Enteropathogenic Escherichia Coli Infection Research Articles

Bioprospecting of inhibitors of EPEC virulence from metabolites of marine actinobacteria from the Arctic Sea.

A considerable number of antibacterial agents are derived from bacterial metabolites. Similarly, numerous known compounds that impede bacterial virulence stem from bacterial metabolites. Enteropathogenic Escherichia coli (EPEC) is a notable human pathogen causing intestinal infections, particularly affecting infant mortality in developing regions. These infections are characterized by microvilli effacement and intestinal epithelial lesions linked with aberrant actin polymerization. This study aimed to identify potential antivirulence compounds for EPEC infections among bacterial metabolites harvested from marine actinobacteria (Kocuria sp. and Rhodococcus spp.) from the Arctic Sea by the application of virulence-based screening assays. Moreover, we demonstrate the suitability of these antivirulence assays to screen actinobacteria extract fractions for the bioassay-guided identification of metabolites. We discovered a compound in the fifth fraction of a Kocuria strain that interferes with EPEC-induced actin polymerization without affecting growth. Furthermore, a growth-inhibiting compound was identified in the fifth fraction of a Rhodococcus strain. Our findings include the bioassay-guided identification, HPLC-MS-based dereplication, and isolation of a large phospholipid and a likely antimicrobial peptide, demonstrating the usefulness of this approach in screening for compounds capable of inhibiting EPEC virulence.

Modulating Liquid-Liquid Phase Separation of Nck Adaptor Protein against Enteropathogenic Escherichia coli Infection.

Signaling proteins often form biomolecular condensates through liquid-liquid phase separation (LLPS) during intracellular signal transduction. Modulating the LLPS property of intracellular protein condensates will redirect intracellular signals and provide a potential way to regulate cellular physiology. Phosphorylation of multiple tyrosine residues of the transmembrane receptor nephrin is known to drive the LLPS of the adaptor protein Nck and neuronal Wiskott-Aldrich Syndrome protein (N-WASP) and form the Nck signaling complex. Phosphorylation of the translocated intimin receptor (Tir) in the host cell may recruit this enteropathogenic Escherichia coli (EPEC) virulence factor to the Nck signaling complex and lead to the entry of EPEC into the intestine cell. In this work, we first identified a phosphotyrosine (pY)-containing peptide 3pY based on the sequence similarity of nephrin and Tir; 3pY promoted the LLPS of Nck and N-WASP, mimicking the role of phosphorylated nephrin. Next, we designed a covalent blocker of Nck, peptide p1 based on the selected pY peptides, which site-selectively reacted with the SH2 domain of Nck (Nck-SH2) at Lys331 through a proximity-induced reaction. The covalent reaction of p1 with Nck blocked the protein binding site of Nck-SH2 and disintegrated the 3pY/Nck/N-WASP condensates. In the presence of membrane-translocating peptide L17E, p1 entered Caco-2 cells in the cytosol, reduced the number of Nck puncta, and rendered Caco-2 cells resistant to EPEC infection. Site-selective covalent blockage of Nck thereby disintegrates intracellular Nck condensates, inhibits actin reorganization, and shuts down the entrance pathway of EPEC. This work showcases the promotion or inhibition of protein phase separation by synthetic peptides and the use of reactive peptides as LLPS disruptors and signal modulators.

Enteropathogenic E. coli infection co-elicits lysosomal exocytosis and lytic host cell death.

Enteropathogenic Escherichia coli (EPEC) infection is a significant cause of gastroenteritis, mainly in children. Therefore, studying the mechanisms of EPEC infection is an important research theme. EPEC modulates its host cell life by injecting via a type III secretion machinery cell death modulating effector proteins. For instance, while EspF and Map promote mitochondrial cell death, EspZ antagonizes cell death. We show that these effectors also control lysosomal exocytosis, i.e., the trafficking of lysosomes to the host cell plasma membrane. Interestingly, the capacity of these effectors to induce or protect against cell death correlates completely with their ability to induce LE, suggesting that the two processes are interconnected. Modulating host cell death is critical for establishing bacterial attachment to the host and subsequent dissemination. Therefore, exploring the modes of LE involvement in host cell death is crucial for elucidating the mechanisms underlying EPEC infection and disease.

