EspFu-Mediated Actin Assembly Enhances Enteropathogenic Escherichia coli Adherence and Activates Host Cell Inflammatory Signaling Pathways

ABSTRACT The translocation of effectors into the host cell through type 3 secretion systems (T3SS) is a sophisticated strategy employed by pathogenic bacteria to subvert host responses and facilitate colonization. Enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC) utilize the Tir and EspFu (also known as TccP) effectors to remodel the host cytoskeleton, culminating in the formation of attaching and effacing (AE) lesions on enterocytes. While some EPEC strains require tyrosine phosphorylation of Tir and recruitment of the host Nck to trigger actin polymerization, EHEC and certain EPEC strains, whose Tir is not phosphorylated, rely on the effector EspFu for efficient actin remodeling. Here, we investigated the role played by Tir-Nck and Tir-EspFu actin polymerization pathways during the infection of epithelial cells, as well as the host transcriptional response to the AE lesion formation induced by EPEC. We found that EspFu-mediated actin assembly promotes bacterial attachment and epithelial colonization more efficiently than Tir-Nck. Moreover, we showed that both actin polymerization mechanisms can activate inflammatory pathways and reverse the anti-inflammatory response induced by EPEC in epithelial cells. However, this activity is remarkably more evident in infections with EspFu-expressing EPEC strains. This study demonstrates the complex interactions between effector-mediated actin remodeling and inflammation. Different strains carry different combinations of these two effectors, highlighting the plasticity of pathogenic E. coli enteric infections. IMPORTANCE EPEC is among the leading causes of diarrheal disease worldwide. The colonization of the gut mucosa by EPEC results in actin pedestal formation at the site of bacterial attachment. These pedestals are referred to as attaching and effacing (AE) lesions. Here, we exploit the different molecular mechanisms used by EPEC to induce AE lesions on epithelial cells, showing that the effector EspFu is associated with increased bacterial attachment and enhanced epithelial colonization compared to the Tir-Nck pathway. Moreover, we also showed that actin pedestal formation can counterbalance the anti-inflammatory activity induced by EPEC, especially when driven by EspFu. Collectively, our findings provide new insights into virulence mechanisms employed by EPEC to colonize epithelial cells, as well as the host response to this enteric pathogen.

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Pathogenic Escherichia coli Hijacks GTPase-Activated p21-Activated Kinase for Actin Pedestal Formation

mBio ◽

10.1128/mbio.01876-19 ◽

2019 ◽

Vol 10 (4) ◽

Cited By ~ 3

Author(s):

Vikash Singh ◽

Anthony Davidson ◽

Peter J. Hume ◽

Vassilis Koronakis

Keyword(s):

Escherichia Coli ◽

Host Cell ◽

Early Stage ◽

Small Gtpases ◽

Bacterial Attachment ◽

Host Cells ◽

Content Type ◽

E Coli ◽

P21 Activated Kinase ◽

Pedestal Formation

ABSTRACT Enteropathogenic Escherichia coli and enterohemorrhagic E. coli (EPEC and EHEC, respectively) are extracellular pathogens that reorganize the host cell cytoskeleton to form “actin pedestals” beneath the tightly adherent bacteria, a critical step in pathogenesis. EPEC and EHEC inject effector proteins that manipulate host cell signaling cascades to trigger pedestal assembly. One such effector, EspG, has been reported to bind and activate p21-activated kinase (PAK), a key cytoskeletal regulator, but the function of this interaction and whether it impacts pedestal assembly are unknown. Here, we demonstrate that deletion of espG significantly impairs pedestal formation and attachment by both EPEC and EHEC. This role of EspG is shown to be dependent on its interaction with PAK. Unexpectedly, EspG was able to subvert PAK only in the presence of Rho family small GTPases, which function to both concentrate PAK at the membrane and stimulate PAK activation. Our findings reveal a novel mechanism by which EspG hijacks PAK and sustains its active state to drive bacterial attachment to host cells. IMPORTANCE Enteropathogenic E. coli and enterohemorrhagic E. coli (EPEC and EHEC, respectively) remain a significant global health problem. Both EPEC and EHEC initiate infection by attaching to cells in the host intestine, triggering the formation of actin-rich “pedestal” structures directly beneath the adherent pathogen. These bacteria inject their own receptor into host cells, which upon binding to a protein on the pathogen surface triggers pedestal formation. Multiple other proteins are also delivered into the cells of the host intestine, but how they contribute to disease is often less clear. Here, we show how one of these injected proteins, EspG, hijacks a host signaling pathway for pedestal production. This provides new insights into this essential early stage in EPEC and EHEC disease.

