scholarly journals Interaction of an Outer Membrane Protein of Enterotoxigenic Escherichia coli with Cell Surface Heparan Sulfate Proteoglycans

2002 ◽  
Vol 70 (3) ◽  
pp. 1530-1537 ◽  
Author(s):  
James M. Fleckenstein ◽  
James T. Holland ◽  
David L. Hasty
Keyword(s):  
Escherichia Coli ◽  
Epithelial Cell ◽  
Epithelial Cells ◽  
Cell Surface ◽  
Heparan Sulfate ◽  
Heparan Sulfate Proteoglycans ◽  
Eukaryotic Cells ◽  
Heparin Binding ◽  
Wild Type ◽  
E Coli

ABSTRACT We have previously shown that enterotoxigenic invasion protein A (Tia), a 25-kDa outer membrane protein encoded on an apparent pathogenicity island of enterotoxigenic Escherichia coli (ETEC) strain H10407, mediates attachment to and invasion into cultured human gastrointestinal epithelial cells. The epithelial cell receptor(s) for Tia has not been identified. Here we show that Tia interacts with cell surface heparan sulfate proteoglycans. Recombinant E. coli expressing Tia mediated invasion into wild-type epithelial cell lines but not invasion into proteoglycan-deficient cells. Furthermore, wild-type eukaryotic cells, but not proteoglycan-deficient eukaryotic cells, attached to immobilized polyhistidine-tagged recombinant Tia (rTia). Binding of epithelial cells to immobilized rTia was inhibited by exogenous heparan sulfate glycosaminoglycans but not by hyaluronic acid, dermatan sulfate, or chondroitin sulfate. Similarly, pretreatment of eukaryotic cells with heparinase I, but not pretreatment of eukaryotic cells with chrondroitinase ABC, inhibited attachment to rTia. In addition, we also observed heparin binding to both immobilized rTia and recombinant E. coli expressing Tia. Heparin binding was inhibited by a synthetic peptide representing a surface loop of Tia, as well as by antibodies directed against this peptide. Additional studies indicated that Tia, as a prokaryotic heparin binding protein, may also interact via sulfated proteoglycan molecular bridges with a number of mammalian heparan sulfate binding proteins. These findings suggest that the binding of Tia to host epithelial cells is mediated at least in part through heparan sulfate proteoglycans and that ETEC belongs on the growing list of pathogens that utilize these ubiquitous cell surface molecules as receptors.

scholarly journals A heparin-binding activity on Leishmania amastigotes which mediates adhesion to cellular proteoglycans.

1993 ◽  
Vol 123 (3) ◽  
pp. 759-766 ◽  
Author(s):  
D C Love ◽  
J D Esko ◽  
D M Mosser
Keyword(s):  
Heparan Sulfate ◽  
Cho Cells ◽  
Mammalian Cells ◽  
Binding Activity ◽  
Heparan Sulfate Proteoglycans ◽  
Heparin Binding ◽  
Wild Type ◽  
Amastigote Form ◽  
High Affinity ◽  
Leishmania Amastigotes

The intracellular amastigote form of leishmania is responsible for the cell-to-cell spread of leishmania infection in the mammalian host. In this report, we identify a high-affinity, heparin-binding activity on the surface of the amastigote form of leishmania. Amastigotes of Leishmania amazonensis bound approximately 120,000 molecules of heparin per cell, with a Kd of 8.8 x 10(-8) M. This heparin-binding activity mediates the adhesion of amastigotes to mammalian cells via heparan sulfate proteoglycans, which are expressed on the surface of mammalian cells. Amastigotes bound efficiently to a variety of adherent cells which express cell-surface proteoglycans. Unlike wild-type CHO cells, which bound amastigotes avidly, CHO cells with genetic deficiencies in heparan sulfate proteoglycan biosynthesis or cells treated with heparitinase failed to bind amastigotes even at high parasite-input dosages. Cells which express normal levels of undersulfated heparan bound amastigotes nearly as efficiently as did wild-type cells. The adhesion of amastigotes to wild-type nonmyeloid cells was almost completely inhibited by the addition of micromolar amounts of soluble heparin or heparan sulfate but not by the addition of other sulfated polysaccharides.l Binding of amastigotes to macrophages, however, was inhibited by only 60% after pretreatment of amastigotes with heparin, suggesting that macrophages have an additional mechanism for recognizing amastigotes. These results suggest that leishmania amastigotes express a high-affinity, heparin-binding activity on their surface which can interact with heparan sulfate proteoglycans on mammalian cells. This interaction may represent an important first step in the invasion of host cells by amastigotes.

