular cGMP accumulation compared to T84 or Caco-2 models. Cyclic nucleotide export/degradation demonstrates additional complexity in the mechanism of ETEC infection and may redirect understanding of diarrheal onset.

Mechanical Stimuli Affect Escherichia coli Heat-Stable Enterotoxin-Cyclic GMP Signaling in a Human Enteroid Intestine-Chip Model.

Modeling host-pathogen interactions with human intestinal epithelia using enteroid monolayers on permeable supports (such as Transwells) represents an alternative to animal studies or use of colon cancer-derived cell lines. However, the static monolayer model does not expose epithelial cells to mechanical forces normally present in the intestine, including luminal flow and serosal blood flow (shear force) or peristaltic forces. To determine the contribution of mechanical forces in the functional response of human small intestine to a virulence factor of a pathogenic intestinal bacterium, human jejunal enteroids were cultured as monolayers in microengineered fluidic-based Organ-Chips (Intestine-Chips) exposed to enterotoxigenic Escherichia coli heat-stable enterotoxin A (ST) and evaluated under conditions of static fluid, apical and basolateral flow, and flow plus repetitive stretch. Application of flow increased epithelial cell height and apical and basolateral secretion of cyclic GMP (cGMP) under baseline, unstimulated conditions. Addition of ST under flow conditions increased apical and basolateral secretion of cGMP relative to the level under static conditions but did not enhance intracellular cGMP accumulation. Cyclic stretch did not have any significant effect beyond that contributed by flow. This study demonstrates that fluid flow application initiates changes in intestinal epithelial cell characteristics relative to those of static culture conditions under both baseline conditions and with exposure to ST enterotoxin and suggests that further investigations of the application of these mechanical forces will provide insights into physiology and pathophysiology that more closely resemble intact intestine than study under static conditions.

Experimental Infection of Human Volunteers with the Heat-Stable Enterotoxin-Producing Enterotoxigenic Escherichia coli Strain TW11681.

Infection with enterotoxigenic Escherichia coli (ETEC) producing the heat-stable enterotoxin (ST) is one of the most important causes of childhood diarrhoea in low- and middle-income countries. Here, we undertook a controlled human infection model (CHIM) study to investigate whether ST-producing ETEC strain TW11681 would be suitable for testing the protective efficacy of new ST-based vaccine candidates in vaccine challenge models. In groups of three, nine volunteers ingested 1 × 106, 1 × 107, or 1 × 108 colony-forming units (CFU) of TW11681. Flow cytometry-based assays were used to measure CD4+ T cell responses and antibody levels targeting virulence factors expressed by the strain. We found that infection with TW11681 elicited few and mild symptoms, including mild diarrhoea in two volunteers, both of whom ingested 1 × 106 CFU. Averaged across all volunteers, the CD4+ T cell responses specific for E. coli YghJ mucinase peaked 10 days after infection (3.2-fold (p = 0.016)), while the CD4+ T cell responses specific for Colonization Factor Antigen I (CFA/I) major fimbrial subunit (CfaB) peaked after 28 days (3.6-fold (p = 0.063)). The serum CfaB-specific anti-IgA and anti-IgG/IgM levels were significantly increased and peaked 3 months after infection. Both remained elevated for the duration of the 12-month follow-up. The corresponding anti-YghJ serological response was strongest after 10 days, although a significant increase was seen only for IgA levels (3.2-fold (p = 0.008)). In conclusion, due to its low diarrhoea attack risk, TW11681 is probably not suitable for testing the efficacy of new vaccines in human challenge studies at doses 1 × 106 to 1 × 108. However, the strain may still be useful in CHIMs for studying ETEC host-pathogen interactions.

Investigating possible association between multidrug resistance and isolate origin with some virulence factors of Escherichia coli strains isolated from infant faeces and fresh green vegetables.