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Posttranscriptional Control of Microbe-Induced Rearrangement of Host Cell Actin

mBio ◽

10.1128/mbio.01025-13 ◽

2014 ◽

Vol 5 (1) ◽

Cited By ~ 35

Author(s):

Charley C. Gruber ◽

Vanessa Sperandio

Keyword(s):

Escherichia Coli ◽

Host Cell ◽

Type Iii Secretion ◽

Cell Biology ◽

Molecular Mechanisms ◽

Bacterial Attachment ◽

Posttranscriptional Control ◽

Content Type ◽

Type Iii ◽

Pedestal Formation

ABSTRACTRemodeling of the host cytoskeleton is a common strategy employed by bacterial pathogens. Although there is vigorous investigation of the cell biology underlying these bacterially mediated cytoskeleton modifications, knowledge of the plasticity and dynamics of the bacterial signaling networks that regulate the expression of genes necessary for these phenotypes is lacking. EnterohemorrhagicEscherichia coliattaches to enterocytes, forming pedestal-like structures. Pedestal formation requires the expression of the locus-of-enterocyte-effacement (LEE) andespFugenes. The LEE encodes a molecular syringe, a type III secretion system (T3SS) used by pathogens to translocate effectors such as EspFu into the host cell. By using a combination of genetic, biochemical, and cell biology approaches, we show that pedestal formation relies on posttranscriptional regulation by two small RNAs (sRNAs), GlmY and GlmZ. The GlmY and GlmZ sRNAs are unique; they have extensive secondary structures and work in concert. Although these sRNAs may offer unique insights into RNA and posttranscriptional biology, thus far, only one target and one mechanism of action (exposure of the ribosome binding site from theglmSgene to promote its translation) has been described. Here we uncovered new targets and two different molecular mechanisms of action of these sRNAs. In the case of EspFu expression, they promote translation by cleavage of the transcript, while in regard to the LEE, they promote destabilization of the mRNA. Our findings reveal that two unique sRNAs act in concert through different molecular mechanisms to coordinate bacterial attachment to mammalian cells.IMPORTANCEPathogens evolve by horizontal acquisition of pathogenicity islands. We describe here how two sRNAs, GlmY and GlmZ, involved in cellular metabolism and cellular architecture, through the posttranscriptional control of GlmS (the previously only known target of GlmY and GlmZ), which controls amino sugar synthesis, have been coopted to modulate the expression of virulence. These sRNAs quickly allow for plasticity in gene expression in order for enterohemorrhagicEscherichia colito fine-tune the expression of its complex type III secretion machinery and its effectors to promote bacterial attachment and subsequent actin rearrangement on host cells. Pedestal formation is a very dynamic process. Many of the genes necessary for pedestal formation are located within the same operon to evolutionarily guarantee that they are inherited together. However, it is worth noting that within these operons, several genes need to yield more proteins than others and that these differences cannot be efficiently regulated at the transcriptional level.

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EPEC Recruits a Cdc42-Specific GEF, Frabin, To Facilitate PAK Activation and Host Cell Colonization

mBio ◽

10.1128/mbio.01423-20 ◽

2020 ◽

Vol 11 (6) ◽

Author(s):

Vikash Singh ◽

Peter J. Hume ◽

Anthony Davidson ◽

Vassilis Koronakis

Keyword(s):

