scholarly journals Structural and Functional Lesions in Brush Border of Human Polarized Intestinal Caco-2/TC7 Cells Infected by Members of the Afa/Dr Diffusely Adhering Family of Escherichia coli

2000 ◽  
Vol 68 (10) ◽  
pp. 5979-5990 ◽  
Author(s):  
Isabelle Peiffer ◽  
Julie Guignot ◽  
Alain Barbat ◽  
Christophe Carnoy ◽  
Steve L. Moseley ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Brush Border ◽  
Glucose Transporter ◽  
Binding Capacity ◽  
Cell Interaction ◽  
Cytoskeletal Proteins ◽  
Wild Type ◽  
Fimbrial Adhesin ◽  
Human Intestinal Cells ◽  
Dependent Mechanism

ABSTRACT Diffusely adhering Escherichia coli (DAEC) strains expressing F1845 fimbrial adhesin or Dr hemagglutinin belonging to the Afa/Dr family of adhesins infect cultured polarized human intestinal cells through recognition of the brush border-associated decay-accelerating factor (DAF; CD55) as a receptor. The wild-type Afa/Dr DAEC strain C1845 has been shown to induce brush border lesions by an adhesin-dependent mechanism triggering apical F-actin rearrangements. In the present study, we undertook to further characterize cell injuries following the interaction of wild-type Afa/Dr DAEC strains C1845 and IH11128 expressing fimbrial F1845 adhesin and Dr hemagglutinin, respectively, with polarized, fully differentiated Caco-2/TC7 cells. In both cases, bacterium-cell interaction was followed by rearrangement of the major brush border-associated cytoskeletal proteins F-actin, villin, and fimbrin, proteins which play a pivotal role in brush border assembly. In contrast, distribution of G-actin, actin-depolymerizing factor, and tubulin was not modified. Using draE mutants, we found that a mutant in which cysteine replaces aspartic acid at position 54 conserved binding capacity but failed to induce F-actin disassembly. Accompanying the cytoskeleton injuries, we found that the distribution of brush border-associated functional proteins sucrase-isomaltase (SI), dipeptidylpeptidase IV (DPPIV), glucose transporter SGLT1, and fructose transporter GLUT5 was dramatically altered. In parallel, SI and DPPIV enzyme activity decreased.

Enteropathogenic Escherichia coli adherence to intestinal epithelial monolayers diminishes barrier function

1995 ◽  
Vol 268 (2) ◽  
pp. G374-G379 ◽  
Author(s):  
J. Spitz ◽  
R. Yuhan ◽  
A. Koutsouris ◽  
C. Blatt ◽  
J. Alverdy ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Host Cell ◽  
Barrier Function ◽  
Cell Function ◽  
Cytoskeletal Proteins ◽  
Calcium Stores ◽  
Intestinal Epithelial ◽  
Enteropathogenic Escherichia Coli ◽  
Wild Type ◽  
Cell Monolayers

The mechanism by which enteropathogenic Escherichia coli (EPEC) causes diarrhea remains elusive. Several alterations within the host cell have been demonstrated to occur following EPEC attachment including increases in intracellular Ca2+ concentration and rearrangement and phosphorylation of several cytoskeletal proteins. The consequences of these intracellular perturbations on host cell function, however, have not been determined. The aim of this study was to examine the effect of EPEC adherence on intestinal epithelial barrier function. T84 cell monolayers were infected with either wild-type EPEC or a nonadherent isogenic derivative. Transepithelial electrical resistance, a measure of barrier function, decreased 33.5 +/- 6.4% after a 6-h incubation with the wild-type strain. Electron microscopy revealed ultrastructurally normal cells, and lactate dehydrogenase release assays failed to demonstrate cytotoxicity. Dual 22Na+ and [3H]mannitol flux studies localized the permeability defect to tight junctions. In addition, cumulative flux of the paracellular marker mannitol was four- to fivefold greater across monolayers infected with wild-type EPEC. Sequestration of intracellular calcium stores by dantrolene completely abrogated the resistance drop associated with EPEC attachment. These data demonstrate that adherence of EPEC to intestinal epithelial cell monolayers disrupts tight junction barrier function via a calcium-requiring event.