Virtual Screening Assisted Search for Inhibitors of the Translocated Intimin Receptor of Enteropathogenic Escherichia Coli.

This study aimed to identify inhibitors of the translocated intimin receptor (Tir) of enteropathogenic Escherichia coli (EPEC). EPEC is an intestinal pathogen that causes diarrhea and is a major health concern worldwide. Because Tir is a key virulence factor involved in EPEC pathogenesis, inhibiting its function is a potential strategy for controlling EPEC infections. Virtual screening was applied to chemical libraries to search for compounds that inhibit Tir-mediated bacterial adherence to host cells. Three sites were targeted using the cocrystal structure published earlier. A selection of compounds was then assessed in a cell-based infection model and fluorescence microscopy assay. The results of this study provide a basis for further optimization and testing of Tir inhibitors as potential therapeutic agents for EPEC infections.

STAT1/MUC4 activation promotes antimicrobial peptide production to reduce intestinal epithelium barrier injury caused by enteropathogenic Escherichia coli infection

IntroductionAntimicrobial peptides (AMPs) are endogenous peptides that have been identified to alleviate intestinal epithelial barrier inflammation and dysfunction caused by enteropathogenic Escherichia coli (EPEC) infection; nonetheless, the upstream molecular mechanism of the production of AMPs is poorly understood.Material and methodsThe binding of signal transducer and activator of transcription (STAT) 1 (STAT1) to mucin 4 (MUC4) was examined by Co-Immunoprecipitation assay. To detect the influence of STAT1 and MUC4 expression, C57BL/6 mice model of EPEC infection in vivo and EPEC infected intestinal epithelial cells (IEC) in vitro model was established. Expressions of STAT1, MUC4, phosphorylated (p)-STAT1, proinflammatory cytokines, zonula occludens-1 (ZO-1) and AMP-related genes in mouse ileum and/or IEC were analyzed by immunohistochemical test, immunofluorescence assay, Western blot, and/or qRT-PCR. Meanwhile, IEC viability and apoptosis were measured using CCK-8 assay and flow cytometry.Resultsp-STAT1, MUC4, ZO-1 and AMP-related gene were lowly expressed in the ileum of EPEC-infected mice. p-STAT1 and MUC4 bound to each other. The expressions of STAT1 and MUC4 were decreased in EPEC-infected IEC. Overexpression of STAT1 resisted EPEC-induced viability decrease, apoptosis promotion, ZO-1 activity inhibition, release of proinflammatory cytokines, and downregulation of MUC4 and AMP-related genes in IEC. MUC4 knockdown partly counteracted the effect of STAT1 overexpression, but did not affect the forced STAT1 overexpression in EPEC-infected IEC.ConclusionsSTAT1/MUC4 pathway activation promotes AMP production to mitigate intestinal epithelium barrier injury caused by EPEC infection.

The role of Nod-like receptors in modulating the microbiota-gut-brain axis following neonatal bacterial infection

