scholarly journals Participation of Integrinα5β1 in the Fibronectin-Mediated Adherence of EnteroaggregativeEscherichia colito Intestinal Cells

2014 ◽  
Vol 2014 ◽  
pp. 1-8 ◽  
Author(s):  
Mariana Izquierdo ◽  
Alejandra Alvestegui ◽  
James P. Nataro ◽  
Fernando Ruiz-Perez ◽  
Mauricio J. Farfan
Keyword(s):  
Escherichia Coli ◽  
Intestinal Cells ◽  
Cellular Receptor ◽  
T84 Cells ◽  
Intestinal Epithelia ◽  
Eaec Strain ◽  
Aggregative Adherence ◽  
Direct Binding ◽  
Rgd Site ◽  
Interaction With Receptors

Adherence to the intestinal epithelia is a key feature in enteroaggregativeEscherichia coli(EAEC) infection. The aggregative adherence fimbriae (AAFs) are involved in EAEC interaction with receptors at the surface of intestinal cells. We and others have demonstrated that fibronectin is a receptor for AAF/II fimbriae. Considering that the major cellular receptor of fibronectin is integrinα5β1, in this study we evaluated the participation of this receptor in the fibronectin-mediated adherence of EAEC strain 042 to intestinal cells. We found that EAEC strain 042 has the ability to bind directly and indirectly to integrinα5β1; direct binding was not mediated by AAF/II fimbriae and indirect binding was mediated by AAF/II and fibronectin. Coimmunoprecipitation assays confirmed the formation of the complex AafA/fibronectin/integrinα5β1. To evaluate EAEC adherence to intestinal cells, T84 cells were incubated with fibronectin and an antibody that blocks the interaction region of integrinα5β1to fibronectin, the RGD site. Under these conditions, we found the number of adherent bacteria to epithelial cells significantly reduced. Additionally, fibronectin-mediated adherence of EAEC strain 042 was abolished in HEp-2 cells transfected with integrinα5shRNA. Altogether, our data support the involvement of integrinα5β1in the fibronectin-mediated EAEC binding to intestinal cells.

scholarly journals Identification of Cell Surface-Exposed Proteins Involved in the Fimbria-Mediated Adherence of Enteroaggregative Escherichia coli to Intestinal Cells

2014 ◽  
Vol 82 (4) ◽  
pp. 1719-1724 ◽  
Author(s):  
Mariana Izquierdo ◽  
Fernando Navarro-Garcia ◽  
Raul Nava-Acosta ◽  
James P. Nataro ◽  
Fernando Ruiz-Perez ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Prototype Strain ◽  
Intestinal Cells ◽  
Intestinal Epithelial ◽  
T84 Cells ◽  
Content Type ◽  
Intestinal Epithelia ◽  
Liquid Chromatography Tandem Mass ◽  
Interfering Rna

ABSTRACTFimbria-mediated adherence to the intestinal epithelia is a key step in enteroaggregativeEscherichia coli(EAEC) pathogenesis. To date, four fimbriae have been described for EAEC; aggregative adherence fimbria II (AAF/II) is the most important adherence factor for EAEC prototype strain 042. Previously, we described results showing that extracellular matrix (ECM) components might be involved in the recognition of AAF/II fimbriae by intestinal cells. In this study, we sought to identify novel potential receptors on intestinal epithelial cells recognized by the AAF/II fimbriae. Purified AafA-dscprotein, the major subunit of AAF/II fimbriae, was incubated with a monolayer of T84 cells, cross-linked to the surface-exposed T84 cell proteins, and immunoprecipitated by using anti-AafA antibodies. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of cellular proteins bound to AafA-dscprotein identified laminin (previously recognized as a potential receptor for AAF/II) and cytokeratin 8 (CK8). Involvement of the major subunit of AAF/II fimbriae (AafA protein) in the binding to recombinant CK8 was confirmed by adherence assays with purified AAF/II fimbriae, AafA-dscprotein, and strain 042. Moreover, HEp-2 cells transfected with CK8 small interfering RNA (siRNA) showed reduced 042 adherence compared with cells transfected with scrambled siRNA as a control. Adherence of 042 to HEp-2 cells preincubated with antibodies against ECM proteins or CK8 was substantially reduced. Altogether, our results supported the idea of a role of CK8 as a potential receptor for EAEC.

scholarly journals Enteroaggregative Escherichia coli Disrupts Epithelial Cell Tight Junctions