scholarly journals Cloning, Expression, and Characterization of Fimbrial Operon F9 from Enterohemorrhagic Escherichia coli O157:H7

2006 ◽  
Vol 74 (4) ◽  
pp. 2233-2244 ◽  
Author(s):  
Alison S. Low ◽  
Francis Dziva ◽  
Alfredo G. Torres ◽  
Jessenya L. Martinez ◽  
Tracy Rosser ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Epithelial Cells ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Enterohemorrhagic Escherichia Coli ◽  
Wild Type ◽  
Induced Expression ◽  
E Coli ◽  
Extracellular Matrix Components ◽  
K 12

ABSTRACT Recent transposon mutagenesis studies with two enterohemorrhagic Escherichia coli (EHEC) strains, a sero- type O26:H- strain and a serotype O157:H7 strain, led to identification of a putative fimbrial operon that promotes colonization of young calves (1 to 2 weeks old). The distribution of the gene encoding the major fimbrial subunit present in O-island 61 of EHEC O157:H7 in a characterized set of 78 diarrheagenic E. coli strains was determined, and this gene was found in 87.2% of the strains and is therefore not an EHEC-specific region. The cluster was amplified by long-range PCR and cloned into the inducible expression vector pBAD18. Induced expression in E. coli K-12 led to production of fimbriae, as demonstrated by transmission electron microscopy and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. The fimbriae were purified, and sera to the purified major subunit were raised and used to demonstrate expression from wild-type E. coli O157:H7 strains. Induced expression of the fimbriae, designated F9 fimbriae, was used to characterize binding to bovine epithelial cells, bovine gastrointestinal tissue explants, and extracellular matrix components. The fimbriae promoted increases in the levels of E. coli K-12 binding only to bovine epithelial cells. In contrast, induced expression of F9 fimbriae in E. coli O157:H7 significantly reduced adherence of the bacteria to bovine gastrointestinal explant tissue. This may have been due to physical hindrance of type III secretion-dependent attachment. The main F9 subunit gene was deleted in E. coli O157:H7, and the resulting mutant was compared with the wild-type strain for colonization in weaned cattle. While the shedding levels of the mutant were reduced, the animals were still colonized at the terminal rectum, indicating that the adhesin is not responsible for the rectal tropism observed but may contribute to colonization at other sites, as demonstrated previously with very young animals.

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

2012 ◽  
Vol 78 (7) ◽  
pp. 2179-2189 ◽  
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.

scholarly journals CD14- and Toll-Like Receptor-Dependent Activation of Bladder Epithelial Cells by Lipopolysaccharide and Type 1 Piliated Escherichia coli

2003 ◽  
Vol 71 (3) ◽  
pp. 1470-1480 ◽  
Author(s):  
Joel D. Schilling ◽  
Steven M. Martin ◽  
David A. Hunstad ◽  
Kunal P. Patel ◽  
Matthew A. Mulvey ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Epithelial Cell ◽  
Epithelial Cells ◽  
Cell Lines ◽  
Epithelial Cell Line ◽  
Renal Epithelial Cell ◽  
E Coli ◽  
Epithelial Cell Lines ◽  
Renal Epithelial Cell Line

