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Background Effective treatment options for recurrent Clostridioides difficile infection (rCDI) are limited, with high recurrence rates associated with the current standard of care. Herein we report results from an open-label Phase 2 trial to evaluate the safety, efficacy, and durability of RBX2660—a standardized microbiota-based investigational live biotherapeutic—and a closely-matched historical control cohort. Methods This prospective, multicenter, open-label Phase 2 study enrolled patients who had experienced either ≥ 2 recurrences of CDI, treated by standard-of-care antibiotic therapy, after a primary CDI episode, or ≥ 2 episodes of severe CDI requiring hospitalization. Participants received up to 2 doses of RBX2660 rectally administered with doses 7 days apart. Treatment success was defined as the absence of CDI diarrhea without the need for retreatment for 8 weeks after completing study treatment. A historical control group with matched inclusion and exclusion criteria was identified from a retrospective chart review of participants treated with standard-of-care antibiotics for recurrent CDI who matched key criteria for the study. The primary objective was to compare treatment success of RBX2660 to the historical control group. A key secondary outcome was the safety profile of RBX2660, including adverse events and CDI occurrence through 24 months after treatment. In addition, fecal samples from RBX2660-treated participants were sequenced to evaluate microbiome composition and functional changes from before to after treatment. Results In this Phase 2 open-label clinical trial, RBX2660 demonstrated a 78.9% (112/142) treatment success rate compared to a 30.7% (23/75) for the historical control group (p Conclusions In this Phase 2 trial, RBX2660 was safe and effective for reducing rCDI recurrence as compared to a historical control group. Microbiome changes are consistent with restorative changes implicated in resisting C. difficile recurrence. Clinical Trials Registration NCT02589847 (10/28/2015)

The leading risk factor for Clostridioides (Clostridium) difficile infection (CDI) is broad-spectrum antibiotics, which lead to low microbial diversity, or dysbiosis. Current therapeutic strategies for CDI are insufficient, as they do not address the key role of the microbiome in preventing C. difficile spore germination into toxin-producing vegetative bacteria, which leads to symptomatic disease. Fecal microbiota transplant (FMT) appears to reduce the risk of recurrent CDI through microbiome restoration. However, a wide range of efficacy rates have been reported, and few placebo-controlled trials have been conducted, limiting our understanding of FMT efficacy and safety. We discuss the current knowledge gaps driven by questions around the quality and consistency of clinical trial results, patient selection, diagnostic methodologies, use of suppressive antibiotic therapy, and methods for adverse event reporting. We provide specific recommendations for future trial designs of FMT to provide improved quality of the clinical evidence to better inform treatment guidelines.

Enteropathogenic Escherichia coli (EPEC) uses a type three secretion system to inject effector proteins into host intestinal epithelial cells, causing diarrhea. EPEC induces the formation of pedestals underlying attached bacteria, disrupts tight junction (TJ) structure and function, and alters apico-basal polarity by redistributing the polarity proteins Crb3 and Pals1, although the mechanisms are unknown. Here we investigate the temporal relationship of PAR polarity complex and TJ disruption following EPEC infection. EPEC recruits active aPKC&zeta, a PAR polarity protein, to actin within pedestals and at the plasma membrane prior to disrupting TJ. The EPEC effector EspF binds the endocytic protein sorting nexin 9 (SNX9). This interaction impacts actin pedestal organization, recruitment of active aPKC&zeta, to actin at cell&ndash, cell borders, endocytosis of JAM-A S285 and occludin, and TJ barrier function. Collectively, data presented herein support the hypothesis that EPEC-induced perturbation of TJ is a downstream effect of disruption of the PAR complex and that EspF binding to SNX9 contributes to this phenotype. aPKC&zeta, phosphorylates polarity and TJ proteins and participates in actin dynamics. Therefore, the early recruitment of aPKC&zeta, to EPEC pedestals and increased interaction with actin at the membrane may destabilize polarity complexes ultimately resulting in perturbation of TJ.