Escherichia Coli ◽

Host Cell ◽

Small Gtpases ◽

Bacterial Attachment ◽

Host Protein ◽

Host Cells ◽

Target Cells ◽

Content Type ◽

Activated State ◽

Pedestal Formation

ABSTRACT Enteropathogenic Escherichia coli (EPEC) is an extracellular pathogen that tightly adheres to host cells by forming “actin pedestals” beneath the bacteria, a critical step in pathogenesis. EPEC injects effector proteins that manipulate host cell signaling cascades to trigger pedestal assembly. We have recently shown that one such effector, EspG, hijacks p21-activated kinase (PAK) and sustains its activated state to drive the cytoskeletal changes necessary for attachment of the pathogen to target cells. This EspG subversion of PAK required active Rho family small GTPases in the host cell. Here we show that EPEC itself promotes the activation of Rho GTPases by recruiting Frabin, a host guanine nucleotide exchange factor (GEF) for the Rho GTPase Cdc42. Cells devoid of Frabin showed significantly lower EPEC-induced PAK activation, pedestal formation, and bacterial attachment. Frabin recruitment to sites of EPEC attachment was driven by EspG and required localized enrichment of phosphatidylinositol 4,5-bisphosphate (PIP2) and host Arf6. Our findings identify Frabin as a key target for EPEC to ensure the activation status of cellular GTPases required for actin pedestal formation. IMPORTANCE Enteropathogenic Escherichia coli (EPEC) is a leading cause of diarrhea in children, especially in the developing world. EPEC initiates infection by attaching to cells in the host intestine, triggering the formation of actin-rich “pedestal” structures directly beneath the adherent pathogen. These bacteria inject their own receptor into host cells, which upon binding to a protein on the pathogen surface triggers pedestal formation. Multiple other proteins are also delivered into the cells of the host intestine, which work together to hijack host signaling pathways to drive pedestal production. Here we show how EPEC hijacks a host protein, Frabin, which creates the conditions in the cell necessary for the pathogen to manipulate a specific pathway that promotes pedestal formation. This provides new insights into this essential early stage in disease caused by EPEC.

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The interaction of Escherichia coli O157 :H7 and Salmonella Typhimurium flagella with host cell membranes and cytoskeletal components

Microbiology ◽

10.1099/mic.0.000959 ◽

2020 ◽

Vol 166 (10) ◽

pp. 947-965 ◽

Cited By ~ 1

Author(s):

Eliza B. Wolfson ◽

Johanna Elvidge ◽

Amin Tahoun ◽

Trudi Gillespie ◽

Judith Mantell ◽

...

Keyword(s):

Electron Microscopy ◽

Escherichia Coli ◽

Host Cell ◽

Type Species ◽

Actin Polymerization ◽

Cell Membranes ◽

Content Type ◽

E Coli ◽

Link Type ◽

Host Cell Membranes

Bacterial flagella have many established roles beyond swimming motility. Despite clear evidence of flagella-dependent adherence, the specificity of the ligands and mechanisms of binding are still debated. In this study, the molecular basis of Escherichia coli O157:H7 and Salmonella enterica serovar Typhimurium flagella binding to epithelial cell cultures was investigated. Flagella interactions with host cell surfaces were intimate and crossed cellular boundaries as demarcated by actin and membrane labelling. Scanning electron microscopy revealed flagella disappearing into cellular surfaces and transmission electron microscopy of S. Typhiumurium indicated host membrane deformation and disruption in proximity to flagella. Motor mutants of E. coli O157:H7 and S. Typhimurium caused reduced haemolysis compared to wild-type, indicating that membrane disruption was in part due to flagella rotation. Flagella from E. coli O157 (H7), EPEC O127 (H6) and S. Typhimurium (P1 and P2 flagella) were shown to bind to purified intracellular components of the actin cytoskeleton and directly increase in vitro actin polymerization rates. We propose that flagella interactions with host cell membranes and cytoskeletal components may help prime intimate attachment and invasion for E. coli O157:H7 and S. Typhimurium, respectively.

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

mBio ◽

10.1128/mbio.00025-15 ◽

2015 ◽

Vol 6 (2) ◽

Cited By ~ 33

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.

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Comparative Analysis of EspF Variants in Inhibition of Escherichia coli Phagocytosis by Macrophages and Inhibition of E. coli Translocation through Human- and Bovine-Derived M Cells

Infection and Immunity ◽

10.1128/iai.00023-11 ◽

2011 ◽

Vol 79 (11) ◽

pp. 4716-4729 ◽

Cited By ~ 19

Author(s):

Amin Tahoun ◽

Gabriella Siszler ◽

Kevin Spears ◽

Sean McAteer ◽

Jai Tree ◽

...

