scholarly journals Enteropathogenic Escherichia coli O125:H6 Triggers Attaching and Effacing Lesions on Human Intestinal Biopsy Specimens Independently of Nck and TccP/TccP2

2007 ◽  
Vol 76 (1) ◽  
pp. 361-368 ◽  
Author(s):  
Li Bai ◽  
Stephanie Schüller ◽  
Andrew Whale ◽  
Aurelie Mousnier ◽  
Olivier Marches ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Actin Polymerization ◽  
Cultured Cells ◽  
Intestinal Biopsy ◽  
Pathogenic Potential ◽  
Mucosal Surfaces ◽  
Culture Models ◽  
Gut Mucosa ◽  
Syndrome Protein

ABSTRACT Typical enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC) employ either Nck, TccP/TccP2, or Nck and TccP/TccP2 pathways to activate the neuronal Wiskott-Aldrich syndrome protein (N-WASP) and to trigger actin polymerization in cultured cells. This phenotype is used as a marker for the pathogenic potential of EPEC and EHEC strains. In this paper we report that EPEC O125:H6, which represents a large category of strains, lacks the ability to utilize either Nck or TccP/TccP2 and hence triggers actin polymerization in vitro only inefficiently. However, we show that infection of human intestinal biopsies with EPEC O125:H6 results in formation of typical attaching and effacing lesions. Expression of TccP in EPEC O125:H6, which harbors an EHEC O157-like Tir, resulted in efficient actin polymerization in vitro and enhanced colonization of human intestinal in vitro organ cultures with detectable N-WASP and electron-dense material at the site of bacterial adhesion. These results show the existence of a natural category of EPEC that colonizes the gut mucosa using Nck- and TccP-independent mechanisms. Importantly, the results highlight yet again the fact that conclusions made on the basis of in vitro cell culture models cannot be extrapolated wholesale to infection of mucosal surfaces and that the ability to induce actin polymerization on cultured cells should not be used as a definitive marker for EPEC and EHEC virulence.

scholarly journals Cortactin Recruitment by Enterohemorrhagic Escherichia coli O157:H7 during Infection In Vitro and Ex Vivo

2008 ◽  
Vol 76 (10) ◽  
pp. 4669-4676 ◽  
Author(s):  
Aurelie Mousnier ◽  
Andrew D. Whale ◽  
Stephanie Schüller ◽  
John M. Leong ◽  
Alan D. Phillips ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Actin Polymerization ◽  
Cultured Cells ◽  
Ex Vivo ◽  
Enterohemorrhagic Escherichia Coli ◽  
T3ss Effector ◽  
Mucosal Surfaces ◽  
N Terminus

ABSTRACT Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is an important human pathogen that colonizes the gut mucosa via attaching and effacing (A/E) lesions; A/E lesion formation in vivo and ex vivo is dependent on the type III secretion system (T3SS) effector Tir. Infection of cultured cells by EHEC leads to induction of localized actin polymerization, which is dependent on Tir and a second T3SS effector protein, TccP, also known as EspFU. Recently, cortactin was shown to bind both the N terminus of Tir and TccP via its SH3 domain and to play a role in EHEC-triggered actin polymerization in vitro. In this study, we investigated the recruitment of cortactin to the site of EHEC adhesion during infection of in vitro-cultured cells and mucosal surfaces ex vivo (using human terminal ileal in vitro organ cultures [IVOC]). We have shown that cortactin is recruited to the site of EHEC adhesion in vitro downstream of TccP and N-WASP. Deletion of the entire N terminus of Tir or replacing the N-terminal polyproline region with alanines did not abrogate actin polymerization or cortactin recruitment. In contrast, recruitment of cortactin to the site of EHEC adhesion in IVOC is TccP independent. These results imply that cortactin is recruited to the site of EHEC adhesion in vitro and ex vivo by different mechanisms and suggest that cortactin might have a role during EHEC infection of mucosal surfaces.