Enteropathogenic Escherichia coli (EPEC) is a foodborne pathogen that uses a type III secretion system to translocate effector molecules into host intestinal epithelial cells (IECs) subverting several host cell processes and signaling cascades. Interestingly, EPEC infection induces only modest intestinal inflammation in the host. The homologous EPEC effector proteins, NleH1 and NleH2, suppress the nuclear factor-κB (NF-κB) pathway and apoptosis in vitro. Increased apoptosis and activation of NF-κB and MAP kinases (MAPK) contribute to the pathogenesis of inflammatory bowel diseases (IBD). The aim of this study was to determine if NleH1 and NleH2 also block MAPK pathways in vitro and in vivo, and to compare the effects of these bacterial proteins on a murine model of colitis. Cultured IECs were infected with various strains of EPEC expressing NleH1 and NleH2, or not, and the activation of ERK1/2 and p38 was determined. In addition, the impact of infection with various strains of EPEC on murine DSS colitis was assessed by change in body weight, colon length, histology, and survival. Activation of apoptosis and MAPK signaling were also evaluated. Our data show that NleH1, but not NleH2, suppresses ERK1/2 and p38 activation in vitro. Interestingly, NleH1 affords significantly greater protection against and hastens recovery from dextran sodium sulfate (DSS)-induced colitis compared to NleH2. Strikingly, colitis-associated mortality was abolished by infection with EPEC strains expressing NleH1. Interestingly, in vivo NleH1 suppresses activation of ERK1/2 and p38 and blocks apoptosis independent of the kinase domain that inhibits NF-κB. In contrast, NleH2 suppresses only caspase-3 and p38, but not ERK1/2. We conclude that NleH1 affords greater protection against and improves recovery from DSS colitis compared to NleH2 due to its ability to suppress ERK1/2 in addition to NF-κB, p38, and apoptosis. These findings warrant further investigation of anti-inflammatory bacterial proteins as novel treatments for IBD.

Epithelial cells constitute a physical barrier that aids in protecting the host from microbial pathogens. Polarized epithelial cells contain distinct apical and basolateral membrane domains separated by intercellular junctions, including tight junctions (TJs), which contribute to the maintenance of apical-basal polarity. Polarity complexes also contribute to the establishment of TJ formation. Several pathogens perturb epithelial TJ barrier function and structure in addition to causing a loss of apical-basal polarity. Here, we review the impact of pathogenic bacteria on the disruption of cell-cell junctions and epithelial polarity.

Enteropathogenic Escherichia coli (EPEC), one of the diarrheagenic E. coli pathotypes, is among the most important food-borne pathogens infecting children worldwide. Inhibition of serotonin transporter (SERT), which regulates extracellular availability of serotonin (5-HT), has been implicated previously in EPEC-associated diarrhea. EPEC was shown to inhibit SERT via activation of protein tyrosine phosphatase (PTPase), albeit the specific PTPase involved is not known. Current studies aimed to identify EPEC-activated PTPase and its role in SERT inhibition. Infection of Caco-2 monolayers with EPEC strain E2348/69 for 30 min increased the activity of Src-homology-2 domain containing PTPase (SHP2) but not SHP1 or PTPase 1B. Similarly, Western blot studies showed increased tyrosine phosphorylation of (p-tyrosine) SHP2, indicative of its activation. Concomitantly, EPEC infection decreased SERT p-tyrosine levels. This was associated with increased interaction of SHP2 with SERT, as evidenced by coimmunoprecipitation studies. To examine whether SHP2 directly influences SERT phosphorylation status or function, SHP2 cDNA plasmid constructs (wild type, constitutively active, or dominant negative) were overexpressed in Caco-2 cells by Amaxa electroporation. In the cells overexpressing constitutively active SHP2, SERT polypeptide showed complete loss of p-tyrosine. In addition, there was a decrease in SERT function, as measured by Na+Cl−-sensitive [3H]5-HT uptake, and an increase in association of SERT with SHP2 in Caco-2 cells expressing constitutively active SHP2 compared with dominant-negative SHP2. Our data demonstrate that intestinal SERT is a target of SHP2 and reveal a novel mechanism by which a common food-borne pathogen uses cellular SHP2 to inhibit SERT. NEW & NOTEWORTHY The data presented in the current study reveal that intestinal serotonin transporter (SERT) is a target of the tyrosine phosphatase SHP2 and show a novel mechanism by which a common diarrheagenic pathogen, EPEC, activates cellular SHP2 to inhibit SERT function. These studies highlight host-pathogen interactions, which may be of therapeutic relevance in the management of diarrhea associated with enteric infections.

Background & Aims Sperm flagellar 1 (also called CLAMP) is a microtubule-associated protein that regulates microtubule dynamics and planar cell polarity in multi-ciliated cells. We investigated the localization and function of sperm flagellar 1, or CLAMP, in human intestinal epithelia cells (IECs). Methods We performed studies with SKCO-15 and human intestinal enteroids established from biopsies from different intestinal segments (duodenal, jejunum, ileal, and colon) of a single donor. Enteroids were induced to differentiation after incubation with growth factors. The distribution of endogenous CLAMP in IECs was analyzed by immunofluorescence microscopy using total internal reflection fluorescence-ground state depletion and confocal microscopy. CLAMP localization was followed during the course of intestinal epithelial cell polarization as cells progressed from flat to compact, confluent monolayers. Protein interactions with endogenous CLAMP were determined in SKCO-15 cells using proximity ligation assays and co-immunoprecipitation. CLAMP was knocked down in SKCO-15 monolayers using small hairpin RNAs and cells were analyzed by immunoblot and immunofluorescence microscopy. The impact of CLAMP knock-down in migrating SKCO-15 cells was assessed using scratch-wound assays. Results CLAMP bound to actin and apical junctional complex proteins but not microtubules in IECs. In silico analysis predicted the calponin-homology domain of CLAMP to contain conserved amino acids required for actin binding. During IEC polarization, CLAMP distribution changed from primarily basal stress fibers and cytoplasm in undifferentiated cells to apical membranes and microvilli in differentiated monolayers. CLAMP accumulated in lamellipodia and filopodia at the leading edge of migrating cells in association with actin. CLAMP knock-down reduced the number of filopodia, perturbed filopodia polarity, and altered the organization of actin filaments within lamellipodia. Conclusions CLAMP is an actin-binding protein, rather than a microtubule-binding protein, in IECs. CLAMP distribution changes during intestinal epithelial cell polarization, regulates the formation of filopodia, and appears to assist in the organization of actin bundles within lamellipodia of migrating IECs. Studies are needed to define the CLAMP domains that interact with actin and whether its loss from IECs affects intestinal function.