AIMS: In this study, the association between multidrug resistance (MDR) and the expression of some virulence factors were evaluated in Escherichia coli strains isolated from infant faeces and fresh green vegetables. The effect of isolate origin on associated virulence factors was evaluated. In addition, genetic fingerprinting of a sample of these isolates (10 isolates from each group) was studied in order to detect any genetic relatedness among these isolates. METHODS AND RESULTS: Escherichia coli isolates were divided into four groups based on their origin (human faeces or plant) and their antibiotic resistance (multiresistance or susceptible). PCR was used to investigate heat-labile and heat-stable enterotoxin genes, and four siderophore genes (aerobactin, enterobactin, salmochelin and yersiniabactin). Genetic fingerprinting of the isolates was performed using enterobacterial repetitive intergenic consensus PCR. Siderophore production was measured by a colorimetric method. Biofilm formation was evaluated by a crystal violet assay. The results of the study showed that the expression of MDR is not significantly associated with an increase in these virulence factors or with biofilm formation. However, the origin of isolates had a significant association with siderophore gene availability and consequently on the concentrations of siderophores released. Genetic fingerprinting indicated that human and plant isolates have the same clonal origin, suggesting their circulation among humans and plants. CONCLUSION: Antibiotic-susceptible strains of E. coli may be as virulent as MDR strains. Results also suggest that the environment can play a potential role in selection of strains with specific virulence factors. SIGNIFICANCE AND IMPACT OF THE STUDY: Antibiotic-susceptible isolates of Escherichia coli from plant or human origin can be as virulent as the multidrug resistance (MDR) ones. Genetic relatedness was detected among the isolates of plant and human origin, indicating the circulation of these bacteria among human and plants. This could imply a potential role for environmental antimicrobial resistant bacteria in human infection.

Evolution of diarrheagenic Escherichia coli pathotypes in India.

CONTEXT: Diarrheagenic Escherichia coli (DEC) is the leading cause of infectious diarrhea in developing countries. On the basis of virulence and phenotypic characteristics, the DEC is categorized into multiple pathotypes. Each pathotype has different pathogenesis and geographical distribution. Thus, the proper management of disease relies on rapid and accurate identification of DEC pathotypes. AIMS: The aim of the study was to determine the prevalence of DEC pathotypes in India. MATERIALS AND METHODS: A cross-sectional study was carried out between January 2008 and December 2012 at Jawaharlal Nehru Medical College and KLES Dr. Prabhakar Kore Hospital and Medical Research Center, Belgaum (Karnataka), India. A total of 300 stool samples were collected from diarrhea patients with age >3 months. The DEC was identified by both conventional and molecular methods. RESULTS: Of 300 samples, E. coli were detected in 198 (66%) and 170 (56.6%) samples by culture and polymerase chain reaction, respectively. Among DEC (n = 198) isolates, eae gene (59.5%) was the most prevalent followed by stx (27.7%), east (27.2%), elt (12.6%), est (10.6%), ipaH (5.5%), and eagg (1.5%) genes. On the basis of virulence genes, enteropathogenic E. coli (33.8%) was the most common pathotype followed by Shiga toxin-producing E. coli (STEC, 23.2%), enterotoxigenic E. coli (ETEC, 13.6%), enteroinvasive E. coli (5.5%), enteroaggregative heat-stable enterotoxin 1-harboring E. coli (EAST1EC, 4.5%), STEC/ETEC (3.5%), STEC/enteroaggregative E. coli (STEC/EAEC, 1.0%), and EAEC (0.05%). CONCLUSIONS: The hybrid DEC is potentially more virulent than basic pathotypes. The pathotyping should be included in clinical settings for the proper management of DEC-associated diarrhea.

Proliferation of enterotoxigenic Escherichia coli strain TW11681 in stools of experimentally infected human volunteers.