2010 ◽  
Vol 78 (11) ◽  
pp. 4958-4964 ◽  
Author(s):  
Maura C. Strauman ◽  
Jill M. Harper ◽  
Susan M. Harrington ◽  
Erik Juncker Boll ◽  
James P. Nataro
Keyword(s):  
Escherichia Coli ◽  
Tight Junction ◽  
Tight Junctions ◽  
Cell Model ◽  
Barrier Dysfunction ◽  
T84 Cells ◽  
E Coli ◽  
Eaec Strain ◽  
Epithelial Barrier Dysfunction

ABSTRACT Enteroaggregative Escherichia coli (EAEC) is responsible for inflammatory diarrhea in diverse populations, but its mechanisms of pathogenesis have not been fully elucidated. We have used a previously characterized polarized intestinal T84 cell model to investigate the effects of infection with EAEC strain 042 on tight junction integrity. We find that infection with strain 042 induces a decrease in transepithelial electrical resistance (TER) compared to uninfected controls and to cells infected with commensal E. coli strain HS. When the infection was limited after 3 h by washing and application of gentamicin, we observed that the TER of EAEC-infected monolayers continued to decline, and they remained low even as long as 48 h after the infection. Cells infected with the afimbrial mutant strain 042aafA exhibited TER measurements similar to those seen in uninfected monolayers, implicating the aggregative adherence fimbriae II (AAF/II) as necessary for barrier dysfunction. Infection with wild-type strain 042 induced aberrant localization of the tight junction proteins claudin-1 and, to a lesser degree, occludin. EAEC-infected T84 cells exhibited irregular shapes, and some cells became elongated and/or enlarged; these effects were not observed after infection with commensal E. coli strain HS or 042aafA. The effects on tight junctions were also observed with AAF/I-producing strain JM221, and an afimbrial mutant was similarly deficient in inducing barrier dysfunction. Our results show that EAEC induces epithelial barrier dysfunction in vitro and implicates the AAF adhesins in this phenotype.

scholarly journals Novel Aggregative Adherence Fimbria Variant of Enteroaggregative Escherichia coli

2015 ◽  
Vol 83 (4) ◽  
pp. 1396-1405 ◽  
Author(s):  
Rie Jønsson ◽  
Carsten Struve ◽  
Nadia Boisen ◽  
Ramona Valentina Mateiu ◽  
Araceli E. Santiago ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Gene Cluster ◽  
Gene Encoding ◽  
Content Type ◽  
Fimbrial Subunit ◽  
Eaec Strain ◽  
Aggregative Adherence ◽  
In The Beginning ◽  
Important Variant ◽  
Pcr Targeting

EnteroaggregativeEscherichia coli(EAEC) organisms belong to a diarrheagenic pathotype known to cause diarrhea and can be characterized by distinct aggregative adherence (AA) in a stacked-brick pattern to cultured epithelial cells. In this study, we investigated 118 EAEC strains isolated from the stools of Danish adults with traveler's diarrhea. We evaluated the presence of the aggregative adherence fimbriae (AAFs) by a multiplex PCR, targeting the four known major subunit variants as well as their usher-encoding genes. Almost one-half (49/118) of the clinical isolates did not possess any known AAF major fimbrial subunit, despite the presence of other AggR-related loci. Further investigation revealed the presence of an AAF-related gene encoding a yet-uncharacterized adhesin, termedagg5A. The sequence of theagg5DCBAgene cluster shared fimbrial accessory genes (usher, chaperone, and minor pilin subunit genes) with AAF/III, as well as the signal peptide present in the beginning of theagg3Agene. The completeagg5DCBAgene cluster from a clinical isolate, EAEC strain C338-14, with the typical stacked-brick binding pattern was cloned, and deletion of the cluster was performed. Transformation to a nonadherentE. coliHB101 and complementation of the nonadherent C338-14 mutant with the complete gene cluster restored the AA adhesion. Overall, we found theagg5Agene in 12% of the 118 strains isolated from Denmark, suggesting that this novel adhesin represents an important variant.

scholarly journals Aggregative Adherence Fimbriae II of Enteroaggregative Escherichia coli Are Required for Adherence and Barrier Disruption during Infection of Human Colonoids

2020 ◽  
Vol 88 (9) ◽  
Author(s):  
Laura A. Gonyar ◽  
Rachel M. Smith ◽  
Jorge A. Giron ◽  
Nicholas C. Zachos ◽  
Fernando Ruiz-Perez ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Asymptomatic Infection ◽  
Gastrointestinal Mucosa ◽  
Epithelial Surface ◽  
Content Type ◽  
Barrier Disruption ◽  
Eaec Strain ◽  
Growth Faltering ◽  
Aggregative Adherence

