scholarly journals Efficient Translocation of EspC into Epithelial Cells Depends on Enteropathogenic Escherichia coli and Host Cell Contact

2006 ◽  
Vol 74 (4) ◽  
pp. 2293-2303 ◽  
Author(s):  
Jorge E. Vidal ◽  
Fernando Navarro-García
Keyword(s):  
Escherichia Coli ◽  
Epithelial Cells ◽  
Host Cell ◽  
Cytopathic Effect ◽  
Cell Interaction ◽  
Cell Contact ◽  
Internalization Mechanism ◽  
Cell Cytosol ◽  
Cytoskeletal Disruption

ABSTRACT EspC is an autotransporter protein secreted by enteropathogenic Escherichia coli (EPEC). The pathogenic role of EspC in EPEC infection is unknown. We have shown that the purified EspC produces enterotoxicity and cytotoxicity; for the latter effect, EspC must be internalized. However, the internalization mechanism is unknown. Here we show that azithromycin (an inhibitor of pinocytosis), but not drugs affecting caveole-, clathrin-, or receptor-mediated endocytosis, inhibited purified EspC internalization and cytoskeletal disruption, suggesting that purified EspC is internalized by pinocytosis. Furthermore, unlike in cholera toxin, we were unable to detect a receptor on epithelial cells by pretreatment at 4°C. Upon EspC entry, it is delivered directly into the cell cytosol, as shown by the fact that drugs that inhibit intracellular trafficking had no effect on cytoskeletal disruption. All these data suggest that purified EspC internalization is not a physiological internalization mechanism; hence, we explored EspC internalization during the infection of epithelial cells by EPEC. Like other EPEC virulence factors, EspC secretion is stimulated by EPEC when it is grown in cell culture medium and enhanced by the presence of epithelial cells. Physiologically secreted EspC was efficiently internalized during EPEC and host cell interaction. Additionally, the lack of EspC internalization caused by using an isogenic mutant prevented the cytopathic effect caused by EPEC. These data suggest that EPEC uses an efficient mechanism to internalize milieu-secreted EspC into epithelial cells; once inside the cells, EspC is able to induce the cytopathic effect caused by EPEC.

scholarly journals Bacterial Secretions of Nonpathogenic Escherichia coli Elicit Inflammatory Pathways: a Closer Investigation of Interkingdom Signaling

mBio ◽  
2015 ◽  
Vol 6 (2) ◽  
Author(s):  
Amin Zargar ◽  
David N. Quan ◽  
Karen K. Carter ◽  
Min Guo ◽  
Herman O. Sintim ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Epithelial Cells ◽  
Negative Feedback ◽  
Cell Contact ◽  
Bacterial Cells ◽  
Content Type ◽  
Phenotypic Differences ◽  
E Coli ◽  
Autoinducer 2 ◽  
Human Epithelial Cells

ABSTRACTThere have been many studies on the relationship between nonpathogenic bacteria and human epithelial cells; however, the bidirectional effects of the secretomes (secreted substances in which there is no direct bacterium-cell contact) have yet to be fully investigated. In this study, we use a transwell model to explore the transcriptomic effects of bacterial secretions from two different nonpathogenicEscherichia colistrains on the human colonic cell line HCT-8 using next-generation transcriptome sequencing (RNA-Seq).E. coliBL21 and W3110, while genetically very similar (99.1% homology), exhibit key phenotypic differences, including differences in their production of macromolecular structures (e.g., flagella and lipopolysaccharide) and in their secretion of metabolic byproducts (e.g., acetate) and signaling molecules (e.g., quorum-sensing autoinducer 2 [AI-2]). After analysis of differential epithelial responses to the respective secretomes, this study shows for the first time that a nonpathogenic bacterial secretome activates the NF-κB-mediated cytokine-cytokine receptor pathways while also upregulating negative-feedback components, including the NOD-like signaling pathway. Because of AI-2's relevance as a bacterium-bacterium signaling molecule and the differences in its secretion rates between these strains, we investigated its role in HCT-8 cells. We found that the expression of the inflammatory cytokine interleukin 8 (IL-8) responded to AI-2 with a pattern of rapid upregulation before subsequent downregulation after 24 h. Collectively, these data demonstrate that secreted products from nonpathogenic bacteria stimulate the transcription of immune-related biological pathways, followed by the upregulation of negative-feedback elements that may serve to temper the inflammatory response.IMPORTANCEThe symbiotic relationship between the microbiome and the host is important in the maintenance of human health. There is a growing need to further understand the nature of these relationships to aid in the development of homeostatic probiotics and also in the design of novel antimicrobial therapeutics. To our knowledge, this is the first global-transcriptome study of bacteria cocultured with human epithelial cells in a model to determine the transcriptional effects of epithelial cells in which epithelial and bacterial cells are allowed to “communicate” with each other only through diffusible small molecules and proteins. By beginning to demarcate the direct and indirect effects of bacteria on the gastrointestinal (GI) tract, two-way interkingdom communication can potentially be mediated between host and microbe.