scholarly journals Modelling of Infection by Enteropathogenic Escherichia coli Strains in Lineages 2 and 4 Ex Vivo and In Vivo by Using Citrobacter rodentium Expressing TccP

2009 ◽  
Vol 77 (4) ◽  
pp. 1304-1314 ◽  
Author(s):  
Francis Girard ◽  
Valérie F. Crepin ◽  
Gad Frankel
Keyword(s):  
Escherichia Coli ◽  
Actin Polymerization ◽  
Ex Vivo ◽  
In Vivo Studies ◽  
Citrobacter Rodentium ◽  
T3ss Effector ◽  
Gut Mucosa ◽  
Colonization Dynamics

ABSTRACT Enteropathogenic Escherichia coli (EPEC) strains colonize the human gut mucosa via attaching-and-effacing (A/E) lesion formation, while in vitro they employ diverse strategies to trigger actin polymerization. Strains belonging to the EPEC-1 lineage trigger strong actin polymerization via tyrosine phosphorylation of the type III secretion system (T3SS) effector Tir, recruitment of Nck, and activation of N-WASP. Strains belonging to EPEC-2 and EPEC-4 can trigger strong actin polymerization by dual mechanisms, since while employing the Tir-Nck pathway they can additionally activate N-WASP via the T3SS effectors TccP2 and TccP, respectively. It is currently not known if the ability to trigger actin polymerization by twin mechanisms increases in vivo virulence or fitness. Since mice are resistant to EPEC infection, in vivo studies are frequently done using the murine model pathogen Citrobacter rodentium, which shares with EPEC-1 strains the ability to induce A/E lesions and trigger strong actin polymerization via the Tir:Nck pathway. In order to model infections with EPEC-2 and EPEC-4, we constructed C. rodentium strains expressing TccP. Using a mouse intestinal in vitro organ culture model and oral gavage into C57BL/6 mice, we have shown that TccP can cooperate with Tir of C. rodentium. The recombinant strains induced typical A/E lesions ex vivo and in vivo. Expression of TccP did not alter C. rodentium colonization dynamics or pathology. In competition with the wild-type strain, expression of TccP in C. rodentium did not confer a competitive advantage.

scholarly journals Interactions of Enteropathogenic Escherichia coli with Pediatric and Adult Intestinal Biopsy Specimens during Early Adherence

2009 ◽  
Vol 77 (10) ◽  
pp. 4463-4468 ◽  
Author(s):  
Romney M. Humphries ◽  
Christopher C. M. Waterhouse ◽  
George Mulvey ◽  
Paul Beck ◽  
Glen D. Armstrong
Keyword(s):  
Escherichia Coli ◽  
Age Groups ◽  
Watery Diarrhea ◽  
Intestinal Biopsy ◽  
Epec Strain ◽  
Type Iv ◽  
Biopsy Specimens ◽  
Cultured Epithelial Cells ◽  
Glycan Receptors

ABSTRACT Enteropathogenic Escherichia coli (EPEC) strains cause watery diarrhea almost exclusively in young children. The basis for this age discrimination has never been determined, but it may be related to host cell receptors. During infection, EPEC strains express type IV bundle-forming pili composed of repeating subunits of the protein called bundlin. The very first interaction between EPEC and in vitro-cultured epithelial cells is mediated by the binding of α-bundlin to a carbohydrate receptor that contains, at a minimum, the N-acetyllactosamine (LacNAc) glycan sequence. However, bundlins expressed from the β-bundlin allele do not bind LacNAc glycan sequences. Herein, we investigated whether EPEC strains use α-bundlin to mediate early adherence to human intestinal biopsy specimens cultured in vitro by assessing the ability of isogenic EPEC mutants expressing either the α1- or β1-bundlin allele or a bundlin-deficient EPEC strain to bind to these specimens. Furthermore, we directly compared the abilities of a wild-type EPEC strain to bind to the epithelial surfaces of both human adult and pediatric biopsy specimens. Our results demonstrate that β-bundlin does not act as an adhesin during early EPEC adherence to adult duodenal biopsy specimens. The results also indicate that EPEC binds equally well to adult and pediatric biopsy specimens in an early adherence assay. This result is supported by the finding that the early adherence of EPEC to both adult and pediatric biopsy specimens was inhibited by LacNAc neoglycoconjugates, suggesting that organisms expressing α-bundlin-type bundle-forming pili initially bind to related glycan receptors in both age groups.