Objective: Pediatric infection with diarrheal pathogens causes cognitive deficits in adulthood via an unknown mechanism. Neonatal infection with a bacterial enteric pathogen disrupts the development of the microbiota-gut-brain (MGB) axis in adult mice. Given the role of pattern recognition receptors, including Nucleotide Oligomerization Domain 1 (Nod1) in innate immune responses to infection, our objective was to establish their role in disrupted MGB axis communication following neonatal enteropathogenic Escherichia coli (EPEC) infection. Hypothesis: Intestinal epithelial cell (IEC) Nod1 modulates disrupted MGB axis communication following neonatal EPEC infection. Methods: C57BL/6 (male/female) IEC Nod1 conditional knockout mice (IEC cKO; Cre+) and wild type (WT) littermate controls (Cre-) were used. Neonatal mice were infected on postnatal day 7 (P7) with EPEC (10 5 CFU; strain e2348/69; orogastric gavage), or vehicle control (Luria broth). Colonization was assessed at P14 and P21 (MacConkey agar plating) and disease monitored via weight loss and ileal inflammation (qPCR). Behavior and neuroinflammation were assessed in adulthood (6-8 weeks). Behavioral tests included the novel object recognition task (NOR) for cognitive function, the light/dark (L/D) box for anxiety-like behavior, and open field test (OFT) for general and locomotor behavior. Results: Neonatally EPEC-infected Nod1 cKO mice demonstrated blunted ileal inflammation, with similar decreased expression of Il6, Tlr2, Tlr4, and Nod2 in cKO vs WT EPEC-infected mice ( P = NS, n = 4-6 ), with no evidence of inflammation in uninfected controls (WT or cKO). In contrast, evidence of hippocampal neuroinflammation was present in EPEC-infected Nod1 cKO mice, with increased expression of Iba1, Il6, and Il22 ( P < 0.0001, n = 4-6), compared to sham-infected Nod1 cKO mice or uninfected control mice. In addition, EPEC-infected Nod1 cKO mice demonstrated decreased hippocampal expression of the neurogenesis marker Bdnf ( P < 0.0001, n ≥ 4) compared to EPEC-infected WT mice, or uninfected mice. With respect to behavior, EPEC-infected WT mice had a lower exploration ratio (49.5 ± 1.7% vs. 54.6 ± 1.7%, P = 0.0035, n ≥ 8), whereas EPEC-infected Nod1 cKO mice displayed normal exploration of a novel object compared to uninfected Nod1 cKO mice ( P = NS, n = 10-12), without impacting anxiety-like behavior ( P = NS, n = 10-12). Conclusions: IEC Nod1 is important for maintaining MGB axis communication following neonatal EPEC infection. We speculate that decreased ileal inflammation due to absence of Nod1 IEC expression prevents cognitive deficits in adulthood, despite the evidence of neuroinflammation and impaired neurogenesis. Inflammation in the gut and brain will be further characterized by immunofluorescence in ongoing studies. NIH R01AT009365; NIH R21MH108154. Olivia Orahood is an active-duty member of the United States Space Force. The views expressed in this article are those of the author and do not reflect the official policy or position of the United States Space Force, United States Air Force, Department of Defense, or the U.S. Government This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Pathophysiology of Enteropathogenic Escherichia coli-induced Diarrhea.

Enteropathogenic Escherichia coli (EPEC) are important diarrheal pathogens of infants and young children. Since the availability of molecular diagnosis methods, we now have new insights into the incidence and prevalence of these infections. Recent epidemiological studies indicate that atypical EPEC (aEPEC) are seen more frequently than typical EPEC (tEPEC) worldwide, including in both endemic diarrhea and diarrhea outbreaks. Therefore, it is important to further characterize the pathogenicity of these emerging strains. The virulence mechanisms and pathophysiology of the attaching and effacing lesion (A/E) and the type-three-secretion-system (T3SS) are complex but well-studied. A/E strains use their pool of locus of enterocyte effacement (LEE)-encoded and non-LEE-encoded effector proteins to subvert and modulate cellular and barrier properties of the host. However, the exact mechanisms of diarrhea in EPEC infection are not completely understood. From the clinical perspective, there is a need for fast, easy, and inexpensive diagnostic methods to define optimal treatment and prevention for children in endemic areas. In this article, we present a review of the classification of EPEC, epidemiology, pathogenesis of the disease caused by these bacteria, determinants of virulence, alterations in signaling, determinants of colonization vs. those of disease, and the limited information we have on the pathophysiology of EPEC-induced diarrhea. This article combines peer-reviewed evidence from our own studies and the results of an extensive literature search in the databases PubMed, EMBASE, and Scopus.

The Gut Microbiome of an Indigenous Agropastoralist Population in a Remote Area of Colombia with High Rates of Gastrointestinal Infections and Dysbiosis.