Keyword(s):

Escherichia Coli ◽

Epithelial Cells ◽

Cellular Localization ◽

M Cells ◽

Binding Assays ◽

M Cell ◽

Content Type ◽

E Coli ◽

Coculture System

ABSTRACTThe EspF protein is secreted by the type III secretion system of enteropathogenic and enterohemorrhagicEscherichia coli(EPEC and EHEC, respectively). EspF sequences differ between EHEC O157:H7, EHEC O26:H11, and EPEC O127:H6 in terms of the number of SH3-binding polyproline-rich repeats and specific residues in these regions, as well as residues in the amino domain involved in cellular localization. EspFO127is important for the inhibition of phagocytosis by EPEC and also limits EPEC translocation through antigen-sampling cells (M cells). EspFO127has been shown to have effects on cellular organelle function and interacts with several host proteins, including N-WASP and sorting nexin 9 (SNX9). In this study, we compared the capacities of differentespFalleles to inhibit (i) bacterial phagocytosis by macrophages, (ii) translocation through an M-cell coculture system, and (iii) uptake by and translocation through cultured bovine epithelial cells. TheespFgene fromE. coliserotype O157 (espFO157) allele was significantly less effective at inhibiting phagocytosis and also had reduced capacity to inhibitE. colitranslocation through a human-derivedin vitroM-cell coculture system in comparison toespFO127andespFO26. In contrast,espFO157was the most effective allele at restricting bacterial uptake into and translocation through primary epithelial cells cultured from the bovine terminal rectum, the predominant colonization site of EHEC O157 in cattle and a site containing M-like cells. Although LUMIER binding assays demonstrated differences in the interactions of the EspF variants with SNX9 and N-WASP, we propose that other, as-yet-uncharacterized interactions contribute to the host-based variation in EspF activity demonstrated here.

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The Legionella Kinase LegK2 Targets the ARP2/3 Complex To Inhibit Actin Nucleation on Phagosomes and Allow Bacterial Evasion of the Late Endocytic Pathway

mBio ◽

10.1128/mbio.00354-15 ◽

2015 ◽

Vol 6 (3) ◽

Cited By ~ 36

Author(s):

Céline Michard ◽

Daniel Sperandio ◽

Nathalie Baïlo ◽

Javier Pizarro-Cerdá ◽

Lawrence LeClaire ◽

...

Keyword(s):

Protein Kinase ◽

Host Cell ◽

Legionella Pneumophila ◽

Actin Polymerization ◽

Secretion System ◽

Endocytic Pathway ◽

Specific Chemical ◽

Actin Remodeling ◽

Content Type

ABSTRACTLegionella pneumophila, the etiological agent of legionellosis, replicates within phagocytic cells. Crucial to biogenesis of the replicative vacuole is the Dot/Icm type 4 secretion system, which translocates a large number of effectors into the host cell cytosol. Among them is LegK2, a protein kinase that plays a key role inLegionellainfection. Here, we identified the actin nucleator ARP2/3 complex as a target of LegK2. LegK2 phosphorylates the ARPC1B and ARP3 subunits of the ARP2/3 complex. LegK2-dependent ARP2/3 phosphorylation triggers global actin cytoskeleton remodeling in cells, and it impairs actin tail formation byListeria monocytogenes, a well-known ARP2/3-dependent process. During infection, LegK2 is addressed to theLegionella-containing vacuole surface and inhibits actin polymerization on the phagosome, as revealed by legK2 gene inactivation. Consequently, LegK2 prevents late endosome/lysosome association with the phagosome and finally contributes to remodeling of the bacterium-containing phagosome into a replicative niche. The inhibition of actin polymerization by LegK2 and its effect on endosome trafficking are ARP2/3 dependent since it can be phenocopied by a specific chemical inhibitor of the ARP2/3 complex. Thus, LegK2-ARP2/3 interplay highlights an original mechanism of bacterial virulence with an unexpected role in local actin remodeling that allows bacteria to control vesicle trafficking in order to escape host defenses.IMPORTANCEDeciphering the individual contribution of each Dot/Icm type 4 secretion system substrate to the intracellular life-style ofL. pneumophilaremains the principal challenge in understanding the molecular basis ofLegionellavirulence. Our finding that LegK2 is a Dot/Icm effector that inhibits actin polymerization on theLegionella-containing vacuole importantly contributes to the deciphering of the molecular mechanisms evolved byLegionellato counteract the endocytic pathway. Indeed, our results highlight the essential role of LegK2 in preventing late endosomes from fusing with the phagosome. More generally, this work is the first demonstration of local actin remodeling as a mechanism used by bacteria to control organelle trafficking. Further, by characterizing the role of the bacterial protein kinase LegK2, we reinforce the concept that posttranslational modifications are key strategies used by pathogens to evade host cell defenses.

