Tight Junctions In Epithelial Cells Research Articles

Striatin is a novel modulator of cell adhesion.

The adherens junctions (AJs) and tight junctions (TJs) provide critical adhesive contacts between neighboring epithelial cells and are crucial for epithelial adhesion, integrity, and barrier functions in a wide variety of tissues and organisms. The striatin protein family, which are part of the striatin interaction phosphatases and kinases complex, are multidomain scaffolding proteins that play important biologic roles. We have previously shown that striatin colocalizes with the tumor suppressor protein adenomatous polyposis coli in the TJs of epithelial cells. Here we show that striatin affects junction integrity and cell migration, probably through a mechanism that involves the adhesion molecule E-cadherin. Cells engaged in cell-cell adhesion expressed a high MW-modified form of striatin that forms stable associations with detergent-insoluble, membrane-bound cellular fractions. In addition, striatin has recently been suggested to be a target of the poly (ADP-ribose) polymerases Tankyrase 1, and we have found that striatin interacts with Tankyrase 1 and is subsequently poly-ADP-ribosylated. Taken together, our results suggest that striatin is a novel cell-cell junctional protein that functions to maintain correct cell adhesion and may have a role in establishing the relationship between AJs and TJs that is fundamental for epithelial cell-cell adhesion.-Lahav-Ariel, L., Caspi, M., Nadar-Ponniah, P. T., Zelikson, N., Hofmann, I., Hanson, K. K., Franke, W. W., Sklan, E. H., Avraham, K. B., Rosin-Arbesfeld, R. Striatin is a novel modulator of cell adhesion.

Structure characterization of a polysaccharide extracted from noni (Morinda citrifolia L.) and its protective effect against DSS-induced bowel disease in mice

Inflammatory bowel disease (IBD), including Crohn's disease and ulcerative colitis, has gradually emerged as a public health challenge worldwide. Morinda citrifolia (noni) has been used to treat various ailments by Pacific Islanders for centuries. In this work, noni fruit polysaccharide (NFP) was obtained by hot water extraction, and its structure was initially explored. The molecular weight of NFP was approximately 456 kDa, and its monosaccharide components predominantly consisted of Gala (58.44%), Gal (4.44%), Glu (21.13%), Rha (4.84%), and Ara (2.16%) to form galacturonic acid, rhamnogalacturonan-I, arabinogalactan, and arabinan. The protective effect of NFP on the intestinal mucosal barrier was then investigated using DSS-induced IBD model mice. Whereas DSS treatment inhibited intestinal mucosal expression and destroyed the tight junction of epithelial cells, by contrast, NFP promoted mucosal and tight junction protein (zonula occludens-1 and occludin) expression. In conclusion, NFP improved the clinical diagnosis of acute colitis in IBD mice and may present a potential source of IBD treatment.

Correction: Proteomic screening identifies the zonula occludens protein ZO-1 as a new partner for ADAM12 in invadopodia-like structures.

[This corrects the article DOI: 10.18632/oncotarget.25106.].

Evaluation of immunoresponses and cytotoxicity from skin exposure to metallic nanoparticles.

Nanotechnology is an interdisciplinary science that has developed rapidly in recent years. Metallic nanoparticles (NPs) are increasingly utilized in dermatology and cosmetology, because of their unique properties. However, skin exposure to NPs raises concerns regarding their transdermal toxicity. The tight junctions of epithelial cells form the skin barrier, which protects the host against external substances. Recent studies have found that NPs can pass through the skin barrier into deeper layers, indicating that skin exposure is a means for NPs to enter the body. The distribution and interaction of NPs with skin cells may cause toxic side effects. In this review, possible penetration pathways and related toxicity mechanisms are discussed. The limitations of current experimental methods on the penetration and toxic effects of metallic NPs are also described. This review contributes to a better understanding of the risks of topically applied metallic NPs and provides a foundation for future studies.

Supplementation of p40, a Lactobacillus rhamnosus GG-derived protein, in early life promotes epidermal growth factor receptor-dependent intestinal development and long-term health outcomes.