An Indigenous agropastoralist population called the Wiwa from the Sierra Nevada de Santa Marta, in North-East Colombia, shows high rates of gastrointestinal infections. Chronic gut inflammatory processes and dysbiosis could be a reason, suggesting an influence or predisposing potential of the gut microbiome composition. The latter was analyzed by 16S rRNA gene amplicon next generation sequencing from stool samples. Results of the Wiwa population microbiomes were associated with available epidemiological and morphometric data and compared to control samples from a local urban population. Indeed, locational-, age-, and gender-specific differences in the Firmicutes/Bacteriodetes ratio, core microbiome, and overall genera-level microbiome composition were shown. Alpha- and ß-diversity separated the urban site from the Indigenous locations. Urban microbiomes were dominated by Bacteriodetes, whereas Indigenous samples revealed a four times higher abundance of Proteobacteria. Even differences among the two Indigenous villages were noted. PICRUSt analysis identified several enriched location-specific bacterial pathways. Moreover, on a general comparative scale and with a high predictive accuracy, we found Sutterella associated with the abundance of enterohemorrhagic Escherichia coli (EHEC), Faecalibacteria associated with enteropathogenic Escherichia coli (EPEC) and helminth species Hymenolepsis nana and Enterobius vermicularis. Parabacteroides, Prevotella, and Butyrivibrio are enriched in cases of salmonellosis, EPEC, and helminth infections. Presence of Dialister was associated with gastrointestinal symptoms, whereas Clostridia were exclusively found in children under the age of 5 years. Odoribacter and Parabacteroides were exclusively identified in the microbiomes of the urban population of Valledupar. In summary, dysbiotic alterations in the gut microbiome in the Indigenous population with frequent episodes of self-reported gastrointestinal infections were confirmed with epidemiological and pathogen-specific associations. Our data provide strong hints of microbiome alterations associated with the clinical conditions of the Indigenous population.

Burden of disease estimation based on Escherichia coli quantification in ready-to-eat meals served in Portuguese institutional canteens

Shiga toxin-producing E. coli (STEC), enterotoxigenic E. coli (ETEC) and enteropathogenic E. coli (EPEC) are associated with the onset of gastroenteritis with different severities, and STEC has been associated with other sequelae, such as hemolytic uremic syndrome and end stage renal disease. The main goal of this study was to estimate the annual foodborne burden of disease associated with STEC, ETEC and EPEC infection based on E. coli quantification results obtained in the routine analysis of ready-to-eat meals served in institutional canteens from 2018 to 2019. A stochastic Quantitative Microbial Risk Assessment (QMRA) model was used to estimate the expected number of cases per health outcome and Disability Adjusted Life Years (DALYs). Assuming a daily consumption of a whole meal portion (450 g), the estimated burden was of 4.2 x 10−3 DALYs/person/year for STEC infection, 2.82 x 10−4 DALYs/person/year for ETEC infection and 7.91 x 10−6 DALYs/person/year for EPEC infection. Additionally, using the Sobol method, the sensitivity analysis revealed that the factors with higher influence on the final output (DALYs) were the pathotype's prevalence for the STEC model, the number of people exposed to the hazard for the ETEC model and E. coli concentrations for the EPEC model.

Antigenic evaluation of extracted fimbrial protein obtained from pathogenic Escherichia coli isolated from diarrheic camel neonates

Enteropathogenic Escherichia coli (EPEC) were considered prime reason of diarrhea among neonatal livestock animals in developing countries and could be of public health importance via contaminated milk and meat. Continual attempts were conducted to combat this illness using various antigenic determinants. This study was performed on four E. coli serotypes O26, O45, O103 and O111, which were previously recovered from diarrheic camel calves in Giza, Egypt. Extraction of fimbrial proteins was carried out through dialysis then evaluation of their immunogenic activity was preceded. SDS-PAGE electrophoresis was performed on crude extracted fimbrial proteins and revealed single band for each isolate ranged from 22 to 33kDa. Immunobloting was implemented on the extracted crude fimbrial proteins against E.coli O26 antisera formerly prepared in rabbit.These findings suggested that the fimbrial proteins are of immunogenicity importance and can serve as a protective passive vaccine antigen in prevention of diarrhea caused by EPEC and ETEC infection in camel calves.