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Formin FHOD1 regulates the size of EPEC pedestals

10.1101/2020.06.15.149344 ◽

2020 ◽

Author(s):

Xuyao Priscilla Liu ◽

Mrinal Shah ◽

Linda J. Kenney

Keyword(s):

Surface Area ◽

Actin Filaments ◽

Actin Polymerization ◽

Src Family Kinases ◽

Bacterial Attachment ◽

E Coli ◽

Pedestal Formation

SummaryEnteropathogenic E. coli (EPEC) is an extracellular pathogen that causes polymerization of actin filaments at the site of bacterial attachment, referred to as ‘actin pedestals’. Actin polymerization in the pedestal was believed to be solely regulated via the Nck-WASp-Arp2/3 pathway before formins were recently discovered to be associated with pedestals. Herein, we explored the collaborative role of formins in contributing to EPEC pedestal formation. In particular, we discovered that the formin FHOD1 preferentially localized to the pedestal base and its knockdown drastically reduced pedestal surface area. The pedestal localization of formin FHOD1 was found to be dependent on Tir phosphorylation at Y474, and on FHOD1 phosphorylation at Y99 from host Src family kinases (SFKs). Interestingly, differences in Arp2/3 and FHOD1 dynamics were observed. In large pedestals, Arp3 was nearly absent, but FHOD1 levels were high, suggesting that Arp2/3 and formins were segregated temporally. In line with this observation, as the pedestals grew in size, FHOD1 localization increased, while Arp3 localization decreased along the pedestals. Together, our results suggest that EPEC employs multiple actin nucleators that act at different stages of pedestal formation.Graphical abstract

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Molecular Characterization of the EhaG and UpaG Trimeric Autotransporter Proteins from Pathogenic Escherichia coli

Applied and Environmental Microbiology ◽

10.1128/aem.06680-11 ◽

2012 ◽

Vol 78 (7) ◽

pp. 2179-2189 ◽

Cited By ~ 47

Author(s):

Makrina Totsika ◽

Timothy J. Wells ◽

Christophe Beloin ◽

Jaione Valle ◽

Luke P. Allsopp ◽

...

Keyword(s):

Escherichia Coli ◽

Epithelial Cells ◽

Cell Surface ◽

Specific Binding ◽

Negative Regulator ◽

Passenger Domain ◽

Content Type ◽

E Coli ◽

Ecm Proteins

ABSTRACTTrimeric autotransporter proteins (TAAs) are important virulence factors of many Gram-negative bacterial pathogens. A common feature of most TAAs is the ability to mediate adherence to eukaryotic cells or extracellular matrix (ECM) proteins via a cell surface-exposed passenger domain. Here we describe the characterization of EhaG, a TAA identified from enterohemorrhagicEscherichia coli(EHEC) O157:H7. EhaG is a positional orthologue of the recently characterized UpaG TAA from uropathogenicE. coli(UPEC). Similarly to UpaG, EhaG localized at the bacterial cell surface and promoted cell aggregation, biofilm formation, and adherence to a range of ECM proteins. However, the two orthologues display differential cellular binding: EhaG mediates specific adhesion to colorectal epithelial cells while UpaG promotes specific binding to bladder epithelial cells. The EhaG and UpaG TAAs contain extensive sequence divergence in their respective passenger domains that could account for these differences. Indeed, sequence analyses of UpaG and EhaG homologues from severalE. coligenomes revealed grouping of the proteins in clades almost exclusively represented by distinctE. colipathotypes. The expression of EhaG (in EHEC) and UpaG (in UPEC) was also investigated and shown to be significantly enhanced in anhnsisogenic mutant, suggesting that H-NS acts as a negative regulator of both TAAs. Thus, while the EhaG and UpaG TAAs contain some conserved binding and regulatory features, they also possess important differences that correlate with the distinct pathogenic lifestyles of EHEC and UPEC.

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

Infection and Immunity ◽

10.1128/iai.71.3.1396-1404.2003 ◽

2003 ◽

Vol 71 (3) ◽

pp. 1396-1404 ◽

Cited By ~ 30

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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