ABSTRACT Symptomatic and asymptomatic infection with the diarrheal pathogen enteroaggregative Escherichia coli (EAEC) is associated with growth faltering in children in developing settings. The mechanism of this association is unknown, emphasizing a need for better understanding of the interactions between EAEC and the human gastrointestinal mucosa. In this study, we investigated the role of the aggregative adherence fimbriae II (AAF/II) in EAEC adherence and pathogenesis using human colonoids and duodenal enteroids. We found that a null mutant in aafA, the major subunit of AAF/II, adhered significantly less than wild-type (WT) EAEC strain 042, and adherence was restored in a complemented strain. Immunofluorescence confocal microscopy of differentiated colonoids, which produce an intact mucus layer comprised of the secreted mucin MUC2, revealed bacteria at the epithelial surface and within the MUC2 layer. The WT strain adhered to the epithelial surface, whereas the aafA deletion strain remained within the MUC2 layer, suggesting that the presence or absence of AAF/II determines both the abundance and location of EAEC adherence. In order to determine the consequences of EAEC adherence on epithelial barrier integrity, colonoid monolayers were exposed to EAEC constructs expressing or lacking aafA. Colonoids infected with WT EAEC had significantly decreased epithelial resistance, an effect that required AAF/II, suggesting that binding of EAEC to the epithelium is necessary to impair barrier function. In summary, we show that production of AAF/II is critical for adherence and barrier disruption in human colonoids, suggesting a role for this virulence factor in EAEC colonization of the gastrointestinal mucosa.

scholarly journals Organization of Biogenesis Genes for Aggregative Adherence Fimbria II Defines a Virulence Gene Cluster in Enteroaggregative Escherichia coli

1999 ◽  
Vol 181 (6) ◽  
pp. 1779-1785 ◽  
Author(s):  
Waldir P. Elias ◽  
John R. Czeczulin ◽  
Ian R. Henderson ◽  
Luiz R. Trabulsi ◽  
James P. Nataro
Keyword(s):  
Escherichia Coli ◽  
Insertional Mutagenesis ◽  
Virulence Gene ◽  
Transcriptional Activator ◽  
Nucleotide Sequence Analysis ◽  
Virulence Plasmid ◽  
Virulence Determinants ◽  
Eaec Strain ◽  
Distinct Cluster ◽  
Aggregative Adherence

ABSTRACT Several virulence-related genes have been described for prototype enteroaggregative Escherichia coli (EAEC) strain 042, which has been shown to cause diarrhea in human volunteers. Among these factors are the enterotoxins Pet and EAST and the fimbrial antigen aggregative adherence fimbria II (AAF/II), all of which are encoded on the 65-MDa virulence plasmid pAA2. Using nucleotide sequence analysis and insertional mutagenesis, we have found that the genes required for the expression of each of these factors, as well as the transcriptional activator of fimbrial expression AggR, map to a distinct cluster on the pAA2 plasmid map. The cluster is 23 kb in length and includes two regions required for expression of the AAF/II fimbria. These fimbrial biogenesis genes feature a unique organization in which the chaperone, subunit, and transcriptional activator lie in one cluster, whereas the second, unlinked cluster comprises a silent chaperone gene, usher, and invasin reminiscent of Dr family fimbrial clusters. This plasmid-borne virulence locus may represent an important set of virulence determinants in EAEC strains.

scholarly journals Long Polar Fimbriae of Enterohemorrhagic Escherichia coli O157:H7 Bind to Extracellular Matrix Proteins

2011 ◽  
Vol 79 (9) ◽  
pp. 3744-3750 ◽  
Author(s):  
Mauricio J. Farfan ◽  
Lidia Cantero ◽  
Roberto Vidal ◽  
Douglas J. Botkin ◽  
Alfredo G. Torres
Keyword(s):  
Escherichia Coli ◽  
Extracellular Matrix ◽  
Enterohemorrhagic Escherichia Coli ◽  
Matrix Proteins ◽  
Intestinal Cells ◽  
T84 Cells ◽  
Content Type ◽  
Fimbrial Subunit ◽  
Ecm Proteins

