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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) 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.

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.