Interactions Between Salmonella Typhimurium, Enteropathogenic Escherichia Coli (EPEC), and Host Epithelial Cells

1995 ◽  
Vol 9 (1) ◽  
pp. 31-36 ◽  
Author(s):  
B.B. Finlay
Keyword(s):  
Escherichia Coli ◽  
Epithelial Cells ◽  
Salmonella Typhimurium ◽  
Host Cell ◽  
Inositol Phosphate ◽  
Enteric Pathogens ◽  
Host Protein ◽  
Host Cells ◽  
Enteropathogenic Escherichia Coli ◽  
Sequence Of Events

The interactions that occur between pathogenic micro-organisms and their host cells are complex and intimate. We have used two enteric pathogens, Salmonella typhimurium and enteropathogenic Escherichia coli (EPEC), to examine the interactions that occur between these organisms and epithelial cells. Although these are enteric pathogens, the knowledge and techniques developed from these systems may be applied to the study of dental pathogens. Both S. typhimurium and EPEC disrupt epithelial monolayer integrity, although by different mechanisms. Both pathogens cause loss of microvilli and re-arrangement of the underlying host cytoskeleton. Despite these similarities, both organisms send different signals into the host cell. EPEC signal transduction involves generation of intracellular calcium and inositol phosphate fluxes, and activation of host tyrosine kinases that results in tyrosine phosphorylation of a 90-kDa host protein. Bacterial mutants have been identifed that are deficient in signaling to the host. We propose a sequence of events that occur when EPEC interacts with epithelial cells. Once inside a host cell, S. typhimurium remains within a vacuole. To define some of the parameters of the intracellular environment, we constructed genetic fusions of known genes with lacZ, and used these fusions as reporter probes of the intracellular vacuolar environment. We have also begun to examine the bacterial and host cell factors necessary for S. typhimurium to multiply within epithelial cells. We found that this organism triggers the formation of novel tubular lysosomes, and these structures are linked with intracellular replication.

scholarly journals Cross-Talk between Adherens Junctions and Desmosomes Depends on Plakoglobin

1997 ◽  
Vol 136 (4) ◽  
pp. 919-934 ◽  
Author(s):  
Jani E. Lewis ◽  
James K. Wahl ◽  
Kristin M. Sass ◽  
Pamela J. Jensen ◽  
Keith R. Johnson ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Cell Line ◽  
Adherens Junctions ◽  
Adherens Junction ◽  
Epithelial Cell Line ◽  
Cell Contact ◽  
Common Component ◽  
Classical Cadherins ◽  
E Cadherin

Squamous epithelial cells have both adherens junctions and desmosomes. The ability of these cells to organize the desmosomal proteins into a functional structure depends upon their ability first to organize an adherens junction. Since the adherens junction and the desmosome are separate structures with different molecular make up, it is not immediately obvious why formation of an adherens junction is a prerequisite for the formation of a desmosome. The adherens junction is composed of a transmembrane classical cadherin (E-cadherin and/or P-cadherin in squamous epithelial cells) linked to either β-catenin or plakoglobin, which is linked to α-catenin, which is linked to the actin cytoskeleton. The desmosome is composed of transmembrane proteins of the broad cadherin family (desmogleins and desmocollins) that are linked to the intermediate filament cytoskeleton, presumably through plakoglobin and desmoplakin. To begin to study the role of adherens junctions in the assembly of desmosomes, we produced an epithelial cell line that does not express classical cadherins and hence is unable to organize desmosomes, even though it retains the requisite desmosomal components. Transfection of E-cadherin and/or P-cadherin into this cell line did not restore the ability to organize desmosomes; however, overexpression of plakoglobin, along with E-cadherin, did permit desmosome organization. These data suggest that plakoglobin, which is the only known common component to both adherens junctions and desmosomes, must be linked to E-cadherin in the adherens junction before the cell can begin to assemble desmosomal components at regions of cell–cell contact. Although adherens junctions can form in the absence of plakoglobin, making use only of β-catenin, such junctions cannot support the formation of desmosomes. Thus, we speculate that plakoglobin plays a signaling role in desmosome organization.