scholarly journals Interaction of Enteropathogenic and Shiga Toxin-Producing Escherichia coli and Porcine Intestinal Mucosa: Role of Intimin and Tir in Adherence

2005 ◽  
Vol 73 (9) ◽  
pp. 6005-6016 ◽  
Author(s):  
Francis Girard ◽  
Isabelle Batisson ◽  
Gad M. Frankel ◽  
Josée Harel ◽  
John M. Fairbrother
Keyword(s):  
Escherichia Coli ◽  
Shiga Toxin ◽  
Actin Polymerization ◽  
Animal Species ◽  
Ex Vivo ◽  
Epec Strain ◽  
E Coli ◽  
In Vitro Organ Culture

ABSTRACT The ileal in vitro organ culture (IVOC) model using tissues originating from colostrum-deprived newborn piglets has proven to be an effective way to study the attaching and effacing (A/E) phenotype of porcine enteropathogenic Escherichia coli (EPEC) ex vivo. The aim of this study was to investigate the role of intimin subtype and Tir in the adherence of EPEC and Shiga-toxin-producing E. coli (STEC), isolated from different animal species, to porcine intestinal IVOC. Moreover, the role of intimin in Tir-independent adherence of the human EPEC strain E2348/69 was investigated using intimin and Tir-deficient derivatives. Our results demonstrated that A/E E. coli strains (AEEC) from various animal species and humans induce the A/E phenotype in porcine ileal IVOC and that intimin subtype influences intestinal adherence and tropism of AEEC strains. We also showed that a tir mutant of EPEC strain E2348/69 demonstrates close adherence to the epithelial cells of porcine ileal IVOC segments, with microvillous effacement but with no evidence of actin polymerization or pedestal formation, and that intimin seems to be involved in this phenotype. Overall, this study provides further evidence for the existence of one or more host-cell-encoded intimin receptor(s) in the pig gut.

scholarly journals Genetic Diversity of Heat-Labile Toxin Expressed by Enterotoxigenic Escherichia coli Strains Isolated from Humans

2008 ◽  
Vol 190 (7) ◽  
pp. 2400-2410 ◽  
Author(s):  
M. A. Lasaro ◽  
J. F. Rodrigues ◽  
C. Mathias-Santos ◽  
B. E. C. Guth ◽  
A. Balan ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cultured Cells ◽  
Rflp Analysis ◽  
Restriction Enzymes ◽  
Amino Acid Sequences ◽  
Enterotoxigenic Escherichia Coli ◽  
Heat Labile ◽  
Natural Diversity