The beneficial effects of the gut microbiota on growth in early life are well known. However, knowledge about the mechanisms underlying regulating intestinal development by the microbiota is limited. p40, a Lactobacillus rhamnosus GG-derived protein, transactivates epidermal growth factor receptor (EGFR) in intestinal epithelial cells for protecting the intestinal epithelium against injury and inflammation. Here, we developed p40-containing pectin/zein hydrogels for targeted delivery of p40 to the small intestine and the colon. Treatment with p40-containing hydrogels from postnatal day 2 to 21 significantly enhanced bodyweight gain prior to weaning and functional maturation of the intestine, including intestinal epithelial cell proliferation, differentiation, and tight junction formation, and IgA production in early life in wild-type mice. These p40-induced effects were abolished in mice with specific deletion of EGFR in intestinal epithelial cells, suggesting that transactivation of EGFR in intestinal epithelial cells may mediate p40-regulated intestinal development. Furthermore, neonatal p40 treatment reduced the susceptibility to intestinal injury and colitis and promoted protective immune responses, including IgA production and differentiation of regulatory T cells, in adult mice. These findings reveal novel roles of neonatal supplementation of probiotic-derived factors in promoting EGFR-mediated maturation of intestinal functions and innate immunity, which likely promote long-term beneficial outcomes.

The effects of hydrocortisone on tight junction genes in an in vitro model of the human fallopian epithelial cells

The tight junction between epithelial cells helps making connections in the fallopian tube and contributes to successful fertilization. Breaking the tight junction complex induces various diseases such as the EP. Previous studies have shown that glucocorticoids are effective in repairing and maintaining intercellular tight junctions in epithelial cells of the fallopian tube, although their mechanism is still unknown. This research is a genomic study of hydrocortisone’s effect on epithelial cells of the fallopian tube. Using the human fallopian tube, epithelial cell line (OE-E6/E7) was cultured in four concentrations of hydrocortisone (0 nM, 50 nM, 100 nM and 200 nM) for three durations (24 h, 48 h and 72 h). Glucocorticoids are effective on the expression of Zona occluding-1(ZO-1), Claudin 4, Claudin3, Desmoglein and E-cadherin genes involved in the tight junctions of the fallopian tube. The expression of all genes was up-regulated in the concentrations of 100 nM after 48 h treatment, as compared with the control (0 nM). However, their expression was down-regulated significantly after 72 h treatment (P < 0.05).The present study showed that treatment of epithelial cells of the fallopian tube with glucocorticoid increased the expression of genes involved in tight junctions, including claudin-3, claudin-4, E-cadherin, zona occludin-1 and Desmoglein-1. The obtained data suggests that a new mechanism is developed for glucocorticoid induction of tight junctions by increasing the expression of claudin-3, claudin-4, E-cadherin, zona occludin-1 and Desmoglein-1 genes.

Avian Influenza Virus Subtype H9N2 Affects Intestinal Microbiota, Barrier Structure Injury, and Inflammatory Intestinal Disease in the Chicken Ileum.

Avian influenza virus subtype H9N2 (H9N2 AIV) has caused significant losses to the poultry industry due to the high mortality associated with secondary infections attributable to E. coli. This study tries to address the underlying secondary mechanisms after H9N2 AIV infection. Initially, nine day-old specific pathogen-free chickens were assigned to control (uninfected) and H9N2-infected groups, respectively. Using Illumina sequencing, histological examination, and quantitative real-time PCR, it was found that H9N2 AIV caused intestinal microbiota disorder, injury, and inflammatory damage to the intestinal mucosa. Notably, the genera Escherichia, especially E. coli, significantly increased (p < 0.01) at five days post-infection (dpi), while Lactobacillus, Enterococcus, and other probiotic organisms were significantly reduced (p < 0.01). Simultaneously, the mRNA expression of tight junction proteins (ZO-1, claudin 3, and occludin), TFF2, and Muc2 were significantly reduced (p < 0.01), indicating the destruction of the intestinal epithelial cell tight junctions and the damage of mucin layer construction. Moreover, the mRNA expression of proinflammatory cytokines IFN-γ, IL-22, IFN-α, and IL-17A in intestinal epithelial cells were significantly upregulated, resulting in the inflammatory response and intestinal injury. Our findings may provide a theoretical basis for observed gastroenteritis-like symptoms such as diarrhea and secondary E. coli infection following H9N2 AIV infection.