BACKGROUND: As part of the effort to develop an enterotoxigenic Escherichia coli (ETEC) human challenge model for testing new heat-stable toxin (ST)-based vaccine candidates, a controlled human infection model study based on the ST-producing ETEC strain TW11681 was undertaken. Here, we estimate stool TW11681 DNA concentration and evaluate its association with dose, clinical symptoms, and with levels of antibodies targeting the CfaB subunit of the ETEC Colonization Factor Antigen I and the E. coli mucinase YghJ. Nine volunteers ingested different doses of the strain and were subsequently followed for 9 days with daily stool specimen collection and clinical examination. Stool DNA was purified by using a newly developed microplate-based method, and DNA originating from TW11681 was quantified by using a probe-based quantitative PCR assay. Antibody levels against CfaB and YghJ were measured in serum collected before and 10 and 28 days after TW11681 was ingested by using a bead-based flow cytometry immunoassay. RESULTS: For 6 of the 9 volunteers, the stool TW11681 DNA concentration increased sharply a median 3.5 (range 2-5) days after dose ingestion, peaking at a median of 5.4% (range 3.3-8.2%) of the total DNA in the specimen. The concentration then fell sharply during the subsequent days, sometimes even before the onset of antibiotic treatment. The size or timing of these proliferation peaks did not seem to be associated with the number of TW11681 bacteria ingested, but the 2 volunteers who developed diarrhea and all five who experienced abdominal pains or cramps had these peaks. The 3 volunteers who did not have the proliferation peaks experienced fewer symptoms and they generally had relatively low CfaB- and YghJ-specific antibody levels before ingesting the strain and subsequently weaker responses than the other volunteers afterwards. CONCLUSIONS: Since the lack of proliferation peaks appears to be associated with fewer clinical symptoms and lower serum antibody responses to virulence factors of the infecting strain, it may be important to account for proliferation peaks when explaining results from controlled human infection model studies and for improving the accuracy of protective efficacy estimates when testing new ETEC diarrhea vaccine candidates.

Guanylate Cyclase C: A Current Hot Target, from Physiology to Pathology.

BACKGROUND: Guanylate cyclase C (GC-C) receptor is a transmembrane receptor, predominantly expressed in intestinal epithelial cells, which is considered to play a main role in homeostasis and function of the digestive tract. The endogenous ligands for this receptor are the paracrine hormones uroguanylin and guanylin. Upon ligand binding, GC-C receptors increase cyclic guanosine monophosphate (cGMP) levels, regulating a variety of key cell-type specific processes such as chloride and bicarbonate secretion, epithelial cell growth, regulation of intestinal barrier integrity and visceral sensitivity. It has been suggested that GC-C acts as an intestinal tumor suppressor with the potential to prevent the initiation and progression of colorectal cancer. In fact, loss of ligand expression is a universal step in sporadic colorectal carcinogenesis. Interestingly, the role of GC-C is not limited to the digestive tract but it has been extended to several other systems such as the cardiovascular system, kidney, and the central nervous system, where it has been involved in a gut-hypothalamus endocrine axis regulating appetite. Objetive: In this review we summarize the physiology of the GC-C receptor and its ligands, focusing on newly developed drugs like linaclotide, and their suggested role to reverse/prevent the diseases in which the receptor is involved. CONCLUSION: Available data points toward a relationship between uroguanylin and guanylin and their receptor and pathological processes like gastrointestinal and renal disorders, colorectal cancer, obesity, metabolic syndrome and mental disorders among others. Recent pharmacological developments in the regulation of GC-receptor may involve further improvements in the treatment of relevant diseases.

Injury and mechanism of recombinant E. coli expressing STa on piglets colon.

Enterotoxigenic Escherichia coli (ETEC) is primary pathogenic bacteria of piglet diarrhea, over two thirds of piglets diarrhea caused by ETEC are resulted from STa-producing ETEC strains. This experiment was conducted to construct the recombinant E. coli expressing STa and study the injury and mechanism of recombinant E. coli expressing STa on 7 days old piglets colon. Twenty-four 7 days old piglets were allotted to four treatments: control group, STa group (2 × 109 CFU E. coli LMG194-STa), LMG194 group (2 × 109 CFU E. coli LMG194) and K88 group (2 × 109 CFU E. coli K88). The result showed that E. coli infection significantly increased diarrhea rates; changed DAO activity in plasma and colon; damaged colonic mucosal morphology including crypt depth, number of globet cells, density of lymphocytes and lamina propria cell density; substantially reduced antioxidant capacity by altering activities of GSH-Px, SOD, and TNOS and productions of MDA and H2O2; obviously decreased AQP3, AQP4 and KCNJ13 protein expression levels; substantially altered the gene expression levels of inflammatory cytokines. Conclusively, STa group had the biggest effect on these indices in four treatment groups. These results suggested that the recombinant strain expressed STa can induce piglets diarrhea and colonic morphological and funtional damage by altering expression of proteins connect to transportation function and genes associated with intestinal injury and inflammatory cytokines.