Infectious diarrhea causes approximately 179 million illnesses annually in the US. Multiplex PCR assays for enteric pathogens detect enteropathogenic Escherichia coli (EPEC) in 12–29% of diarrheal stool samples from all age groups in developed nations. The aim of this study was to isolate and characterize EPEC from diarrhea samples identified as EPEC positive by BioFire Gastrointestinal Panel (GIP). EPEC is the second most common GIP-detected pathogen, equally present in sole and mixed infections peaking during summer months. EPEC bacterial load is higher in samples with additional pathogens. EPEC-GIP-positive stool samples were cultured on MacConkey II agar and analyzed by colony PCR for eaeA and bfpA to identify and classify EPEC isolates as typical (tEPEC) or atypical (aEPEC). EPEC were not recovered from the majority of stool samples with only 61 isolates obtained from 277 samples; most were aEPEC from adults. bfpA-mRNA was severely diminished in 3 of 4 bfpA-positive isolates. HeLa and SKCO-15 epithelial cells were infected with EPEC isolates and virulence-associated phenotypes, including adherence pattern, attachment level, pedestal formation, and tight junction disruption, were assessed. All aEPEC adherence patterns were represented with diffuse adherence predominating. Attachment rates of isolates adhering with defined adherence patterns were higher than tEPEC lacking bfpA (ΔbfpA). The majority of isolates formpedestals. All but one isolate initially increases but ultimately decreases transepithelial electrical resistance of SKCO-15 monolayers, similar to ΔbfpA. Most isolates severely disrupt occludin; ZO-1 disruption is variable. Most aEPEC isolates induce more robust virulence-phenotypes in vitro than ΔbfpA, but less than tEPEC-E2348/69.

Enteropathogenic Escherichia coli (EPEC) is a food-borne pathogen that causes infantile diarrhea worldwide. EPEC decreases the activity and surface expression of the key intestinal Cl−/HCO3− exchanger SLC26A3 [downregulated in adenoma (DRA)], contributing to the pathophysiology of early diarrhea. Little is known about the mechanisms governing membrane recycling of DRA. In the current study, Caco-2 cells were used to investigate DRA trafficking under basal conditions and in response to EPEC. Apical Cl−/HCO3− exchange activity was measured as DIDS-sensitive 125I− uptake. Cell surface biotinylation was performed to assess DRA endocytosis and exocytosis. Inhibition of clathrin-mediated endocytosis by chlorpromazine (60 μM) increased apical Cl−/HCO3− exchange activity. Dynasore, a dynamin inhibitor, also increased function and surface levels of DRA via decreased endocytosis. Perturbation of microtubules by nocodazole revealed that intact microtubules are essential for basal exocytic (but not endocytic) DRA recycling. Mice treated with colchicine showed a decrease in DRA surface levels as visualized by confocal microscopy. In response to EPEC infection, DRA surface expression was reduced partly via an increase in DRA endocytosis and a decrease in exocytosis. These effects were dependent on the EPEC virulence genes espG1 and espG2. Intriguingly, the EPEC-induced decrease in DRA function was unaltered in the presence of dynasore, suggesting a clathrin-independent internalization of surface DRA. In conclusion, these studies establish the role of clathrin-mediated endocytosis and microtubules in the basal surface expression of DRA and demonstrate that the EPEC-mediated decrease in DRA function and apical expression in Caco-2 cells involves decreased exocytosis.

Infections of the gastrointestinal tract (GIT) by pathogenic microorganisms such as bacteria, viruses, or protozoan parasites impose a huge burden on society. Despite intensive research to combat infections, increasing antibiotic resistance and newly emerging infectious diseases continue to pose a challenge in developing effective therapeutic interventions. This necessitates increased understanding of how these organisms alter host cell physiology to cause disease. Dissecting the complex and dynamic interactions between the host immune systems, epithelial responses and pathogens will more clearly elucidate the mechanisms of pathogenesis. Significant advances in our understanding of important aspects of host-pathogen interactions have been made during the last decades utilizing both in vivo animal models as well as recently using human organoid cultures. This chapter aims to highlight common human pathogens that infect the GIT with an emphasis on the microbial-epithelial interactions that may lead to the identification of novel drug targets to prevent or treat such infections and the associated consequences.