Proteomic screening identifies the zonula occludens protein ZO-1 as a new partner for ADAM12 in invadopodia-like structures.

The epithelial mesenchymal transition (EMT) is a key process for cancer cell invasion and migration. This complex program whereby epithelial tumor cells loose polarity and acquire mesenchymal phenotype is driven by the regulation of cell-cell adhesion and cell-substrate interactions. We recently described the association of ADAM12 with EMT and we now use immunoprecipitation and proteomic approaches to identify interacting partners for ADAM12 during EMT. We identify twenty proteins that are involved in molecular mechanisms associated with adhesion/invasion processes. Integrative network analyses point out the zonula occludens protein ZO-1, as a new potential partner for ADAM12. In silico screening demonstrates that ZO-1 and ADAM12 are coexpressed in breast cancer cell lines sharing EMT signature. We validate the interaction between ZO-1 and ADAM12 in invasive breast cancer cell lines and show that ZO-1 and ADAM12 co-localize in actin- and cortactin-rich structures. Silencing either ADAM12 or ZO-1 inhibits gelatin degradation demonstrating that both proteins are required for matrix degradation. We further show that matrix metalloprotease 14, known to mediate degradation of collagen in invadopodia-like structures interacts with ZO-1. Depletion of PKCε that regulates the recruitment of ADAM12 and ZO-1 to cell membranes induces a decrease in ADAM12 and ZO-1 at invadopodia-like structures and degradation activity. Together our data provide evidence for a new interaction between ADAM12, a mesenchymal marker induced during TGF-β-dependent EMT and ZO-1, a scaffolding protein expressed in tight junctions of epithelial cells, both proteins being redistributed at the invadopodia-like structures of mesenchymal invasive cells to promote PKCε-dependent matrix degradation.

DNA demethylation of claudin-4 suppresses migration and invasion in laryngeal squamous carcinoma cells

Claudin-4 (CLDN4) is a member of the claudin transmembrane protein family, which consists of integral membrane proteins that are components of the epithelial cell tight junctions; these tight junctions regulate movement of solutes and ions through the paracellular space. CLDN4 is also a differentiation marker and is believed to indicate an epithelial phenotype. However, the role of CLDN4 in laryngeal squamous carcinoma is still unclear. Here, we showed that CLDN4 expression was down-regulated in laryngeal squamous carcinoma tissues and negatively correlated with methyl-CpG-binding protein 2. In addition, CLDN4 was hypermethylated in HEp-2 cells. DNA demethylation of CLDN4 by 5-aza-2'-deoxycytidine suppressed migration and invasion of HEp-2 cells, whereas CLDN4 silencing restored the migration and invasion of HEp-2 cells. Therefore, CLDN4 plays a key role in laryngeal squamous carcinoma progression.

The bile salt sodium taurocholate induces Campylobacter jejuni outer membrane vesicle production and increases OMV-associated proteolytic activity.