scholarly journals Villin and actin in the mouse kidney brush-border membrane bind to and are degraded by meprins, an interaction that contributes to injury in ischemia-reperfusion

2011 ◽  
Vol 301 (4) ◽  
pp. F871-F882 ◽  
Author(s):  
Elimelda Moige Ongeri ◽  
Odinaka Anyanwu ◽  
W. Brian Reeves ◽  
Judith S. Bond
Keyword(s):  
Brush Border ◽  
Knockout Mice ◽  
Brush Border Membrane ◽  
Ischemia Reperfusion ◽  
Kidney Injury ◽  
Mouse Kidney ◽  
Cytoskeletal Proteins ◽  
Kidney Tubules ◽  
Wild Type

Meprins, metalloproteinases abundantly expressed in the brush-border membranes (BBMs) of rodent proximal kidney tubules, have been implicated in the pathology of renal injury induced by ischemia-reperfusion (IR). Disruption of the meprin β gene and actinonin, a meprin inhibitor, both decrease kidney injury resulting from IR. To date, the in vivo kidney substrates for meprins are unknown. The studies herein implicate villin and actin as meprin substrates. Villin and actin bind to the cytoplasmic tail of meprin β, and both meprin A and B are capable of degrading villin and actin present in kidney proteins as well as purified recombinant forms of these proteins. The products resulting from degradation of villin and actin were unique to each meprin isoform. The meprin B cleavage site in villin was Glu744-Val745. Recombinant forms of rat meprin B and homomeric mouse meprin A had Km values for villin and actin of ∼1 μM (0.6–1.2 μM). The kcat values varied substantially (0.6–128 s−1), resulting in different efficiencies for cleavage, with meprin B having the highest kcat/ Km values (128 M−1·s−1 × 106). Following IR, meprins and villin redistributed from the BBM to the cytosol. A 37-kDa actin fragment was detected in protein fractions from wild-type, but not in comparable preparations from meprin knockout mice. The levels of the 37-kDa actin fragment were significantly higher in kidneys subjected to IR. The data establish that meprins interact with and cleave villin and actin, and these cytoskeletal proteins are substrates for meprins.

scholarly journals Recruitment of CD55 and CD66e Brush Border-Associated Glycosylphosphatidylinositol-Anchored Proteins by Members of the Afa/Dr Diffusely Adhering Family of Escherichia coli That Infect the Human Polarized Intestinal Caco-2/TC7 Cells

2000 ◽  
Vol 68 (6) ◽  
pp. 3554-3563 ◽  
Author(s):  
Julie Guignot ◽  
Isabelle Peiffer ◽  
Marie-Françoise Bernet-Camard ◽  
Douglas M. Lublin ◽  
Christophe Carnoy ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Brush Border ◽  
Intestinal Epithelial Cells ◽  
Binding Capacity ◽  
Deletion Mutants ◽  
Stable Transfection ◽  
Intestinal Epithelial ◽  
Inhibition Assay ◽  
Short Consensus Repeat ◽  
Bacteria Inhibition

ABSTRACT The Afa/Dr family of diffusely adhering Escherichia coli (Afa/Dr DAEC) includes bacteria expressing afimbrial adhesins (AFA), Dr hemagglutinin, and fimbrial F1845 adhesin. We show that infection of human intestinal Caco-2/TC7 cells by the Afa/Dr DAEC strains C1845 and IH11128 is followed by clustering of CD55 around adhering bacteria. Mapping of CD55 epitopes involved in CD55 clustering by Afa/Dr DAEC was conducted using CD55 deletion mutants expressed by stable transfection in CHO cells. Deletion in the short consensus repeat 1 (SCR1) domain abolished Afa/Dr DAEC-induced CD55 clustering. In contrast, deletion in the SCR4 domain does not modify Afa/Dr DAEC-induced CD55 clustering. We show that the brush border-associated glycosylphosphatidylinositol (GPI)-anchored protein CD66e (carcinoembryonic antigen) is recruited by the Afa/Dr DAEC strains C1845 and IH11128. This conclusion is based on the observations that (i) infection of Caco-2/TC7 cells by Afa/Dr DAEC strains is followed by clustering of CD66e around adhering bacteria and (ii) Afa/Dr DAEC strains bound efficiently to stably transfected HeLa cells expressing CD66e, accompanied by CD66e clustering around adhering bacteria. Inhibition assay using monoclonal antibodies directed against CD55 SCR domains, and polyclonal anti-CD55 and anti-CD66e antibodies demonstrate that CD55 and CD66e function as a receptors for the C1845 and IH11128 bacteria. Moreover, using structural draE gene mutants, we found that a mutant in which cysteine replaced aspartic acid at position 54 displayed conserved binding capacity but failed to induce CD55 and CD66e clustering. Taken together, these data give new insights into the mechanisms by which Afa/Dr DAEC induces adhesin-dependent cross talk in the human polarized intestinal epithelial cells by mobilizing brush border-associated GPI-anchored proteins known to function as transducing molecules.