Background Numerous microbiota-based therapies are being evaluated for prevention of C. difficile infection (rCDI), a public health threat with high recurrence rates associated with the current standard of care. RBX2660, a standardized microbiota-based drug, was efficacious for preventing rCDI in a double-blinded Phase 2b clinical study (PUNCH CD 2). Herein we report the durability of RBX2660 beyond the initial primary clinical end-point of a subsequent Phase 2 open-label study, demonstrating rCDI prevention at 6 months post-treatment. Methods This prospective, multi-center, open-label Phase 2 study enrolled subjects who had experienced either ≥2 recurrences of CDI following standard-of-care antibiotic therapy or ≥2 episodes of severe CDI requiring hospitalization. Participants received up to two doses of RBX2660 delivered via enema with doses 7 days apart. The primary endpoint of the open-label clinical study defined efficacy as absence of CDI at 8 weeks from the last dose. Safety follow-ups and durability assessments occurred via telephone at 3, 6, 12, and 24 months. The study is ongoing, and not all subjects have completed their assessments. Results This study included 149 RBX2660-treated subjects and 110 historical control subjects from 31 and 4 centers, respectively, in the United States and Canada. At 8-weeks post-treatment, RBX2660’s efficacy in preventing rCDI (79.9%; 119/149) was higher than CDI-free rates in the historical control group (51.8%, 57/110; P < 0.001). Of the 119 subjects who were determined to be treatment success at 8 weeks, 117 have data through 6 months, of which 8 were exited for non-CDI reasons. Of those 109 subjects through the 6-month follow-up, 3 (2.8%) had a new CDI beyond 8 weeks after enema. The 6-month long-term CDI-free rate was 97.2% (106/109) (median follow-up: 182 days; mean: 177 days). Conclusion RBX2660, a microbiota-based drug, was efficacious for the prevention of recurrent CDI with long-term durability at 6-months post-treatment; a result consistent with 6-month rCDI prevention reported for the Phase 2b PUNCH CD 2 trial. Long-term follow-up of RBX2660 safety and efficacy 24 months is ongoing. This analysis was funded by Rebiotix Inc., Roseville, MN. Disclosures S. Mische, Rebiotix, Inc.: Employee, Salary. R. Orenstein, Rebiotix, Inc.: Scientific Advisor, Consulting fee. E. R. Dubberke, Rebiotix, Inc.: Scientific Advisor, Consulting fee. S. Khanna, Rebiotix, Inc.: Scientific Advisor, Consulting fee and Research support. G. Hecht, Rebiotix, Inc.: Scientific Advisor, Consulting fee. H. Dupont, Rebiotix, Inc.: Investigator, Research support. C. Lee, Rebiotix, Inc.: Scientific Advisor, Consulting fee. K. Blount, Rebiotix, Inc.: Employee, Salary.

Enteropathogenic Escherichia coli (EPEC) uses a type III secretion system to inject effector proteins into host intestinal epithelial cells causing diarrhoea. EPEC infection redistributes basolateral proteins β1-integrin and Na+ /K+ ATPase to the apical membrane of host cells. The Crumbs (Crb) polarity complex (Crb3/Pals1/Patj) is essential for epithelial cell polarisation and tight junction (TJ) assembly. Here, we demonstrate that EPEC displaces Crb3 and Pals1 from the apical membrane to the cytoplasm of cultured intestinal epithelial cells and colonocytes of infected mice. In vitro studies show that EspF, but not Map, alters Crb3, whereas both effectors modulate Pals1. EspF perturbs polarity formation in cyst morphogenesis assays and induces endocytosis and apical redistribution of Na+ /K+ ATPase. EspF binds to sorting nexin 9 (SNX9) causing membrane remodelling in host cells. Infection with ΔespF/pespFD3, a mutant strain that ablates EspF binding to SNX9, or inhibition of dynamin, attenuates Crb3 endocytosis caused by EPEC. In addition, infection with ΔespF/pespFD3 has no impact on Na+ /K+ ATPase endocytosis. These data support the hypothesis that EPEC perturbs apical-basal polarity in an EspF-dependent manner, which would contribute to EPEC-associated diarrhoea by disruption of TJ and altering the crucial positioning of membrane transporters involved in the absorption of ions and solutes.