ABSTRACTAdherence to intestinal cells is a key process in infection caused by enterohemorrhagicEscherichia coli(EHEC). Several adhesion factors that mediate the binding of EHEC to intestinal cells have been described, but the receptors involved in their recognition are not fully characterized. Extracellular matrix (ECM) proteins might act as receptors involved in the recognition of enteric pathogens, including EHEC. In this study, we sought to characterize the binding of EHEC O157:H7 to ECM proteins commonly present in the intestine. We found that EHEC prototype strains as well as other clinical isolates adhered more abundantly to surfaces coated with fibronectin, laminin, and collagen IV. Further characterization of this phenotype, by using antiserum raised against the LpfA1 putative major fimbrial subunit and by addition of mannose, showed that a reduced binding of EHEC to ECM proteins was observed in a long polar fimbria (lpf) mutant. We also found that the two regulators, H-NS and Ler, had an effect in EHEC Lpf-mediated binding to ECM, supporting the roles of these tightly regulated fimbriae as adherence factors. Purified Lpf major subunit bound to all of the ECM proteins tested. Finally, increased bacterial adherence was observed when T84 cells, preincubated with ECM proteins, were infected with EHEC. Taken together, these findings suggest that the interaction of Lpf and ECM proteins contributes to the EHEC colonization of the gastrointestinal tract.

scholarly journals Entero-Aggregative- Haemorrhagic Escherichia coli (EAHEC) Serotype O104:H4 -The Evolving "Superbug".

1970 ◽  
Vol 11 (1) ◽  
pp. 4-11
Author(s):  
CM Biju ◽  
AY Befekadu ◽  
SD Reji ◽  
K Afework ◽  
WC Lan ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Large Scale ◽  
High Throughput Sequencing ◽  
Outbreak Strain ◽  
Beta Lactamases ◽  
Sequencing Technologies ◽  
Eaec Strain ◽  
Aggregative Adherence ◽  
Shiga Toxin 2 ◽  
Uremic Syndrome

The rare, E.coli strain O104:H4 has been identified as the causative agent of one of the largest ever reported food-borneoutbreaks of gastroenteritis and Hemolytic-uremic syndrome (HUS) in Germany this year. This hypervirulent pathotype possess a unique combination of two pathogens: enterohemorrhagicE.coli (EHEC) and enteroaggregative E.coli (EAEC) strains. The serotype has rarely been described previously in humans and never associated with any earlier large scale EHEC outbreaks. It is now being referred to as the Entero-Aggregative-Haemorrhagic Escherichia coli (EAHEC).Advances in high-throughput sequencing technologies helped in rapid complete genome sequencing of the outbreak strains by different laboratories.Comparison of the genome sequence of the outbreak strain with other diarrhea-associated EAEC serotype O104:H4 indicate that the chromosome of the outbreak strain is most similar to that of an early isolated EAEC strain 55989 and has evolved to become more virulent by the acquisition of a Shiga toxin 2 encoding prophage, a plasmid encoding CTX-M beta-lactamases, and substituting the aggregative adherence fimbria III (AAF/III)with the rarer aggregative adherence fimbria I (AAF/I). The present article reviews the virulent traits ofthe outbreak strain, and also presents an update of the different intervention strategies that are being tested for the treatment of infections by such highly pathogenic strains. DOI: http://dx.doi.org/10.3329/bjms.v11i1.9815 BJMS 2012; 11(1): 4-11

scholarly journals Longus, a Type IV Pilus of Enterotoxigenic Escherichia coli, Is Involved in Adherence to Intestinal Epithelial Cells

2010 ◽  
Vol 192 (11) ◽  
pp. 2791-2800 ◽  
Author(s):  
Karina Mazariego-Espinosa ◽  
Ariadnna Cruz ◽  
Maria A. Ledesma ◽  
Sara A. Ochoa ◽  
Juan Xicohtencatl-Cortes
Keyword(s):  
Escherichia Coli ◽  
Epithelial Cells ◽  
Growth Conditions ◽  
Enterotoxigenic Escherichia Coli ◽  
Intestinal Cells ◽  
Intestinal Epithelial ◽  
T84 Cells ◽  
Type Iv ◽  
Colonic Cells ◽  
Ht 29

ABSTRACT Enterotoxigenic Escherichia coli (ETEC) is the leading bacterial cause of diarrhea in the developing world, as well as the most common cause of traveler's diarrhea. The main hallmarks of this type of bacteria are the expression of one or more enterotoxins and fimbriae used for attachment to host intestinal cells. Longus is a pilus produced by ETEC. These bacteria grown in pleuropneumonia-like organism (PPLO) broth at 37°C and in 5% CO2 produced longus, showing that the assembly and expression of the pili depend on growth conditions and composition of the medium. To explore the role of longus in the adherence to epithelial cells, quantitative and qualitative analyses were done, and similar levels of adherence were observed, with values of 111.44 × 104 CFU/ml in HT-29, 101.33 × 104 CFU/ml in Caco-2, and 107.11 × 104 CFU/ml in T84 cells. In addition, the E9034AΔlngA strain showed a significant reduction in longus adherence of 32% in HT-29, 22.28% in Caco-2, and 21.68% in T84 cells compared to the wild-type strain. In experiments performed with nonintestinal cells (HeLa and HEp-2 cells), significant differences were not observed in adherence between E9034A and derivative strains. Interestingly, the E9034A and E9034AΔlngA(pLngA) strains were 30 to 35% more adherent in intestinal cells than in nonintestinal cells. Twitching motility experiments were performed, showing that ETEC strains E9034A and E9034AΔlngA(pLngA) had the capacity to form spreading zones while ETEC E9034AΔlngA does not. In addition, our data suggest that longus from ETEC participates in the colonization of human colonic cells.