Targeting of microvillus protein Eps8 by the NleH effector kinases from enteropathogenic E. coli

Attaching and effacing (AE) lesion formation on enterocytes by enteropathogenic Escherichia coli (EPEC) requires the EPEC type III secretion system (T3SS). Two T3SS effectors injected into the host cell during infection are the atypical kinases, NleH1 and NleH2. However, the host targets of NleH1 and NleH2 kinase activity during infection have not been reported. Here phosphoproteomics identified Ser775 in the microvillus protein Eps8 as a bona fide target of NleH1 and NleH2 phosphorylation. Both kinases interacted with Eps8 through previously unrecognized, noncanonical "proline-rich" motifs, PxxDY, that bound the Src Homology 3 (SH3) domain of Eps8. Structural analysis of the Eps8 SH3 domain bound to a peptide containing one of the proline-rich motifs from NleH showed that the N-terminal part of the peptide adopts a type II polyproline helix, and its C-terminal "DY" segment makes multiple contacts with the SH3 domain. Ser775 phosphorylation by NleH1 or NleH2 hindered Eps8 bundling activity and drove dispersal of Eps8 from the AE lesion during EPEC infection. This finding suggested that NleH1 and NleH2 altered the cellular localization of Eps8 and the cytoskeletal composition of AE lesions during EPEC infection.

Site specific incidence rate of genomic subtypes of enteropathogenic Escherichia coli and association with enteric inflammation and child growth

There is a lack of information highlighting the possible association between the genomic subtypes of enteropathogenic Escherichia coli (EPEC) on environmental enteric dysfunction (EED) and on linear growth during childhood. Genomic subtypes of EPEC from stool samples collected from 1705 children enrolled in the MAL-ED birth cohort were detected by TaqMan Array Cards. We measured site-specific incidence rate by using Poisson regression models, identified the risk factors and estimated the association of genomic subtypes of EPEC with the composite EED score and linear growth at 24 months of age. In general, the highest incidence rate (39%) was found among children having aEPEC infection, which was the greatest in Tanzania (54%). Exclusive breastfeeding and having an improved sanitation facility were found to be protective factors against EPEC infection. In the multivariate models, in overall effect after adjusting for the potential covariates aEPEC showed strong positive associations with the EED scores and tEPEC showed a positive association with poor linear growth at 24 months of age. Our analyses may lay the cornerstone for a prospective epidemiologic investigation for a potential vaccine development aimed at reducing the burden of EPEC infections and combat childhood malnutrition.

Pathophysiology of enteropathogenic Escherichia coli during a host infection.

Enteropathogenic Escherichia coli (EPEC) is a major cause of infantile diarrhea in developing countries. However, sporadic outbreaks caused by this microorganism in developed countries are frequently reported recently. As an important zoonotic pathogen, EPEC is being monitored annually in several countries. Hallmark of EPEC infection is formation of attaching and effacing (A/E) lesions on the small intestine. To establish A/E lesions during a gastrointestinal tract (GIT) infeciton, EPEC must thrive in diverse GIT environments. A variety of stress responses by EPEC have been reported. These responses play significant roles in helping E. coli pass through GIT environments and establishing E. coli infection. Stringent response is one of those responses. It is mediated by guanosine tetraphosphate. Interestingly, previous studies have demonstrated that stringent response is a universal virulence regulatory mechanism present in many bacterial pathogens including EPEC. However, biological signficance of a bacterial stringent response in both EPEC and its interaction with the host during a GIT infection is unclear. It needs to be elucidated to broaden our insight to EPEC pathogenesis. In this review, diverse responses, including stringent response, of EPEC during a GIT infection are discussed to provide a new insight into EPEC pathophysiology in the GIT.

Exopolysaccharides from Bifidobacterium animalis Ameliorate Escherichia coli-Induced IPEC-J2 Cell Damage via Inhibiting Apoptosis and Restoring Autophagy.