Attachment of enterohemorrhagic Escherichia coli (EHEC) to intestinal epithelial cells is critical for colonization and is associated with localized actin assembly beneath bound bacteria. The formation of these actin “pedestals” is dependent on the translocation of effectors into mammalian cells via a type III secretion system (T3SS). Tir, an effector required for pedestal formation, localizes in the host cell plasma membrane and promotes attachment of bacteria to mammalian cells by binding to the EHEC outer surface protein Intimin. Actin pedestal formation has been shown to foster intestinal colonization by EHEC in some animal models, but the mechanisms responsible for this remain undefined. Investigation of the role of Tir-mediated actin assembly promoting host cell binding is complicated by other, potentially redundant EHEC-encoded binding pathways, so we utilized cell binding assays that specifically detect binding mediated by Tir-Intimin interaction. We also assessed the role of Tir-mediated actin assembly in two-step assays that temporally segregated initial translocation of Tir from subsequent Tir-Intimin interaction, thereby permitting the distinction of effects on translocation from effects on cell attachment. In these experimental systems, we compromised Tir-mediated actin assembly by chemically inhibiting actin assembly or by infecting mammalian cells with EHEC mutants that translocate Tir but are specifically defective in Tir-mediated pedestal formation. We found that an inability of Tir to promote actin assembly resulted in a significant and striking decrease in bacterial binding mediated by Tir and Intimin. Bacterial mutants defective for pedestal formation translocated type III effectors to mammalian cells with reduced efficiency, but the decrease in translocation could be entirely accounted for by the decrease in host cell attachment.

Summary Enteropathogenic Escherichia coli (EPEC) uses a type 3 secretion system to transfer effector proteins into the host intestinal epithelial cell. Several effector molecules contribute to tight junction disruption including EspG1 and its homologue EspG2 via a mechanism thought to involve microtubule destruction. The aim of this study was to investigate the contribution of EspG-mediated microtubule disruption to TJ perturbation. We demonstrate that wild type EPEC infection disassembles microtubules and induces the progressive movement of occludin away from the membrane and into the cytosol. Deletion of espG1/G2 attenuates both of these phenotypes. In addition, EPEC infection impedes barrier recovery from calcium switch, suggesting that inhibition of TJ restoration, not merely disruption, prolongs barrier loss. TJs recover more rapidly following infection with ΔespG1/G2 than with wild type EPEC, demonstrating that EspG1/G2 perpetuate barrier loss. Although EspG regulates ADP-ribosylation factor (ARF) and p21-activated kinase (PAK), these activities are not necessary for microtubule destruction or perturbation of TJ structure and function. These data strongly support a role for EspG1/G2 and its associated effects on microtubules in delaying the recovery of damaged tight junctions caused by EPEC infection.

Purpose of review The purpose of this study is to summarize the recent developments in small intestinal bacterial infections. Recent findings This review focuses on aspects of intestinal bacterial infection concerning research developments related to pathogenesis, new therapeutic agents and approaches, as well as potential new vaccine targets. Summary In terms of drug utilization, azithromycin was successfully used to eradicate a Shiga toxin producing Escherichia coli (enterohemorrhagic E. coli) without harmful effects. In the case of Clostridium difficile, fidaxomicin was found to be comparable to or superior to vancomycin depending on study conditions and whether there was concomitant antibiotic use. A novel research finding is the role of galectin 8, which is a danger-sensing lectin, which plays a role in targeting Salmonella for autophagy. In addition, several enteropathogenic E. coli and Shigella effectors were found to inactivate members of the nuclear factor kappa B pathway.

The brain-gut axis allows bidirectional communication between the central nervous system (CNS) and the enteric nervous system (ENS), linking emotional and cognitive centers of the brain with peripheral intestinal functions. Recent experimental work suggests that the gut microbiota have an impact on the brain-gut axis. A group of experts convened by the International Scientific Association for Probiotics and Prebiotics (ISAPP) discussed the role of gut bacteria on brain functions and the implications for probiotic and prebiotic science. The experts reviewed and discussed current available data on the role of gut microbiota on epithelial cell function, gastrointestinal motility, visceral sensitivity, perception and behavior. Data, mostly gathered from animal studies, suggest interactions of gut microbiota not only with the enteric nervous system but also with the central nervous system via neural, neuroendocrine, neuroimmune and humoral links. Microbial colonization impacts mammalian brain development in early life and subsequent adult behavior. These findings provide novel insights for improved understanding of the potential role of gut microbial communities on psychological disorders, most particularly in the field of psychological comorbidities associated with functional bowel disorders like irritable bowel syndrome (IBS) and should present new opportunity for interventions with pro- and prebiotics.

Enteropathogenic E. coli (EPEC) infection is a major cause of infantile diarrhea in the developing world. Using a type-three secretion system, bacterial effector proteins are transferred to the host cell cytosol where they affect multiple physiological functions, ultimately leading to diarrheal disease. Disruption of intestinal epithelial cell tight junctions is a major consequence of EPEC infection and is mediated by multiple effector proteins, among them EspG1 and its homologue EspG2. EspG1/G2 contribute to loss of barrier function via an undefined mechanism that may be linked to their disruption of microtubule networks. Recently new investigations have identified additional roles for EspG. Sequestration of active ADP-ribosylating factor (ARF) proteins and promotion of p21-activated kinase (PAK) activity as well as inhibition of Golgi-mediated protein secretion have all been linked to EspG. In this review, we examine the functions of EspG1/G2 and discuss potential mechanisms of EspG-mediated tight junction disruption.