scholarly journals New Adhesin of Enteroaggregative Escherichia coli Related to the Afa/Dr/AAF Family

2008 ◽  
Vol 76 (7) ◽  
pp. 3281-3292 ◽  
Author(s):  
Nadia Boisen ◽  
Carsten Struve ◽  
Flemming Scheutz ◽  
Karen A. Krogfelt ◽  
James P. Nataro
Keyword(s):  
Escherichia Coli ◽  
Virulence Genes ◽  
Case Control Study ◽  
Pcr Primers ◽  
E Coli ◽  
Eaec Strain ◽  
Aggregative Adherence ◽  
Genes Encoding ◽  
The One ◽  
Control Study

ABSTRACT Enteroaggregative Escherichia coli (EAEC) is an important cause of diarrhea worldwide. We analyzed 17 Danish EAEC strains, isolated in the course of a case control study, for phenotypic and genotypic properties. The strains belonged to at least 14 different serotypes. Using PCR to investigate the prevalence of various putative virulence genes, we found that all but two strains were typical EAEC, as they harbored all or part of the previously described AggR regulon. The majority of the strains harbored genes encoding aggregative adherence fimbriae (AAF). The most common was AAF/I, found in nine strains; eight strains carried no known AAF-related genes. We utilized TnphoA mutagenesis to localize the aggregative adherence (AA) adhesin from one typical EAEC strain, C1010-00, which lacked a known AAF. We identified a TnphoA insertion in a hypothetical Dr-related pilin deposited in GenBank as HdaA. Four additional Danish strains harbored HdaA, and all but one displayed AA to HEp-2 cells. By using PCR primers derived from the pilins and ushers from the three AAF and Hda, we found that 16 of 17 strains exhibited evidence of one of these factors; importantly, the one negative strain also lacked the aggR gene. Cloning of the complete Hda gene cluster and expression in E. coli DH5α resulted in AA and complementation of the C1010-00 nonadherent mutant. Four related adhesins have now been found to confer AA in typical EAEC strains; our data suggest that, together, these variants may account for AA in the large majority of strains.

scholarly journals Heat-Resistant Agglutinin 1 Is an Accessory Enteroaggregative Escherichia coli Colonization Factor

2009 ◽  
Vol 191 (15) ◽  
pp. 4934-4942 ◽  
Author(s):  
Samhita Bhargava ◽  
Brandon B. Johnson ◽  
Jennifer Hwang ◽  
Tamia A. Harris ◽  
Anu S. George ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Biofilm Formation ◽  
Wild Type ◽  
E Coli ◽  
Heat Resistant ◽  
Colonization Factor ◽  
Eaec Strain ◽  
Aggregative Adherence ◽  
Adherence Pattern ◽  
Colonization Model

ABSTRACT Enteroaggregative Escherichia coli (EAEC) is an important cause of acute and persistent diarrhea. The defining stacked brick adherence pattern of Peruvian EAEC isolate 042 has previously been attributed to aggregative adherence fimbriae II (AAF/II), which confer aggregative adherence on laboratory E. coli strains. EAEC strains also show exceptional autoaggregation and biofilm formation, other phenotypes that have hitherto been ascribed to AAF/II. We report that EAEC 042 carries the heat-resistant agglutinin (hra1) gene, also known as hek, which encodes an outer membrane protein. Like AAF/II, the cloned EAEC 042 hra1 gene product is sufficient to confer autoaggregation, biofilm formation, and aggregative adherence on nonadherent and nonpathogenic laboratory E. coli strains. However, an 042 hra1 deletion mutant is not deficient in these phenotypes compared to the wild type. EAEC strain 042 produces a classic honeycomb or stacked brick pattern of adherence to epithelial cells. Unlike wild-type 042, the hra1 mutant typically does not form a tidy stacked brick pattern on HEp-2 cells in culture, which is definitive for EAEC. Moreover, the hra1 mutant is significantly impaired in the Caenorhabditis elegans slow kill colonization model. Our data suggest that the exceptional colonization of strain 042 is due to multiple factors and that Hra1 is an accessory EAEC colonization factor.

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