Campylobacter jejuni, the leading cause of bacterial acute gastroenteritis worldwide, secretes an arsenal of virulence-associated proteins within outer membrane vesicles (OMVs). C.jejuni OMVs contain three serine proteases (HtrA, Cj0511, and Cj1365c) that cleave the intestinal epithelial cell (IEC) tight and adherens junction proteins occludin and E-cadherin, promoting enhanced C.jejuni adhesion to and invasion of IECs. C.jejuni OMVs also induce IECs innate immune responses. The bile salt sodium taurocholate (ST) is sensed as a host signal to coordinate the activation of virulence-associated genes in the enteric pathogen Vibrio cholerae. In this study, the effect of ST on C.jejuni OMVs was investigated. Physiological concentrations of ST do not have an inhibitory effect on C.jejuni growth until the early stationary phase. Coculture of C.jejuni with 0.1% or 0.2% (w/v) ST stimulates OMV production, increasing both lipid and protein concentrations. C.jejuni ST-OMVs possess increased proteolytic activity and exhibit a different protein profile compared to OMVs isolated in the absence of ST. ST-OMVs exhibit enhanced cytotoxicity and immunogenicity to T84 IECs and enhanced killing of Galleria mellonella larvae. ST increases the level of mRNA transcripts of the OMVs-associated serine protease genes and the cdtABC operon that encodes the cytolethal distending toxin. Coculture with ST significantly enhances the OMVs-induced cleavage of E-cadherin and occludin. C.jejuni OMVs also cleave the major endoplasmic reticulum chaperone protein BiP/GRP78 and this activity is associated with the Cj1365c protease. These data suggest that C.jejuni responds to the presence of physiological concentrations of the bile salt ST that increases OMV production and the synthesis of virulence-associated factors that are secreted within the OMVs. We propose that these events contribute to pathogenesis.

Treatment-related survival associations of claudin-2 expression in fibroblasts of colorectal cancer

Claudin-2 is a trans-membrane protein—component of tight junctions in epithelial cells. Elevated claudin-2 expression has been reported in colorectal cancer (CRC). The aim of this study was to investigate the expression patterns of claudin-2 in human CRC samples and analyze its association with clinical characteristics and treatment outcome. TMAs of primary tumors from two cohorts of metastatic CRC (mCRC) were used. Claudin-2 IHC staining was evaluated in a semi-quantitative manner in different regions and cell types. Claudin-2 expression was also analyzed by immunofluorescence in primary cultures of human CRC cancer-associated fibroblasts (CAFs). Initial analyses identified previously unrecognized expression patterns of claudin-2 in CAFs of human CRC. Claudin-2 expression in CAFs of the invasive margin was associated with shorter progression-free survival. Subgroup analyses demonstrated that the survival associations occurred among cases that received 5-FU+oxaliplatin combination treatment, but not in patients receiving 5-FU±irinotecan. The finding was validated by analyses of the independent cohort. In summary, previously unreported stromal expression of claudin-2 in CAFs of human CRC was detected together with significant association between high claudin-2 expression in CAFs and shorter survival in 5-FU+oxaliplatin-treated mCRC patients.

EPEC effector EspF promotes Crumbs3 endocytosis and disrupts epithelial cell polarity.

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.

Functional Modulation of Tight Junction to Enhance the Permeability of Biological Products (Biologics) in Biomembranes.

In recent years, biological products (biologics), including blood components, recombinant therapeutic proteins, antibodies, gene therapeutic materials, and so on, have been produced by biotechnology methods and other novel technologies. These products are essential therapeutic materials in progressive medicine. However, we often encounter the lower permeability of these biologics through biomembranes, due to their high molecular mass. In the last three decades, we have investigated drug delivery systems, including several enhancement methods for the permeability of biologics such as recombinant therapeutic proteins and viral vectors in epithelial cells. This review focuses the development of novel delivery systems for biologics in rectal and nasal administration, and includes an interesting observation of modulators of the tight junction (TJ) function. From cellular biology perspective, the interaction between permeability enhancing materials and targeted molecules in the TJ of epithelial cells was investigated. We elucidated that a TJ modulator will interact with a major constituent protein, for instance claudins, in playing an essential role in the tissue-specific barrier function of the TJ. Furthermore, useful enhancement of gene transfer in cells (for instance, in Caco-2 cells) was found in the combination of an adenovirus vector and capric acid sodium salt (C10), a TJ modulator.