ABSTRACT The natural diversity of the elt operons, encoding the heat-labile toxin LT-I (LT), carried by enterotoxigenic Escherichia coli (ETEC) strains isolated from humans was investigated. For many years, LT was supposed to be represented by a rather conserved toxin, and one derivative, produced by the reference H10407 strain, was intensively studied either as a virulence factor or as a vaccine adjuvant. Amplicons encompassing the two LT-encoding genes (eltA and eltB) of 51 human-derived ETEC strains, either LT+ (25 strains) only or LT+/ST+ (26 strains), isolated from asymptomatic (24 strains) or diarrheic (27 strains) subjects, were subjected to restriction fragment length polymorphism (RFLP) analysis and DNA sequencing. Seven polymorphic RFLP types of the H10407 strain were detected with six (BsaI, DdeI, HhaI, HincII, HphI, and MspI) restriction enzymes. Additionally, the single-nucleotide polymorphic analysis revealed 50 base changes in the elt operon, including 21 polymorphic sites at eltA and 9 at eltB. Based on the deduced amino acid sequences, 16 LT types were identified, including LT1, expressed by the H10407 strain and 23 other strains belonging to seven different serotypes, and LT2, expressed by 11 strains of six different serotypes. In vitro experiments carried out with purified toxins indicated that no significant differences in GM1-binding affinity could be detected among LT1, LT2, and LT4. However, LT4, but not other toxin types, showed reduced toxic activities measured either in vitro with cultured cells (Y-1 cells) or in vivo in rabbit ligated ileal loops. Collectively, these results indicate that the natural diversity of LTs produced by wild-type ETEC strains isolated from human hosts is considerably larger than previously assumed and may impact the pathogeneses of the strains and the epidemiology of the disease.

scholarly journals Targeting the actin nucleation promoting factor WASp provides a therapeutic approach for hematopoietic malignancies

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Guy Biber ◽  
Aviad Ben-Shmuel ◽  
Elad Noy ◽  
Noah Joseph ◽  
Abhishek Puthenveetil ◽  
...  
Keyword(s):  
Small Molecule ◽  
Actin Polymerization ◽  
Hematopoietic Cells ◽  
Actin Nucleation ◽  
Hematopoietic Malignancies ◽  
Nucleation Promoting Factor ◽  
Syndrome Protein ◽  
Cytoskeleton Rearrangement

AbstractCancer cells depend on actin cytoskeleton rearrangement to carry out hallmark malignant functions including activation, proliferation, migration and invasiveness. Wiskott–Aldrich Syndrome protein (WASp) is an actin nucleation-promoting factor and is a key regulator of actin polymerization in hematopoietic cells. The involvement of WASp in malignancies is incompletely understood. Since WASp is exclusively expressed in hematopoietic cells, we performed in silico screening to identify small molecule compounds (SMCs) that bind WASp and promote its degradation. We describe here one such identified molecule; this WASp-targeting SMC inhibits key WASp-dependent actin processes in several types of hematopoietic malignancies in vitro and in vivo without affecting naïve healthy cells. This small molecule demonstrates limited toxicity and immunogenic effects, and thus, might serve as an effective strategy to treat specific hematopoietic malignancies in a safe and precisely targeted manner.

scholarly journals Virulence Potential of a Multidrug-Resistant Escherichia coli Strain Belonging to the Emerging Clonal Group ST101-B1 Isolated from Bloodstream Infection

2020 ◽  
Vol 8 (6) ◽  
pp. 827 ◽  
Author(s):  
Ana Carolina M. Santos ◽  
Rosa M. Silva ◽  
Tiago B. Valiatti ◽  
Fernanda F. Santos ◽  
José F. Santos-Neto ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Antimicrobial Resistance ◽  
Bloodstream Infection ◽  
Galleria Mellonella ◽  
Multidrug Resistant ◽  
Coli Strain ◽  
Pathogenic Potential ◽  
E Coli ◽  
Antimicrobial Resistance Genes

Escherichia coli EC121 is a multidrug-resistant (MDR) strain isolated from a bloodstream infection of an inpatient with persistent gastroenteritis and T-zone lymphoma that died due to septic shock. Despite causing an extraintestinal infection, previous studies showed that it did not have the usual characteristics of an extraintestinal pathogenic E. coli. Instead, it belonged to phylogenetic group B1 and harbored few known virulence genes. To evaluate the pathogenic potential of strain EC121, an extensive genome sequencing and in vitro characterization of various pathogenicity-associated properties were performed. The genomic analysis showed that strain EC121 harbors more than 50 complete virulence genetic clusters. It also displays the capacity to adhere to a variety of epithelial cell lineages and invade T24 bladder cells, as well as the ability to form biofilms on abiotic surfaces, and survive the bactericidal serum complement activity. Additionally, EC121 was shown to be virulent in the Galleria mellonella model. Furthermore, EC121 is an MDR strain harboring 14 antimicrobial resistance genes, including blaCTX-M-2. Completing the scenario, it belongs to serotype O154:H25 and to sequence type 101-B1, which has been epidemiologically linked to extraintestinal infections as well as to antimicrobial resistance spread. This study with E. coli strain EC121 shows that clinical isolates considered opportunistic might be true pathogens that go underestimated.