scholarly journals Mammalian PAR-1 determines epithelial lumen polarity by organizing the microtubule cytoskeleton

2004 ◽  
Vol 164 (5) ◽  
pp. 717-727 ◽  
Author(s):  
David Cohen ◽  
Patrick J. Brennwald ◽  
Enrique Rodriguez-Boulan ◽  
Anne Müsch
Keyword(s):  
Epithelial Cells ◽  
Mdck Cells ◽  
Cell Contact ◽  
Bile Canaliculi ◽  
Madin Darby Canine Kidney ◽  
Lumen Formation ◽  
Contact Sites ◽  
Darby Canine Kidney ◽  
Canine Kidney

Epithelial differentiation involves the generation of luminal surfaces and of a noncentrosomal microtubule (MT) network aligned along the polarity axis. Columnar epithelia (e.g., kidney, intestine, and Madin-Darby canine kidney [MDCK] cells) generate apical lumina and orient MT vertically, whereas liver epithelial cells (hepatocytes and WIFB9 cells) generate lumina at cell–cell contact sites (bile canaliculi) and orient MTs horizontally. We report that knockdown or inhibition of the mammalian orthologue of Caenorhabditis elegans Par-1 (EMK1 and MARK2) during polarization of cultured MDCK and WIFB9 cells prevented development of their characteristic lumen and nonradial MT networks. Conversely, EMK1 overexpression induced the appearance of intercellular lumina and horizontal MT arrays in MDCK cells, making EMK1 the first known candidate to regulate the developmental branching decision between hepatic and columnar epithelial cells. Our experiments suggest that EMK1 primarily promotes reorganization of the MT network, consistent with the MT-regulating role of this gene product in other systems, which in turn controls lumen formation and position.

scholarly journals RpoH Mediates the Expression of Some, but Not All, Genes Induced in Neisseria gonorrhoeae Adherent to Epithelial Cells

2006 ◽  
Vol 74 (5) ◽  
pp. 2767-2776 ◽  
Author(s):  
Ying Du ◽  
Cindy Grove Arvidson
Keyword(s):  
Epithelial Cells ◽  
Neisseria Gonorrhoeae ◽  
Host Cell ◽  
Infection Process ◽  
Host Cells ◽  
Cell Contact ◽  
Transmitted Infection ◽  
New Host ◽  
Sexually Transmitted ◽  
Cell Induction

ABSTRACT Neisseria gonorrhoeae (gonococcus [GC]), is highly adapted to the human host, the only known reservoir for gonococcal infection. However, since it is sexually transmitted, infection of a new host likely requires a regulatory response on the part of the gonococcus to respond to this significant change in environment. We previously showed that adherence of gonococci to epithelial cells results in changes of gene expression in the bacteria that presumably prepare them for subsequent steps in the infection process. Expression of the heat shock sigma factor gene, rpoH, was shown to be important for the invasion step, as gonococci depleted for rpoH were reduced in their ability to invade epithelial cells. Here, we show that of the genes induced in adherent gonococci, two are part of the gonococcal RpoH regulon. When RpoH is depleted, expression of these genes is no longer induced by host cell contact, indicating that RpoH is mediating the host cell induction response of these genes. One RpoH-dependent gene, NGO0376, is shown to be important for invasion of epithelial cells, consistent with earlier observations that RpoH is necessary for this step of infection. Two genes, NGO1684 and NGO0340, while greatly induced by host cell contact, were found to be RpoH independent, indicating that more than one regulator is involved in the response to host cell contact. Furthermore, NGO0340, but not NGO1684, was shown to be important for both adherence and invasion of epithelial cells, suggesting a complex regulatory network in the response of gonococci to contact with host cells.

scholarly journals Probiotics prevent enterohaemorrhagic Escherichia coli O157 : H7-mediated inhibition of interferon-γ-induced tyrosine phosphorylation of STAT-1

Microbiology ◽  
2009 ◽  
Vol 155 (2) ◽  
pp. 531-540 ◽  
Author(s):  
Narveen Jandu ◽  
Zoë Jingjing Zeng ◽  
Kathene C. Johnson-Henry ◽  
Philip M. Sherman
Keyword(s):  
Escherichia Coli ◽  
Surface Layer ◽  
Epithelial Cell ◽  
Epithelial Cells ◽  
Tyrosine Phosphorylation ◽  
Cell Protein ◽  
Cell Contact ◽  
Equal Concentration ◽  
Surface Layer Proteins ◽  
Enterohaemorrhagic Escherichia Coli