Enteropathogenic Escherichia coli (EPEC) is a common zoonotic pathogen that causes acute infectious diarrhea. Probiotics like Bifidobacterium are known to help prevent pathogen infections. The protective effects of Bifidobacterium are closely associated with its secretory products exopolysaccharides (EPS). We explored the effects of the EPS from Bifidobacterium animalis subsp. lactis (B. lactis) on ameliorating the damage of an intestinal porcine epithelial cell line (IPEC-J2) during EPEC infection. Pretreatment with EPS alleviated EPEC-induced apoptosis through the restoration of cell morphology and the downregulation of protein expressions of cleaved-caspase 8, cleaved-caspase 3, and cleaved-PARP. EPS-mediated remission of apoptosis significantly improved cell viability during EPEC infection. EPEC infection also resulted in impaired autophagy, as demonstrated by decreased expressions of autophagy-related proteins Beclin 1, ATG5, and microtubule-binding protein light chain-3B (LC3B) and the increased expression of p62 through western blot analysis. However, EPS reversed these effects which indicated that EPS promoted autophagosome formation. Furthermore, EPS prevented the lysosome damage induced by EPEC as it enhanced lysosomal acidification and raised lysosome-associated protein levels, thus promoted autophagosome degradation. Our findings suggest that the amelioration of EPEC-induced cell damages by EPS is associated with the limitation of detrimental apoptosis and the promotion of autophagy flux.

NleB2 from enteropathogenic Escherichia coli is a novel arginine-glucose transferase effector.

During infection, enteropathogenic Escherichia coli (EPEC) and enterohaemorrhagic E. coli (EHEC) directly manipulate various aspects of host cell function through the translocation of type III secretion system (T3SS) effector proteins directly into the host cell. Many T3SS effector proteins are enzymes that mediate post-translational modifications of host proteins, such as the glycosyltransferase NleB1, which transfers a single N-acetylglucosamine (GlcNAc) to arginine residues, creating an Arg-GlcNAc linkage. NleB1 glycosylates death-domain containing proteins including FADD, TRADD and RIPK1 to block host cell death. The NleB1 paralogue, NleB2, is found in many EPEC and EHEC strains but to date its enzymatic activity has not been described. Using in vitro glycosylation assays combined with mass spectrometry, we found that NleB2 can utilize multiple sugar donors including UDP-glucose, UDP-GlcNAc and UDP-galactose during glycosylation of the death domain protein, RIPK1. Sugar donor competition assays demonstrated that UDP-glucose was the preferred substrate of NleB2 and peptide sequencing identified the glycosylation site within RIPK1 as Arg603, indicating that NleB2 catalyses arginine glucosylation. We also confirmed that NleB2 catalysed arginine-hexose modification of Flag-RIPK1 during infection of HEK293T cells with EPEC E2348/69. Using site-directed mutagenesis and in vitro glycosylation assays, we identified that residue Ser252 in NleB2 contributes to the specificity of this distinct catalytic activity. Substitution of Ser252 in NleB2 to Gly, or substitution of the corresponding Gly255 in NleB1 to Ser switches sugar donor preference between UDP-GlcNAc and UDP-glucose. However, this switch did not affect the ability of the NleB variants to inhibit inflammatory or cell death signalling during HeLa cell transfection or EPEC infection. NleB2 is thus the first identified bacterial Arg-glucose transferase that, similar to the NleB1 Arg-GlcNAc transferase, inhibits host protein function by arginine glycosylation.

Enteropathogenic Escherichia coli Infection Induces Diarrhea, Intestinal Damage, Metabolic Alterations, and Increased Intestinal Permeability in a Murine Model.