Intestinal pathogens have a wide variety of strategies for communicating with host epithelial cells. This review highlights a few key examples of those strategies. Enteropathogenic Escherichia coli (EPEC) use a type III secretion system (T3SS) to alter host ion transport through both transcriptional and post-translational mechanisms. Salmonella use a similar T3SS to invade host cells and modify an intracellular vacuole, which also impacts host vesicle trafficking. Helicobacter pylori use host cell integrins to provide a conformational change which drives the type IV secretion system into the host cell for delivery of CagA. The novel type VI section systems are phage-like apparati that deliver VgrG-1, which causes actin cross-linking and fluid accumulation in a suckling mouse model. An entirely different delivery mechanism is the outer membrane vesicle (OMV) which is composed of bacterial outer membrane wrapped around contents of the periplamsic space. Enterotoxigenic E. coli use OMVs to deliver bundles of heat labile enterotoxin to host cells. Finally we discuss the host responses to these varied methods of communication.

Infectious diarrhea is a major contributor of child morbidity and mortality in developing nations. Murine models to study the pathogenesis of infectious diarrhea caused by organisms such as enteropathogenic E. coli (EPEC) and enterohemorrhagic E. coli (EHEC) are not fully characterized. More emphasis has been placed on infection of mice with the murine specific pathogen Citrobacter rodentium. While these three organisms are genetically related they are not identical. Our goal was to better characterize the murine model of EPEC and EHEC infection by using bioluminescent bacteria to determine temporal and spatial colonization of these two human pathogens. EPEC and EHEC were transformed with a bacterial luciferase expression plasmid containing the constitutive OmpC promoter. C57BL/6 mice were orally inoculated with bioluminescent EPEC or EHEC and bacterial localization in the intestine was monitored ex vivo and in vivo by IVIS. At 3 days after infection, EPEC, EHEC and Citrobacter rodentium were all localized in the cecum and colon. EPEC colonization peaked at day 2-3 and was undetectable by day 7. The bioluminescent EPEC adheres to the cecum and colon of the mouse intestine. However, when EPEC infected mice were administered xylazine/ketamine for in vivo live imaging, the EPEC persisted at high densities for up to 31 days. This is the first report of a bioluminescent imaging of luciferase expressing EPEC in a mouse model.

Fecal microbiota transplantation (FMT) has emerged as an effective treatment for C. difficile infection (CDI) refractory to antibiotic therapy alone. Given the important roles of microbiota in the function of the gastrointestinal tract and other aspects of human physiology, there is also growing interest in studying FMT for other clinical indications. The U.S. Food and Drug Administration has determined that FMT constitutes a drug and is an investigational agent; as such an investigational new drug application (IND) is encouraged in treatment of recurrent CDI and required to administer FMT for any other indications. Most physicians and investigators do not have the regulatory experience necessary to navigate the IND process easily. Our aim is to provide guidance to others who are interested in obtaining an IND to perform or study FMT.

Enteric bacterial pathogens have evolved sophisticated strategies to evade host immune defences. Some pathogens deliver anti-inflammatory effector molecules into the host cell cytoplasm via a type III secretion system (T3SS). Enteropathogenic Escherichia coli (EPEC) inhibits inflammation by an undefined, T3SS-dependent mechanism. Two proteins encoded outside of the EPEC locus of enterocyte effacement (LEE) pathogenicity island, non-LEE-encoded effector H1 (NleH1) and H2 (NleH2), display sequence similarity to Shigella flexneri OspG, which inhibits activation of the pro-inflammatory transcription factor NF-κB. We hypothesized that the anti-inflammatory effects of EPEC were mediated by NleH1 and NleH2. In this study, we examined the effect of NleH1/H2 on the NF-κB pathway. We show that NleH1/H2 are secreted via the T3SS and that transfection of cells with plasmids harbouring nleH1 or nleH2 decreased IKK-β-induced NF-κB activity and attenuated TNF-α-induced degradation of phospho-IκBα by preventing ubiquitination. Serum KC levels were higher in mice infected with ΔnleH1H2 than those infected with WT EPEC, indicating that NleH1/H2 dampen pro-inflammatory cytokine expression. ΔnleH1H2 was cleared more rapidly than WT EPEC while complementation of ΔnleH1H2 with either NleH1 or NleH2 prolonged colonization. Together, these data show that NleH1 and NleH2 function to dampen host inflammation and facilitate EPEC colonization during pathogenesis.