scholarly journals MYH10 governs adipocyte function and adipogenesis through its interaction with GLUT4

2021 ◽  
Author(s):  
N Kislev ◽  
L Mor-Yossef Moldovan ◽  
R Barak ◽  
M Egozi ◽  
D Benayahu
Keyword(s):  
Glucose Transporter ◽  
Cytoskeletal Proteins ◽  
Wild Type ◽  
Cytoskeletal Remodeling ◽  
Kinase C ◽  
Important Regulator ◽  
Insulin Dependent ◽  
And Function ◽  
Cellular Components ◽  
Muscle Myosin

AbstractAdipocyte differentiation is dependent on cytoskeletal remodeling processes that determine and maintain cellular shape and function. In turn, cytoskeletal proteins contribute to the filament-based network responsible for controlling adipocyte’s shape and promoting the intracellular trafficking of key cellular components. Currently, our understanding of these mechanisms remains incomplete. In this study, we identified the non-muscle myosin 10 (MYH10) as an important regulator of adipogenesis and adipocyte function through its interaction with the insulin dependent, Glucose transporter 4 (GLUT4). MYH10 depletion in preadipocytes resulted in impaired adipogenesis, with knockdown cells exhibiting disrupted morphology and reduced molecular adipogenic signals. MYH10 was shown to be in complex with GLUT4 in adipocytes, an interaction regulated by insulin induction. The missing adipogenic capacity of MYH10-KD cells was restored when they uptook GLUT4 vesicles up from neighbor wild-type cells in a co-culture system. Our results provide the first demonstration that MYH10 interacts with GLUT4 in cells and adipose tissue through the insulin pathway. The signaling cascade is regulated by the protein kinase C ζ (PKCζ), which interacts with MYH10 to modify the localization and interaction of both GLUT4 and MYH10 in adipocytes as PKCζ inhibition resulted in reduced GLUT4 and MYH10 translocation and interactions. Overall, our study establishes MYH10 as an essential regulator of GLUT4 translocation, affecting both adipogenesis and adipocyte function, highlighting its importance in future cytoskeleton-based studies in adipocytes.

scholarly journals Role of a novel fimbrial adhesin in acid-induced host adhesion of escherichia coli 0157:H7

2021 ◽  
Author(s):  
Frances B Chingcuanco
Keyword(s):  
Escherichia Coli ◽  
Cell Adhesion ◽  
Acid Stress ◽  
Whole Genome ◽  
Deficient Mutant ◽  
Wild Type ◽  
Array Analysis ◽  
Genome Array ◽  
Fimbrial Adhesin

Enterohemorrhagic Eschericia coli (EHEC) 0157:H7 must be able to survive the acid stress of gastric passage. Previous studies show that adhesion of EHEC 0157:H7 to human epithelial cells is increased if EHEC was pre-exposed to acid. Whole genome array analysis of EHEC reveals that the putative fimbrial gene yadK is significantly upregulated after EHEC exposure to acid treatments. In this study, a YadK deficient mutant (△yadK) in EHEC 0157:H7 wild-type (WT) strain 85-170 was constructed and phenotyped. The results of this study demonstrate that △yadK exposed to acid stress showed no acid-induced increase in bacteria-host cell adhesion observed in similarly acid-induced wild type cells. This study concludes that YadK plays a role in acid-induced host adhesion of EHEC 0157:H7. It also indicates that acid stress, which is a part of the host’s natural assault to resist invasion, may regulate factors responsible for enhanced bacteria-host attachment, resulting in increased EHEC virulence.