Mucoadhesive properties of low molecular weight chitosan- or glycol chitosan- and corresponding thiomer-coated poly(isobutylcyanoacrylate) core-shell nanoparticles

The aim of the present work was to evaluate the mucoadhesive properties of poly(isobutyl cyanoacrylate) (PIBCA) nanoparticles (NPs) coated with Low Molecular Weight (LMW) chitosan (CS)- and glycol chitosan (GCS)-based thiomers as well as with the corresponding LMW unmodified polysaccharides. For this purpose, all the CS- and GCS-based thiomers were prepared under simple and mild conditions starting from the LMW unmodified polymers CS and GCS. The resulting NPs were of spherical shape with diameters ranging from 400 to 600nm and 187 to 309nm, for CS- and GCS-based NPs, respectively. The mucoadhesive characteristics of these core shell NPs were studied in Ussing chambers measuring the percentage of NPs stuck on the mucosal of fresh intestinal tissue after 2h of incubation. Moreover, incubation of nanoparticle formulations with the intestinal tissue induced changes in transmucosal electrical resistance which were measured to gain information into the opening of tight junctions and to control the integrity of the mucosa. Thus, it was found that PIBCA NPs coated with the GCS–Glutathione conjugate (GCGPIBCA NPs) possessed the most favorable mucoadhesive performances. Moreover, both GCGPIBCA- and GCS-N-acetyl-cysteine (GCNPIBCA)-core-shell NPs might induced an enlargement of the epithelial cell tight junctions. In conclusion, coating of PIBCA NPs with GCS-based thiomers may be useful for improving the mucoadhesive and permeation properties of these nanocarriers.

Structurally and functionally characterized in vitro model of rabbit vocal fold epithelium.

In this paper, we describe a method for primary culture of a well differentiated electrically tight rabbit vocal fold epithelial cell multilayer and the measurement of transepithelial electrical resistance (TEER) for the evaluation of epithelial barrier function in vitro. Rabbit larynges were harvested and enzymatically treated to isolate vocal fold epithelial cells and to establish primary culture. Vocal fold epithelial cells were co-cultured with mitomycin C-treated feeder cells on collagen-coated plates. After 10-14 days in primary culture, cells were passaged and cultured until they achieved 70-90% confluence on collagen-coated plates. Epithelial cells were then passaged onto collagen-coated cell culture inserts using 4.5cm2 membrane filters (1.0μm pore size) with 10% fetal bovine serum or 30μg/mL bovine pituitary extract to investigate the effects of growth-promoting additives on TEER. Additional experiments were performed to investigate optimal seeding density (1.1, 2.2, 4.4, or 8.9×105 cells/cm2), the effect of co-culture with feeder cells, and the effect of passage number on epithelial barrier function. Characterization of in vitro cultures was performed using hematoxylin and eosin staining and immunostaining for vocal fold epithelial cell markers and tight junctions. Results revealed higher TEER in cells supplemented with fetal bovine serum compared to bovine pituitary extract. TEER was highest in cells passaged at a seeding density of 2.2×104 cells/cm2, and TEER was higher in cells at passage two than passage three. Ultrastructural experiments revealed a well-differentiated epithelial cell multilayer, expressing the epithelial cell markers CK13, CK14 and the tight junction proteins occludin and ZO-1.

Preterm infant gut microbiota affects intestinal epithelial development in a humanized microbiome gnotobiotic mouse model.