Shiga toxin (Stx) is implicated in the development of hemorrhagic colitis and hemolytic-uremic syndrome, but early symptoms of enterohemorrhagic Escherichia coli (EHEC) infection such as nonbloody diarrhea may be Stx independent. In this study, we defined the effects of EHEC, in the absence of Stx, on the intestinal epithelium using a murine model. EHEC colonization of intestines from two groups of antibiotic-free and streptomycin-treated C57Bl/6J mice were characterized and compared. EHEC colonized the cecum and colon more efficiently than the ileum in both groups; however, greater amounts of tissue-associated EHEC were detected in streptomycin-pretreated mice. Imaging of intestinal tissues of mice infected with bioluminescent EHEC further confirmed tight association of the bacteria with the cecum and colon. Greater numbers of EHEC were also cultured from stool samples obtained from streptomycin-pretreated mice, as compared with those that received no antibiotics. Transmission electron microscopy shows that EHEC infection leads to microvillous effacement of mouse colonocytes. Hematoxylin and eosin staining of the colonic tissues of infected mice revealed a slight increase in the number of lamina propria polymorphonuclear leukocytes. Transmucosal electrical resistance, a measure of epithelial barrier function, was reduced in the colonic tissues of infected animals. Increased mucosal permeability to 4- kDa FITC-dextran was also observed in the colonic tissues of infected mice. Immunofluorescence microscopy showed that EHEC infection resulted in redistribution of the tight junction (TJ) proteins occludin and claudin-3 and increased the expression of claudin-2, whereas ZO-1 localization remained unaltered. Quantitative real-time PCR showed that EHEC altered mRNA transcription of OCLN, CLDN2, and CLDN3. Most notably, claudin-2 expression was significantly increased and correlated with increased intestinal permeability. Our data indicate that C57Bl/6J mice serve as an in vivo model to study the physiological effects of EHEC infection on the intestinal epithelium and suggest that altered transcription of TJ proteins has a role in the increase in intestinal permeability.

Enteropathogenic Escherichia coli (EPEC) infection requires the injection of effector proteins into intestinal epithelial cells (IECs) via type 3 secretion. Type 3-secreted effectors can interfere with IEC signalling pathways via specific protein-protein interactions. For example, E. coli secreted protein F (EspF) binds sorting nexin 9 (SNX9), an endocytic regulator, resulting in tubulation of the plasma membrane. Our aim was to determine the mechanism of EspF/SNX9-induced membrane tubulation. Point mutation of the SNX9 lipid binding domains or truncation of the EspF SNX9 binding domains significantly inhibited tubulation, as did inhibition of clathrin coated pit (CCP) assembly. Although characterized as non-invasive, EPEC are known to invade IECs in vitro and in vivo. Indeed, we found significant invasion of Caco-2 cells by EPEC, which, like tubulation, was blocked by pharmacological inhibition of CCPs. Interestingly, however, inhibition of dynamin activity did not prevent tubulation or EPEC invasion, which is in contrast to Salmonella invasion, which requires dynamin activity. Our data also indicate that EPEC invasion is dependent on EspF and its interaction with SNX9. Together, these findings suggest that EspF promotes EPEC invasion of IECs by harnessing the membrane-deforming activity of SNX9.

Enteropathogenic Escherichia coli (EPEC) is a diarrhoeal pathogen that adheres to epithelial cells of the small intestine and uses a type III secretion system to inject effector proteins into host cells. EPEC infection leads to disruption of host intestinal tight junctions that are important for maintaining intestinal barrier function. This disruption is dependent on the bacterial type III secretion system, as well as the translocated effectors EspF and Map. Here we show that a third type III translocated bacterial effector protein, NleA, is also involved in tight junction disruption during EPEC infection. Using the drug Brefeldin A, we demonstrate that the effect of NleA on tight junction integrity is related to its inhibition of host cell protein trafficking through COPII-dependent pathways. These results suggest that NleA's striking effect on virulence is mediated, at least in part, via its role in disruption of intestinal barrier function.

Background & Aims Serotonin transporter (SERT) plays a critical role in regulating serotonin (5-hydroxytryptamine [5-HT]) availability in the gut. Elevated 5-HT levels are associated with diarrheal conditions such as irritable bowel syndrome and enteric infections. Whether alteration in SERT activity contributes to the pathophysiology of diarrhea induced by the food-borne pathogen enteropathogenic Escherichia coli (EPEC) is not known. The present studies examined the effects of EPEC infection on SERT activity and expression in intestinal epithelial cells and elucidated the underlying mechanisms. Methods Caco-2 cells as a model of human intestinal epithelia and EPEC-infected C57BL/6J mouse model of infection were utilized. SERT activity was measured as Na + and Cl − dependent 3 [H] 5-HT uptake. SERT expression was measured by real-time quantitative reverse-transcription polymerase chain reaction, Western blotting, and immunofluorescence studies. Results Infection of Caco-2 cells with EPEC for 30–120 minutes decreased apical SERT activity ( P max of the transporter; whereas cell surface biotinylation studies revealed no alteration in the cellular or plasma membrane content of SERT in Caco-2 cells. EPEC infection of mice (24 hours) reduced SERT immunostaining with a corresponding decrease in SERT messenger RNA levels, 5-HT uptake, and mucosal 5-HT content in the small intestine. Conclusions Our results demonstrate inhibition of SERT by EPEC and define the mechanisms underlying these effects. These data may aid in the development of a novel pharmacotherapy to modulate the serotonergic system in treatment of infectious diarrheal diseases.