ABSTRACT The gram-negative bacterium Escherichia coli is the leading cause of urinary tract infection. The interaction between type 1 piliated E. coli and bladder epithelial cells leads to the rapid production of inflammatory mediators, such as interleukin-6 (IL-6) and IL-8. Conflicting reports have been published in the literature regarding the mechanism by which uroepithelial cells are activated by type 1 piliated E. coli. In particular, the role of lipopolysaccharide (LPS) in these responses has been an area of significant debate. Much of the data arguing against LPS-mediated activation of bladder epithelial cells have come from studies using a renal epithelial cell line as an in vitro model of the urinary epithelium. In this report, we analyzed three bladder epithelial cell lines and demonstrated that they all respond to LPS. Furthermore, the LPS responsivity of the cell lines directly correlated with their ability to generate IL-6 after E. coli stimulation. The LPS receptor complex utilized by the bladder epithelial cell lines included CD14 and Toll-like receptors, and signaling involved the activation of NF-κB and p38 mitogen-activated protein kinase. Also, reverse transcription-PCR analysis demonstrated that bladder epithelial cells express CD14 mRNA. Thus, the molecular machinery utilized by bladder epithelial cells for the recognition of E. coli is very similar to that described for traditional innate immune cells, such as macrophages. In contrast, the A498 renal epithelial cell line did not express CD14, was hyporesponsive to LPS stimulation, and demonstrated poor IL-6 responses to E. coli.

scholarly journals Increased Expression of Periplasmic Cu,Zn Superoxide Dismutase Enhances Survival of Escherichia coli Invasive Strains within Nonphagocytic Cells

2000 ◽  
Vol 68 (1) ◽  
pp. 30-37 ◽  
Author(s):  
Andrea Battistoni ◽  
Francesca Pacello ◽  
Silvia Folcarelli ◽  
Maria Ajello ◽  
Giovanna Donnarumma ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Superoxide Dismutase ◽  
Epithelial Cells ◽  
Type Strain ◽  
Wild Type Strain ◽  
Yersinia Pseudotuberculosis ◽  
Host Cells ◽  
Wild Type ◽  
Bacterial Killing ◽  
E Coli

ABSTRACT We have studied the influence of periplasmic Cu,Zn superoxide dismutase on the intracellular survival of Escherichia colistrains able to invade epithelial cells by the expression of theinv gene from Yersinia pseudotuberculosis but unable to multiply intracellularly. Intracellular viability assays, confirmed by electron microscopy observations, showed that invasive strains of E. coli engineered to increase Cu,Zn superoxide dismutase production are much more resistant to intracellular killing than strains containing only the chromosomalsodC copy. However, we have found only a slight difference in survival within HeLa cells between a sodC-null mutant and its isogenic wild-type strain. Such a small difference in survival correlates with the very low expression of this enzyme in the wild-type strain. We have also observed that acid- and oxidative stress-sensitiveE. coli HB101(pRI203) is more rapidly killed in epithelial cells than E. coli GC4468(pRI203). The high mortality ofE. coli HB101(pRI203), independent of the acidification of the endosome, is abolished by the overexpression of sodC. Our data suggest that oxyradicals are involved in the mechanisms of bacterial killing within epithelial cells and that high-level production of periplasmic Cu,Zn superoxide dismutase provides bacteria with an effective protection against oxidative damage. We propose that Cu,Zn superoxide dismutase could offer an important selective advantage in survival within host cells to bacteria expressing high levels of this enzyme.

scholarly journals Impact of vaginal douching products on vaginal Lactobacillus, Escherichia coli and epithelial immune responses

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Helai Hesham ◽  
Alissa J. Mitchell ◽  
Agnes Bergerat ◽  
Kristin Hung ◽  
Caroline M. Mitchell
Keyword(s):  
Escherichia Coli ◽  
Cell Death ◽  
Epithelial Cell ◽  
Epithelial Cells ◽  
Immune Responses ◽  
E Coli ◽  
Vaginal Douching ◽  
Vaginal Epithelial Cells ◽  
Epithelial Disruption ◽  
Epithelial Cell Death