Development of the infant small intestine is influenced by bacterial colonization. To promote establishment of optimal microbial communities in preterm infants, knowledge of the beneficial functions of the early gut microbiota on intestinal development is needed. The purpose of this study was to investigate the impact of early preterm infant microbiota on host gut development using a gnotobiotic mouse model. Histological assessment of intestinal development was performed. The differentiation of four epithelial cell lineages (enterocytes, goblet cells, Paneth cells, enteroendocrine cells) and tight junction (TJ) formation was examined. Using weight gain as a surrogate marker for health, we found that early microbiota from a preterm infant with normal weight gain (MPI-H) induced increased villus height and crypt depth, increased cell proliferation, increased numbers of goblet cells and Paneth cells, and enhanced TJs compared with the changes induced by early microbiota from a poor weight gain preterm infant (MPI-L). Laser capture microdissection (LCM) plus qRT-PCR further revealed, in MPI-H mice, a higher expression of stem cell marker Lgr5 and Paneth cell markers Lyz1 and Cryptdin5 in crypt populations, along with higher expression of the goblet cell and mature enterocyte marker Muc3 in villus populations. In contrast, MPI-L microbiota failed to induce the aforementioned changes and presented intestinal characteristics comparable to a germ-free host. Our data demonstrate that microbial communities have differential effects on intestinal development. Future studies to identify pioneer settlers in neonatal microbial communities necessary to induce maturation may provide new insights for preterm infant microbial ecosystem therapeutics.

NQO1-Knockout Mice Are Highly Sensitive to Clostridium Difficile Toxin A-Induced Enteritis.

Clostridium difficile toxin A causes acute gut inflammation in animals and humans. It is known to downregulate the tight junctions between colonic epithelial cells, allowing luminal contents to access body tissues and trigger acute immune responses. However, it is not yet known whether this loss of the barrier function is a critical factor in the progression of toxin A-induced pseudomembranous colitis. We previously showed that NADH:quinone oxidoreductase 1 (NQO1) KO (knockout) mice spontaneously display weak gut inflammation and a marked loss of colonic epithelial tight junctions. Moreover, NQO1 KO mice exhibited highly increased inflammatory responses compared with NQO1 WT (wild-type) control mice when subjected to DSS-induced experimental colitis. Here, we tested whether toxin A could also trigger more severe inflammatory responses in NQO1 KO mice compared with NQO1 WT mice. Indeed, our results show that C. difficile toxin A-mediated enteritis is significantly enhanced in NQO1 KO mice compared with NQO1 WT mice. The levels of fluid secretion, villus disruption, and epithelial cell apoptosis were also higher in toxin A-treated NQO1 KO mice compared with WT mice. The previous and present results collectively show that NQO1 is involved in the formation of tight junctions in the small intestine, and that defects in NQO1 enhance C. difficile toxin A-induced acute inflammatory responses, presumably via the loss of epithelial cell tight junctions.

Alcohol Consumption Increases Claudin‐18 Turnover in Alveolar Epithelial Cells

Alveolar epithelial cell (AEC) tight junctions are crucial for maintaining barrier function between fluid filled tissues and the airspace in the lung. Failure of alveolar tight junctions leads to acute respiratory distress syndrome (ARDS), a frequently lethal condition characterized by airspace flooding in the lung. Chronic alcohol abuse is a great risk factor for ARDS. Alcohol consumption impairs alveolar barrier function by modulation of tight junction proteins, called claudins. Claudins are a family of tetraspan transmembrane proteins that confer specific barrier properties to epithelial cell monolayers by regulating tight junction permeability. We used a rodent model of chronic alcohol ingestion to investigate the effects of alcohol on claudins and barrier function. Primary AECs from alcohol and control fed rats were cultured on Transwell permeable supports and allowed to differentiate into model type I cells. Transepithelial resistance measurements (TER) and dye flux assays were used to determine barrier function in cultured AECs. Tight junction morphology was assessed by immunofluorescence. In response to alcohol, alveolar tight junctions showed altered claudin composition leading to an increase in paracellular leak and formation of projections perpendicular to tight junctions (“spikes”) when immunostained for claudin‐18. Quantification of cells containing tight junction spikes revealed that alcohol increases the portion of cells containing tight junction spikes, which correlated with decreased barrier function. Live cell imaging of AECs expressing YFP‐tagged claudin‐18 confirmed that vesicles containing tagged claudin‐18 fused with and pinched off of tight junction spikes. Therefore, we hypothesized that these tight junction spikes are sites of enhanced protein turnover which might then result in decreased barrier function. As endocytosis is an important regulator of claudin turnover, we examined the effect of treating cells with the inhibitor Dynasore that targets dynamin, a protein which has a role in the scission of endocytic vesicles. Treatment of AECs with Dynasore resulted in a significant decrease in the number of cells containing claudin‐18 spikes and also caused a concomitant decrease in paracellular flux of a 10 kDa marker, Texas Red dextran. Our data support a role for tight junction spikes in regulating paracellular permeability and suggest that dynamin or dynamin‐related proteins may be useful therapeutic targets to stabilize alveolar barrier function and impair the development of ARDS.Support or Funding InformationThis work was supported by Emory Alcohol and Lung Biology Center/National Institutes of Health (NIH) grant P50‐AA013757 (MK), R01‐HL116958 (MK), T32‐AA‐013528 (SAM), the German Academic Exchange Service (DAAD) (BS), R25‐GM099644 (SDW), and Emory+Children's Center of Excellence for Cystic Fibrosis Research (MK).