scholarly journals Differential glycosaminoglycan binding of Chlamydia trachomatis OmcB protein from serovars E and LGV

2008 ◽  
Vol 57 (9) ◽  
pp. 1058-1061 ◽  
Author(s):  
Sanaa Fadel ◽  
Adrian Eley
Keyword(s):  
Escherichia Coli ◽  
Chlamydia Trachomatis ◽  
Heparan Sulphate ◽  
Significant Inhibition ◽  
Host Cells ◽  
Wild Type ◽  
E Coli ◽  
Dependent Mechanism

We recently showed that OmcB protein from Chlamydia trachomatis serovar LGV1 functions as an adhesin. In this study, we produced Escherichia coli expressing OmcB from serovar E and compared this OmcB to OmcB from serovar LGV1. Infectivity inhibition assays carried out with serovars LGV1 and E of C. trachomatis in the presence of recombinant OmcB showed considerable (∼60 %) inhibition of infectivity. In the presence of heparan sulphate, there was significant inhibition (68 %) of adherence of E. coli expressing OmcB from serovar LGV1 only. In a further experiment, recombinant OmcB from serovar LGV1 showed minimal binding to glycosaminoglycan (GAG)-deficient cells, whilst to the same cells, recombinant OmcB from serovar E showed binding equal to that to the wild-type cells. Our experiments strongly suggest that OmcB from serovar E, in contrast to that from serovar LGV1, is not binding to host cells through a GAG-dependent mechanism.

scholarly journals Lsr operon is associated with AI-2 transfer and pathogenicity in avian pathogenic Escherichia coli

2019 ◽  
Vol 50 (1) ◽  
Author(s):  
Jiakun Zuo ◽  
Huifang Yin ◽  
Jiangang Hu ◽  
Jinfeng Miao ◽  
Zhaoguo Chen ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Type Strain ◽  
Wild Type Strain ◽  
Binding Capacity ◽  
Bacterial Load ◽  
Transcriptional Analysis ◽  
Wild Type ◽  
Signal Molecule ◽  
Autoinducer 2

AbstractThe function of Autoinducer-2 (AI-2) which acts as the signal molecule of LuxS-mediated quorum sensing, is regulated through the lsr operon (which includes eight genes: lsrK, lsrR, lsrA, lsrC, lsrD, lsrB, lsrF, and lsrG). However, the functions of the lsr operon remain unclear in avian pathogenic Escherichia coli (APEC), which causes severe respiratory and systemic diseases in poultry. In this study, the presence of the lsr operon in 60 APEC clinical strains (serotypes O1, O2, and O78) was investigated and found to be correlated with serotype and has the highest detection rate in O78. The AI-2 binding capacity of recombinant protein LsrB of APEC (APEC-LsrB) was verified and was found to bind to AI-2 in vitro. In addition, the lsr operon was mutated in an APEC strain (APEC94Δlsr(Cm)) and the mutant was found to be defective in motility and AI-2 uptake. Furthermore, deletion of the lsr operon attenuated the virulence of APEC, with the LD50 of APEC94Δlsr(Cm) decreasing 294-fold compared with wild-type strain APEC94. The bacterial load in the blood, liver, spleen, and kidneys of ducks infected with APEC94Δlsr(Cm) decreased significantly (p < 0.0001). The results of transcriptional analysis showed that 62 genes were up-regulated and 415 genes were down-regulated in APEC94Δlsr(Cm) compared with the wild-type strain and some of the down-regulated genes were associated with the virulence of APEC. In conclusion, our study suggests that lsr operon plays a role in the pathogenesis of APEC.

scholarly journals Alterations in the proteome of the NHERF2 knockout mouse jejunal brush border membrane vesicles

2011 ◽  
Vol 43 (11) ◽  
pp. 674-684 ◽  
Author(s):  
M. Donowitz ◽  
S. Singh ◽  
P. Singh ◽  
M. Chakraborty ◽  
Y. Chen ◽  
...  
Keyword(s):  
Brush Border ◽  
Brush Border Membrane ◽  
Pdz Domain ◽  
Membrane Vesicles ◽  
Signaling Molecules ◽  
Transport Proteins ◽  
Cytoskeletal Proteins ◽  
Brush Border Membrane Vesicles ◽  
Null Mouse ◽  
Wild Type