AbstractWe compared the effect of commercial vaginal douching products on Lactobacillus crispatus, L. jensenii, L. gasseri, L. iners, E. coli, and immortalized vaginal epithelial cells (VK2). All studied douching products (vinegar, iodine and baking soda based) induced epithelial cell death, and all inhibited growth of E. coli. Co-culture of vaginal epithelial cells with any of the lactobacilli immediately following exposure to douching products resulted in a trend to less human cell death. However, co-culture of epithelial cells with L. iners was associated with higher production of IL6 and IL8, and lower IL1RA regardless of presence or type of douching solution. Co-culture with L. crispatus or L. jensenii decreased IL6 production in the absence of douches, but increased IL6 production after exposure to vinegar. Douching products may be associated with epithelial disruption and inflammation, and may reduce the anti-inflammatory effects of beneficial lactobacilli.

scholarly journals Cytokeratin 8 Is an Epithelial Cell Receptor for Pet, a Cytotoxic Serine Protease Autotransporter of Enterobacteriaceae

mBio ◽  
2013 ◽  
Vol 4 (6) ◽  
Author(s):  
Raul Nava-Acosta ◽  
Fernando Navarro-Garcia
Keyword(s):  
Escherichia Coli ◽  
Epithelial Cell ◽  
Epithelial Cells ◽  
Cell Surface ◽  
Serine Protease ◽  
Cell Receptor ◽  
Persistent Diarrhea ◽  
Receptor Ligand ◽  
Content Type ◽  
Class 1

ABSTRACTThe group of proteins known as serine protease autotransporters ofEnterobacteriaceae(SPATE) is a growing family of serine proteases secreted to the external milieu by the type V secretion system. Pet toxin and some other SPATE belong to the class 1 cytotoxic SPATE, which have comparable protease strength on fodrin. Pet is internalized and is directed to its intracellular substrate by retrograde transport. However, the epithelial cell receptor for Pet has yet to be identified. We show that Pet has affinity for the epithelial cell surface until the saturation of the binding sites at 100 nM Pet. Affinity column assays and matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) analysis identified a cytokeratin (CK8) which directly binds to Pet, and both proteins colocalized on the cell surface. Interestingly, CK8 is not present in kidney cell lines, which are not susceptible to Pet. Inhibition experiments by using anti-CK8 andck8small interfering RNA (siRNA) blocked the cytotoxic effect induced by Pet, while exogenous CK8 expression in kidney cells made them susceptible to Pet intoxication. Recombinant CK8 showed a Pet-binding pattern similar to that seen by using fixed cells. Remarkably, Pet colocalized with CK8 and clathrin at early times (receptor-mediated endocytosis), and subsequently, Pet colocalized with CK8 and Rab5b in the early endosomes. These data support the idea that CK8 is an important receptor for Pet on epithelial cells for starting its cytotoxic effects. These data suggest that therapeutics that block Pet-CK8 interaction may improve outcome of diseases caused by Pet-secretingEnterobacteriaceaesuch as enteroaggregativeEscherichia coli.IMPORTANCEReceptor-ligand binding is one mechanism by which cells sense and respond to external cues. Receptors may also be utilized by toxins to mediate their own internalization. Pet toxin is secreted by enteroaggregativeEscherichia coli, an organism that causes persistent diarrhea in children, traveler’s diarrhea, and acute and persistent diarrhea in patients with HIV. Pet is a member of the family of serine protease autotransporters ofEnterobacteriaceae(SPATE). SPATE in different pathogens are virulence factors, and Pet belongs to the class 1 cytotoxic SPATE, which have comparable protease strength on their biological substrate, fodrin (a cytoskeletal protein important for maintaining cell viability). To cleave fodrin, Pet enters the cells by clathrin-mediated endocytosis. This mechanism includes receptor-mediated endocytosis (a receptor-ligand complex triggers the endocytosis). We show that CK8 is an important receptor for Pet on epithelial cells and that it may be useful for identifying molecules that block the interaction of CK8 with Pet.

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