Comparative sequence analysis of enteroaggregative Escherichia coli heat-stable enterotoxin 1 identified in Korean and Japanese Escherichia coli strains.

The aim of this study was to compare the sequence of the astA gene found in 8 Korean and 11 Japanese Escherichia coli isolates. Conventional PCR was used to amplify the astA gene from the chromosomal and plasmid DNA preparation samples of each isolate using commercial DNA extraction kits. Cloning of the PCR products, sequence analysis, and pulse field gel electrophoresis (PFGE) were sequentially performed. An identical copy of astA in each isolate were found for 8 Korean and 8 Japanese E. coli strains isolated from bovine, porcine, and healthy human carriers. Among these, 1 Korean and 4 Japanese isolates carried a stop mutation at residue 16. Three Japanese outbreak strains (V199, V638, and 96-127-23) carried multiple clones of astA gene with multiple amino acids changes at residues 11, 16, 20, 23, 30, 33, and 34. Compared with the non-diarrheal isolates, clonal diversity and sequence variations of the astA gene in outbreak isolates may be associated with virulence potential of EAST1.

Immunogenicity and protective efficacy of a recombinant adenoviral based vaccine expressing heat-stable enterotoxin (STa) and K99 adhesion antigen of enterotoxigenic Escherichia coli in mice.

The diarrheal disease of domestic animals or in humans caused by enterotoxigenic Escherichia coli (ETEC) infections remains a major issue for public health in developing countries. Unfortunately, there is no effective vaccine available for preventing from an ETEC infection. Therefore, the development of a safe and effective vaccine against ETEC is urgently needed. In the present study, A recombinant adenoviral vector Ad5-STa-K99 that capable of expressing a fusion protein of heat-stable enterotoxin (STa) and K99 adhesion antigen of ETEC was generated and its immunogenicity was evaluated in a murine model. The intestinal mucosal secretory IgA(sIgA), serum anti-STa-K99 antibody responses, antigen-specific CD4(+) and CD8(+) T cells frequencies, as well as T-cell proliferation of mice immunized with the viral vector were determined as immunological indexes. The results demonstrated that Ad5-STa-K99 was able to enhance humoral responses with a dramatically augmented antigen-specific serum IgG antibody, and an elevated production of intestinal sIgA in immunized mice, suggesting the elicitation of both of humoral and mucosal immune responses. In addition, this adenoviral vector could significantly promote splenic T cell proliferation and increase the frequencies of CD4(+) and CD8(+) T cell populations in mice, indicative of a capacity to activate T cell responses. More importantly, vaccination of the Ad5-STa-K99 showed a potential to evoke a protective effect from ETEC challenge in mice. These data indicate that the Ad5-STa-K99 is a highly immunogenic vector able to induce a broad range of antigen-specific immune responses in vivo, and evoke a protective immune response against ETEC infections, implying that it may be a novel vaccine candidate warranted for further investigation.

From Escherichia coli heat-stable enterotoxin to mammalian endogenous guanylin hormones

The isolation of heat-stable enterotoxin (STa) from Escherichia coli and cholera toxin from Vibrio cholerae has increased our knowledge of specific mechanisms of action that could be used as pharmacological tools to understand the guanylyl cyclase-C and the adenylyl cyclase enzymatic systems. These discoveries have also been instrumental in increasing our understanding of the basic mechanisms that control the electrolyte and water balance in the gut, kidney, and urinary tracts under normal conditions and in disease. Herein, we review the evolution of genes of the guanylin family and STa genes from bacteria to fish and mammals. We also describe new developments and perspectives regarding these novel bacterial compounds and peptide hormones that act in electrolyte and water balance. The available data point toward new therapeutic perspectives for pathological features such as functional gastrointestinal disorders associated with constipation, colorectal cancer, cystic fibrosis, asthma, hypertension, gastrointestinal barrier function damage associated with enteropathy, enteric infection, malnutrition, satiety, food preferences, obesity, metabolic syndrome, and effects on behavior and brain disorders such as attention deficit, hyperactivity disorder, and schizophrenia.