scholarly journals In Vitro and In Vivo Bacteriolytic Activities of Escherichia coli Phages: Implications for Phage Therapy

2004 ◽  
Vol 48 (7) ◽  
pp. 2558-2569 ◽  
Author(s):  
Sandra Chibani-Chennoufi ◽  
Josette Sidoti ◽  
Anne Bruttin ◽  
Elizabeth Kutter ◽  
Shafiq Sarker ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Drinking Water ◽  
Environmental Water ◽  
Gut Flora ◽  
E Coli ◽  
Adult Mice ◽  
Fecal Recovery ◽  
Gut Mucosa

ABSTRACT Four T4-like coliphages with broad host ranges for diarrhea-associated Escherichia coli serotypes were isolated from stool specimens from pediatric diarrhea patients and from environmental water samples. All four phages showed a highly efficient gastrointestinal passage in adult mice when added to drinking water. Viable phages were recovered from the feces in a dose-dependent way. The minimal oral dose for consistent fecal recovery was as low as 103 PFU of phage per ml of drinking water. In conventional mice, the orally applied phage remained restricted to the gut lumen, and as expected for a noninvasive phage, no histopathological changes of the gut mucosa were detected in the phage-exposed animals. E. coli strains recently introduced into the intestines of conventional mice and traced as ampicillin-resistant colonies were efficiently lysed in vivo by phage added to the drinking water. Likewise, an in vitro phage-susceptible E. coli strain freshly inoculated into axenic mice was lysed in vivo by an orally applied phage, while an in vitro-resistant E. coli strain was not lysed. In contrast, the normal E. coli gut flora of conventional mice was only minimally affected by oral phage application despite the fact that in vitro the majority of the murine intestinal E. coli colonies were susceptible to the given phage cocktail. Apparently, the resident E. coli gut flora is physically or physiologically protected against phage infection.

scholarly journals Temporal Expression of Enteropathogenic Escherichia coli Virulence Genes in an In Vitro Model of Infection

2005 ◽  
Vol 73 (2) ◽  
pp. 1034-1043 ◽  
Author(s):  
Laura Q. Leverton ◽  
James B. Kaper
Keyword(s):  
Escherichia Coli ◽  
In Vitro Model ◽  
Virulence Genes ◽  
Host Cells ◽  
Temporal Expression ◽  
Lesion Formation ◽  
Culture Models ◽  
Vitro Model ◽  
Enterocyte Effacement

ABSTRACT The hallmark of enteropathogenic Escherichia coli (EPEC) infection is the ability of EPEC to cause attaching and effacing (A/E) lesions on intestinal epithelium. This event is reproducible in in vitro tissue culture models of infection. We used real-time PCR to measure transcription from several locus of enterocyte effacement (LEE) operons (LEE1 to LEE5) and from bfp during a 5-h infection of HEp-2 cells with EPEC. We found that after the initial formation of A/E lesions, which occurs as early as 5 min postinfection, EPEC continues to increase transcription from LEE3 to LEE5 as well as from bfp. These levels are maximized by 3 h postinfection and remain constant throughout the course of infection. This increase in transcription from LEE3 to LEE5 occurs when LEE1 (ler) transcription is decreasing. EspA, EspB, intimin, Tir, and bundle-forming pilus expression is detectable during the entire 5-h infection. These results indicate that the EPEC genes involved in localized and intimate adherence are continually expressed after the initial stages of A/E lesion formation on host cells.

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