Secretion of Alpha-Hemolysin by Escherichia coli Disrupts Tight Junctions in Ulcerative Colitis Patients.

Objectives:The potential of Escherichia coli (E. coli) isolated from inflammatory bowel disease (IBD) patients to damage the integrity of the intestinal epithelium was investigated.Methods:E. coli strains isolated from patients with ulcerative colitis (UC) and healthy controls were tested for virulence capacity by molecular techniques and cytotoxic assays and transepithelial electric resistance (TER). E. coli isolate p19A was selected, and deletion mutants were created for alpha-hemolysin (α-hemolysin) (hly) clusters and cytotoxic necrotizing factor type 1 (cnf1). Probiotic E. coli Nissle and pathogenic E. coli LF82 were used as controls.Results:E. coli strains from patients with active UC completely disrupted epithelial cell tight junctions shortly after inoculation. These strains belong to phylogenetic group B2 and are all α-hemolysin positive. In contrast, probiotic E. coli Nissle, pathogenic E. coli LF82, four E. coli from patients with inactive UC and three E. coli strains from healthy controls did not disrupt tight junctions. E. coli p19A WT as well as cnf1, and single loci of hly mutants from cluster I and II were all able to damage Caco-2 (Heterogeneous human epithelial colorectal adenocarcinoma) cell tight junctions. However, this phenotype was lost in a mutant with knockout (Δ) of both hly loci (P<0.001).Conclusions:UC-associated E. coli producing α-hemolysin can cause rapid loss of tight junction integrity in differentiated Caco-2 cell monolayers. This effect was abolished in a mutant unable to express α-hemolysin. These results suggest that high Hly expression may be a mechanism by which specific strains of E. coli pathobionts can contribute to epithelial barrier dysfunction and pathophysiology of disease in IBD.

Group A Streptococcus exploits human plasminogen for bacterial translocation across epithelial barrier via tricellular tight junctions

Group A Streptococcus (GAS) is a human-specific pathogen responsible for local suppurative and life-threatening invasive systemic diseases. Interaction of GAS with human plasminogen (PLG) is a salient characteristic for promoting their systemic dissemination. In the present study, a serotype M28 strain was found predominantly localized in tricellular tight junctions of epithelial cells cultured in the presence of PLG. Several lines of evidence indicated that interaction of PLG with tricellulin, a major component of tricellular tight junctions, is crucial for bacterial localization. A site-directed mutagenesis approach revealed that lysine residues at positions 217 and 252 within the extracellular loop of tricellulin play important roles in PLG-binding activity. Additionally, we demonstrated that PLG functions as a molecular bridge between tricellulin and streptococcal surface enolase (SEN). The wild type strain efficiently translocated across the epithelial monolayer, accompanied by cleavage of transmembrane junctional proteins. In contrast, amino acid substitutions in the PLG-binding motif of SEN markedly compromised those activities. Notably, the interaction of PLG with SEN was dependent on PLG species specificity, which influenced the efficiency of bacterial penetration. Our findings provide insight into the mechanism by which GAS exploits host PLG for acceleration of bacterial invasion into deeper tissues via tricellular tight junctions.