Enteropathogenic E. coli (EPEC) are recognized as one of the leading bacterial causes of infantile diarrhea worldwide. Weaned C57BL/6 mice pretreated with antibiotics were challenged orally with wild-type EPEC or escN mutant (lacking type 3 secretion system) to determine colonization, inflammatory responses and clinical outcomes during infection. Antibiotic disruption of intestinal microbiota enabled efficient colonization by wild-type EPEC resulting in growth impairment and diarrhea. Increase in inflammatory biomarkers, chemokines, cellular recruitment and pro-inflammatory cytokines were observed in intestinal tissues. Metabolomic changes were also observed in EPEC infected mice with changes in tricarboxylic acid (TCA) cycle intermediates, increased creatine excretion and shifts in gut microbial metabolite levels. In addition, by 7 days after infection, although weights were recovering, EPEC-infected mice had increased intestinal permeability and decreased colonic claudin-1 levels. The escN mutant colonized the mice with no weight loss or increased inflammatory biomarkers, showing the importance of the T3SS in EPEC virulence in this model. In conclusion, a murine infection model treated with antibiotics has been developed to mimic clinical outcomes seen in children with EPEC infection and to examine potential roles of selected virulence traits. This model can help in further understanding mechanisms involved in the pathogenesis of EPEC infections and potential outcomes and thus assist in the development of potential preventive or therapeutic interventions.

Fluorescence Detection of Type III Secretion Using a Glu-CyFur Reporter System in Citrobacter rodentium.

Enteropathogenic Escherichia coli (EPEC) is a major cause of infantile diarrhea worldwide. EPEC and the closely related murine model of EPEC infection, Citrobacter rodentium, utilize a type III secretion system (T3SS) to propagate the infection. Since the T3SS is not essential for the bacteria to survive or propagate, inhibiting the virulence factor with a therapeutic would treat the infection without causing harm to commensal bacteria. Studying inhibitors of the T3SS usually requires a BSL-2 laboratory designation and eukaryotic host cells while not indicating the mechanism of inhibition. We have designed a BSL-1 assay using the murine model C. rodentium that does not require mammalian cell culture. This CPG2-reporter assay allows for more rapid analysis of secretion efficiency than Western blotting and is sensitive enough to differentiate between partial and total inhibition of the T3SS. Here we present our method and the results of a small collection of compounds we have screened, including known T3SS inhibitors EGCG, regacin, and aurodox and related quorum sensing inhibitors tannic acid and ellagic acid. We have further characterized EGCG as a T3SS inhibitor and established its IC50 of 1.8 ± 0.4 μM. We also establish tannic acid as a potent inhibitor of the T3SS with an IC50 of 0.65 ± 0.09 μM.

Magnesium Sensing Regulates Intestinal Colonization of Enterohemorrhagic Escherichia coli O157:H7.

The large intestinal pathogen enterohemorrhagic Escherichia coli (EHEC) O157:H7 detects host cues to regulate virulence gene expression during colonization and infection. However, virulence regulatory mechanisms of EHEC O157:H7 in the human large intestine are not fully understood. Herein, we identified a virulence-regulating pathway where the PhoQ/PhoP two-component regulatory system senses low magnesium levels and signals to the O island 119-encoded Z4267 (LmiA; low magnesium-induced regulator A), directly activating loci of enterocyte effacement genes to promote EHEC O157:H7 adherence in the large intestine. Disruption of this pathway significantly decreased EHEC O157:H7 adherence in the mouse intestinal tract. Moreover, feeding mice a magnesium-rich diet significantly reduced EHEC O157:H7 adherence in vivo This LmiA-mediated virulence regulatory pathway is also conserved among several EHEC and enteropathogenic E. coli serotypes; therefore, our findings support the use of magnesium as a dietary supplement and provide greater insights into the dietary cues that can prevent enteric infections.IMPORTANCE Sensing specific gut metabolites is an important strategy for inducing crucial virulence programs by enterohemorrhagic Escherichia coli (EHEC) O157:H7 during colonization and infection. Here, we identified a virulence-regulating pathway wherein the PhoQ/PhoP two-component regulatory system signals to the O island 119-encoded low magnesium-induced regulator A (LmiA), which, in turn, activates locus of enterocyte effacement (LEE) genes to promote EHEC O157:H7 adherence in the low-magnesium conditions of the large intestine. This regulatory pathway is widely present in a range of EHEC and enteropathogenic E. coli (EPEC) serotypes. Disruption of this pathway significantly decreased EHEC O157:H7 adherence in the mouse intestinal tract. Moreover, mice fed a magnesium-rich diet showed significantly reduced EHEC O157:H7 adherence in vivo, indicating that magnesium may help in preventing EHEC and EPEC infection in humans.