To identify additional potential functions for the multi-PDZ domain containing protein Na+/H+exchanger regulatory factor 2 (NHERF2), which is present in the apical domain of intestinal epithelial cells, proteomic studies of mouse jejunal villus epithelial cell brush border membrane vesicles compared wild-type to homozygous NHERF2 knockout FVB mice by a two-dimensional liquid chromatography-tandem mass spectrometry (LC-MS/MS)-iTRAQ approach. Jejunal architecture appeared normal in NHERF2 null in terms of villus length and crypt depth, Paneth cell number, and microvillus structure by electron microscopy. There was also no change in proliferative activity based on BrdU labeling. Four brush border membrane vesicles (BBMV) preparations from wild-type mouse jejunum were compared with four preparations from NHERF2 knockout mice. LC-MS/MS identified 450 proteins in both matched wild-type and NHERF2 null BBMV; 13 proteins were changed in two or more separate BBMV preparations (9 increased and 4 decreased in NHERF2 null mice), while an additional 92 proteins were changed in a single BBMV preparation (68 increased and 24 decreased in NHERF2 null mice). These proteins were categorized as 1) transport proteins (one increased and two decreased in NHERF2 null); 2) signaling molecules (2 increased in NHERF2 null); 3) cytoskeleton/junctional proteins (4 upregulated and 1 downregulated in NHERF2 null); and 4) metabolic proteins/intrinsic BB proteins) (2 upregulated and 1 downregulated in NHERF2 null). Immunoblotting of BBMV was used to validate or extend the findings, demonstrating increase in BBMV of NHERF2 null of MCT1, coronin 3, and ezrin. The proteome of the NHERF2 null mouse small intestinal BB demonstrates up- and downregulation of multiple transport proteins, signaling molecules, cytoskeletal proteins, tight junctional and adherens junction proteins, and proteins involved in metabolism, suggesting involvement of NHERF2 in multiple apical regulatory processes and interactions with luminal contents.

scholarly journals Afa/Dr Diffusely Adhering Escherichia coli C1845 Infection Promotes Selective Injuries in the Junctional Domain of Polarized Human Intestinal Caco-2/TC7 Cells

2000 ◽  
Vol 68 (6) ◽  
pp. 3431-3442 ◽  
Author(s):  
Isabelle Peiffer ◽  
Anne-Béatrice Blanc-Potard ◽  
Marie-Françoise Bernet-Camard ◽  
Julie Guignot ◽  
Alain Barbat ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Brush Border ◽  
Intestinal Cell ◽  
Pathogenic Factor ◽  
Actin Network ◽  
E Coli ◽  
Intestinal Cell Line ◽  
Human Intestinal Cell Line ◽  
Cytoskeleton Rearrangements ◽  
Fimbrial Adhesin

ABSTRACT The Afa/Dr diffusely adhering Escherichia coli (DAEC) C1845 strain harboring the F1845 fimbrial adhesin interacts with the brush border-associated CD55 molecule and promotes elongation of brush border microvilli resulting from rearrangement of the F-actin network. This phenomenon involves the activation of a cascade of signaling coupled to the glycosylphosphatidylinositol-anchored receptor of the F1845 adhesin. We provide evidence that infection of the polarized human intestinal cell line Caco-2/TC7 by strain C1845 is followed by an increase in the paracellular permeability for [3H]mannitol without a decrease of the transepithelial resistance of the monolayers. Alterations in the distribution of tight-junction (TJ)-associated occludin and ZO-1 protein are observed, whereas the distribution of the zonula adherens-associated E-cadherin is not affected. Using the recombinantE. coli strains HB101(pSSS1) and -(pSSS1C) expressing the F1845 fimbrial adhesin, we demonstrate that the adhesin-CD55 interaction is not sufficient for the induction of structural and functional TJ lesions. Moreover, using the actin filament-stabilizing agent Jasplakinolide, we demonstrate that the C1845-induced functional alterations in TJs are independent of the C1845-induced apical cytoskeleton rearrangements. The results indicated that pathogenic factor(s) other than F1845 adhesin may be operant in Afa/Dr DAEC C1845.

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