Enterohaemorrhagic Escherichia coli (EHEC) O157 : H7 inhibits interferon (IFN)-γ-stimulated tyrosine phosphorylation of signal transducer and activator of transcription (STAT)-1 in epithelial cells. We determined the effects of probiotics on EHEC-mediated disruption of IFN-γ-stimulated STAT-1 activation in epithelial cell lines. Confluent Intestine 407, HEp-2 and Caco-2 epithelial cells were pre-treated (3 h) with either probiotics or surface-layer proteins derived from Lactobacillus helveticus R0052 prior to infection with EHEC O157 : H7 strain CL56 (m.o.i. 100 : 1, 6 h, 37 °C in 5 % CO2). Subsequently, cells were washed and stimulated with human recombinant IFN-γ (50 ng ml−1, 0.5 h, 37 °C) followed by whole-cell protein extraction and immunoblotting for tyrosine-phosphorylated STAT-1. Relative to uninfected cells, STAT-1-activation was reduced after EHEC O157 : H7 infection. Pre-incubation with the probiotic L. helveticus R0052 followed by EHEC infection abrogated pathogen-mediated disruption of IFN-γ–STAT-1 signalling. As determined using Transwell inserts, probiotic-mediated protection was independent of epithelial cell contact. In contrast, pre-incubation with boiled L. helveticus R0052, an equal concentration of viable Lactobacillus rhamnosus R0011, or surface-layer proteins (0.14 mg ml−1) did not restore STAT-1 signalling in EHEC-infected cells. The viable probiotic agent L. helveticus R0052 prevented EHEC O157 : H7-mediated subversion of epithelial cell signal transduction responses.

scholarly journals Enterohemorrhagic Escherichia coli O157:H7 Disrupts Stat1-Mediated Gamma Interferon Signal Transduction in Epithelial Cells

2003 ◽  
Vol 71 (3) ◽  
pp. 1396-1404 ◽  
Author(s):  
Peter J. M. Ceponis ◽  
Derek M. McKay ◽  
Joyce C. Y. Ching ◽  
Perpetual Pereira ◽  
Philip M. Sherman
Keyword(s):  
Escherichia Coli ◽  
Signal Transduction ◽  
Epithelial Cells ◽  
Host Cell ◽  
Tyrosine Phosphorylation ◽  
Gamma Interferon ◽  
Enterohemorrhagic Escherichia Coli ◽  
Cell Protein ◽  
E Coli ◽  
Ifn Γ

ABSTRACT Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is a clinically important bacterial enteropathogen that manipulates a variety of host cell signal transduction cascades to establish infection. However, the effect of EHEC O157:H7 on Jak/Stat signaling is unknown. To define the effect of EHEC infection on epithelial gamma interferon (IFN-γ)-Stat1 signaling, human T84 and HEp-2 epithelial cells were infected with EHEC O157:H7 and then stimulated with recombinant human IFN-γ. Cells were also infected with different EHEC strains, heat-killed EHEC, enteropathogenic E. coli (EPEC) O127:H6, and the commensal strain E. coli HB101. Nuclear and whole-cell protein extracts were prepared and were assayed by an electrophoretic mobility shift assay (EMSA) and by Western blotting, respectively. Cells were also processed for immunofluorescence to detect the subcellular localization of Stat1. The EMSA revealed inducible, but not constitutive, Stat1 activation upon IFN-γ treatment of both cell lines. The EMSA also showed that 6 h of EHEC O157:H7 infection, but not 30 min of EHEC O157:H7 infection, prevented subsequent Stat1 DNA binding induced by IFN-γ, whereas infection with EPEC did not. Immunoblotting showed that infection with EHEC, but not infection with EPEC, eliminated IFN-γ-induced Stat1 tyrosine phosphorylation in both dose- and time-dependent fashions and disrupted inducible protein expression of the Stat1-dependent gene interferon regulatory factor 1. Immunofluorescence revealed that EHEC infection did not prevent nuclear accumulation of Stat1 after IFN-γ treatment. Also, Stat1 tyrosine phosphorylation was suppressed by different EHEC isolates, including intimin-, type III secretion- and plasmid-deficient strains, but not by HB101 and heat-killed EHEC. These findings indicate the novel disruption of host cell signaling caused by EHEC infection but not by EPEC infection.

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