EnteropathogenicEscherichia coliInfection Inhibits Intestinal Ascorbic Acid Uptake via Dysregulation of Its Transporter Expression.

Enteropathogenic Escherichia coli (EPEC) infection causes prolonged, watery diarrhea leading to morbidity and mortality. Although EPEC infection impacts nutrient transporter function and expression in intestinal epithelial cells, the effects of EPEC infection on intestinal absorption of ascorbic acid (AA) have not yet been investigated. To investigate the effect of EPEC infection on intestinal AA uptake process and expression of both AA transporters. We used two experimental models: human-derived intestinal epithelial Caco-2 cells and mice. 14C-AA uptake assay, Western blot, RT-qPCR, and promoter assay were performed. EPEC (WT) as well as ΔespF and ΔespG/G2 mutant-infected Caco-2 cells showed markedly inhibited AA uptake, while other mutants (ΔescN, ΔespA, ΔespB, and ΔespD) did not affect AA uptake. Infection also reduced protein and mRNA expression levels for both hSVCT1 and hSVCT2. EPEC-infected mice showed marked inhibitory effect on AA uptake and decreased protein and mRNA expression levels for both mSVCT1 and mSVCT2 in jejunum and colon. MicroRNA regulators of SVCT1 and SVCT2 (miR103a, miR141, and miR200a) were upregulated significantly upon EPEC infection in both Caco-2 and mouse jejunum and colon. In addition, expression of the accessory protein glyoxalate reductase/hydroxypyruvate reductase (GRHPR), which regulates SVCT1 function, was markedly decreased by EPEC infection in both models. These findings suggest that EPEC infection causes inhibition in AA uptake through a multifactorial dysregulation of SVCT1 and SVCT2 expression in intestinal epithelial cells.

Enteropathogenic Escherichia coli (EPEC) infection inhibits intestinal vitamin C transporter function and expression: in vitro and in vivo studies

Enteropathogenic Escherichia coli (EPEC) is a food‐borne pathogen and causes enteric illnesses in children. EPEC infection is often associated with malnutrition of the host, especially in individuals experiencing severe and prolonged infection, and/or in those who are nutritionally compromised. While EPEC infection impacts nutrient transporter function and expression in intestinal epithelial cells, to date the consequences of EPEC infection on intestinal absorption of ascorbic acid (AA) are not known. Therefore, we investigated this issue using human intestinal epithelial Caco‐2 cells (in vitro) and mouse (in vivo) models. Infecting Caco‐2 cells with EPEC (WT) markedly inhibited AA uptake. EPEC mutants (ΔescN, ΔespA, ΔespB and ΔespD) did not affect AA uptake, whereas ΔespF and ΔespG/G2 mutants significantly inhibited AA uptake in Caco‐2 cells. Further, we observed significantly reduced expression of both hSVCT1 and hSVCT2 protein and mRNA levels upon infection. Similarly, infecting mice with EPEC (WT) significantly inhibited AA uptake and also reduced the expression of both mSVCT1 and mSVCT2 protein and mRNA levels in jejunum and colon. Studies have shown that epigenetic mechanisms (microRNA) play a role in mediating responses to bacterial infection in the intestine. Therefore, we determined miR103a, miR141 and miR200a expression levels, which were recently shown to regulate SVCT1 and SVCT2. Data from in vitro and in vivo models showed that all three microRNAs were up‐regulated significantly upon EPEC infection. In addition, expression of the accessory protein human glyoxalate reductase/hydroxypyruvate reductase (hGRHPR), which regulates the SVCT1 function, was also markedly decreased by EPEC in both models. These findings suggest that in intestinal epithelial cells, EPEC infection markedly inhibits AA uptake via dysregulation of both SVCT1 and SVCT2 expression.Support or Funding InformationSupported by NIH grants DK 107474, DK 58057, DK 56057, MH108154, GM088790, and a grant from the DVA