Infection with the gram-negative enteropathogenic Escherichia coli (EPEC), a food-borne pathogen, represents a significant risk to human health. Whereas diarrhea is a major consequence of this infection, malnutrition also occurs especially in severe and prolonged cases, which may aggravate the health status of the infected hosts. Here we examined the effect of EPEC infection on the intestinal uptake of the water-soluble vitamin B1 (thiamin) using an established human intestinal epithelial Caco-2 cell model. The results showed that infecting Caco-2 cells with wild-type EPEC (but not with nonpathogenic E. coli, killed EPEC, or filtered supernatant) leads to a significant ( P < 0.01) inhibition in thiamin uptake. Kinetic parameters of both the nanomolar (mediated by THTR-2) and the micromolar (mediated by THTR-1) saturable thiamin uptake processes were affected by EPEC infection. Cell surface expression of hTHTR-1 and -2 proteins, (determined by the biotinylation method) showed a significantly ( P < 0.01) lower expression in EPEC-treated cells compared with controls. EPEC infection also affected the steady-state mRNA levels as well as promoter activity of the SLC19A2 and SLC19A3 genes. Infecting Caco-2 cells with EPEC mutants that harbor mutations in the escN gene (which encodes a putative ATPase for the EPEC type III secretion system, TTSS) or the espA, espB, or espD genes (which encode structural components of the TTSS) did not affect thiamin uptake. On the other hand, mutations in espF and espH genes (which encode effector proteins) exhibited partial inhibition in thiamin uptake. These results demonstrate for the first time that EPEC infection of human intestinal epithelial cells leads to inhibition in thiamin uptake via effects on physiological and molecular parameters of hTHTR-1 and -2. Furthermore, the inhibition appears to be dependent on a functional TTSS of EPEC.

Epithelial cells lining the colon do not normally express galanin type 1 receptors (Gal1Rs). However, subsequent to infection with enteric pathogens such as Salmonella typhimurium, the Gal1R is rapidly upregulated in colonocytes where it contributes to the excess fluid production associated with diarrhoea. Humans infected with non-typhoid Salmonella respond differently according to age: infants develop diarrhoea but not bacteraemia and survive, while the elderly become bacteraemic and die. Thus the aim of this study was to determine if age-related differences exist in response to S typhimurium infection in mice, and whether these differences are due to altered Gal1R expression. Wild-type C57BL/6J mice that were 2 and 15 months old, as well as 2-month-old Gal1R knockout mice, were infected by gavage. Young wild-type mice expressed Gal1R in response to infection, had increased colonic fluid secretion, low rates of bacteraemia and survived. In contrast, 15-month-old wild-type mice expressed fewer Gal1Rs in response to infection, had attenuated increases in colonic fluid secretion, high rates of bacteraemia and died. A similar profile was noted in 2-month-old Gal1R knockout mice. Addition of polyethylene glycol to the drinking water of 15-month-old wild-type mice increased colonic fluid secretion and reduced rates of bacteraemia to those observed in 2-month-old wild-type mice and eliminated fatalities. The difference in response to S typhimurium infection with age may be due, at least in part, to decreased Gal1R expression and decreased amounts of colonic fluid secretion.

Enteropathogenic E. coli (EPEC) are a leading cause of infantile diarrhea in developing countries, resulting in millions of deaths each year. EPEC secrete virulence factors, also called effectors, directly into host intestinal epithelial cells via type three secretion systems. Secreted effectors then affect host signaling pathways to induce several phenotypes, which ultimately lead to disease. Among the over 20 secreted effectors is E. coli secreted protein F (EspF), a 206 amino acid protein believed to be central to EPEC pathogenesis, as it disrupts tight junction structure and function. Although the mechanism by which this occurs is unknown, EspF has recently been found to contain several protein–protein interaction domains that may be involved. We have shown EspF to interact with the endocytic regulators sorting nexin 9 (SNX9) and N-WASP via non-exclusive binding sites. These interactions induce actin polymerization in vitro, and interaction with SNX9 alters its endocytic activity, as EspF induces the formation of tubular vesicles in a manner dependent upon its interaction with SNX9. EspF, therefore, appears to hijack endocytic regulation via SNX9 and possibly N-WASP interaction, to affect an as yet unidentified pathogenic phenotype.