scholarly journals Effect of Escherichia coli Cytotoxic Necrotizing Factor 1 on Repair of Human Bladder Cell Monolayers In Vitro

1999 ◽  
Vol 67 (7) ◽  
pp. 3657-3661 ◽  
Author(s):  
Michael D. Island ◽  
Xaioling Cui ◽  
John W. Warren
Keyword(s):  
Escherichia Coli ◽  
Bladder Epithelium ◽  
Cell Monolayers ◽  
E Coli ◽  
Cytotoxic Necrotizing Factor ◽  
Bladder Cell ◽  
Bladder Cells ◽  
Vitro Model ◽  
Cytotoxic Necrotizing Factor 1

ABSTRACT We hypothesized that Escherichia coli cytotoxic necrotizing factor 1 (CNF1) might impair migration or proliferation of bladder cells and could potentially interfere with repair of the bladder epithelium. Using experimentally wounded human T24 bladder epithelial cell monolayers as an in vitro model, we found that both the number of T24 cells and the maximum distance they migrated into wounded regions was significantly decreased by bacterial extracts containingE. coli CNF1.

scholarly journals Cytotoxicity of Hemolytic, Cytotoxic Necrotizing Factor 1-Positive and -Negative Escherichia coli to Human T24 Bladder Cells

1998 ◽  
Vol 66 (7) ◽  
pp. 3384-3389 ◽  
Author(s):  
Michael D. Island ◽  
Xiaoling Cui ◽  
Betsy Foxman ◽  
Carl F. Marrs ◽  
Walter E. Stamm ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cell Types ◽  
Blue Staining ◽  
Detectable Effect ◽  
E Coli ◽  
Cytotoxic Necrotizing Factor ◽  
Bladder Cell ◽  
Bladder Cells ◽  
Cytotoxic Necrotizing Factor 1

ABSTRACT Approximately one-half of Escherichia coli isolates from patients with cystitis or pyelonephritis produce the pore-forming cytotoxin hemolysin, a molecule with the capacity to lyse erythrocytes and a range of nucleated cell types. A second toxin, cytotoxic necrotizing factor 1 (CNF1), is found in approximately 70% of hemolytic, but rarely in nonhemolytic, isolates. To evaluate the potential interplay of these two toxins, we used epidemiological and molecular biologic techniques to compare the cytotoxicity of hemolytic, CNF1+, and CNF1− cystitis strains toward human T24 bladder epithelial cells in vitro. A total of 29 isolates from two collections of cystitis-associated E. coli were evaluated by using methylene blue staining of bladder monolayers at 1-h intervals after inoculation with each strain. Most (20 of 29) isolates damaged or destroyed the T24 monolayer (less than 50% remaining) within 4 h after inoculation. As a group, CNF1+ isolates from one collection (11 strains) were less cytotoxic at 4 h than the CNF1− strains in that collection (P = 0.009), but this pattern was not observed among isolates from the second collection (18 strains). To directly evaluate the role of CNF1 in cytotoxicity of hemolytic E. coli without the variables present in multiple clinical isolates, we constructed mutants defective in production of CNF1. Compared to the CNF1+ parental isolates, no change in cytotoxicity was detected in thesecnf1 mutants. Our results indicate that CNF1 does not have a detectable effect on the ability of hemolytic E. coli to damage human bladder cell monolayers in vitro.

scholarly journals Quinolones Induce Partial or Total Loss of Pathogenicity Islands in Uropathogenic Escherichia coli by SOS-Dependent or -Independent Pathways, Respectively

2006 ◽  
Vol 50 (2) ◽  
pp. 649-653 ◽  
Author(s):  
S. M. Soto ◽  
M. T. Jimenez de Anta ◽  
J. Vila
Keyword(s):  
Escherichia Coli ◽  
Virulence Factors ◽  
Reca Protein ◽  
Total Loss ◽  
Pathogenicity Islands ◽  
E Coli ◽  
Cytotoxic Necrotizing Factor ◽  
Tract Infections ◽  
Cytotoxic Necrotizing Factor 1

ABSTRACT Escherichia coli is the most common microorganism causing urinary tract infections. Quinolone-resistant E. coli strains have fewer virulence factors than quinolone-susceptible strains. Several urovirulence genes are located in pathogenicity islands (PAIs). We investigated the capacity of quinolones to induce loss of virulence factors such as hemolysin, cytotoxic necrotizing factor 1, P fimbriae, and autotransporter Sat included in PAIs in three uropathogenic E. coli strains. In a multistep selection, all strains lost hemolytic capacity at between 1 and 4 passages when they were incubated with subinhibitory concentrations of ciprofloxacin, showing a partial or total loss of the PAI containing the hly (hemolysin) and cnf-1 (cytotoxic necrotizing factor 1) genes. RecA− mutants were obtained from the two E. coli strains with partial or total loss of the PAI. The inactivation of the RecA protein affected only the partial loss of the PAI induced by quinolones. No spontaneous loss of PAIs was observed on incubation in the absence of quinolones in either the wild-type or mutant E. coli strains. Quinolones induce partial or total loss of PAIs in vitro in uropathogenic E. coli by SOS-dependent or -independent pathways, respectively.

scholarly journals Characterization of cytotoxic necrotizing factor 1-producing Escherichia coli strains from faeces of healthy macaques

2009 ◽  
Vol 58 (10) ◽  
pp. 1354-1358 ◽  
Author(s):  
Heather R. Martin ◽  
Nancy S. Taylor ◽  
Ellen M. Buckley ◽  
Robert P. Marini ◽  
Mary M. Patterson ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cytopathic Effect ◽  
Repetitive Element ◽  
E Coli ◽  
Cytotoxic Necrotizing Factor ◽  
Clinically Normal ◽  
Cytotoxic Necrotizing Factor 1 ◽  
Element Sequence

Twenty-five (27 %) of 92 clinically normal macaques were found to have β-haemolytic Escherichia coli isolated from their faeces. Five of six isolates chosen for further characterization had multiple antibiotic resistance and were PCR-positive for cytotoxic necrotizing factor 1 (cnf1) with a demonstrated cytopathic effect in vitro. By repetitive element sequence-based PCR genotyping, genetic similarity was established for selected isolates. We believe this to be the first report of E. coli strains producing CNF1 in non-human primates.

Loss of an Intimin-Like Protein Encoded on a Uropathogenic E. coli Pathogenicity Island Reduces Inflammation and Affects Interactions with the Urothelium

2021 ◽  
Author(s):  
Allyson E. Shea ◽  
Jolie A. Stocki ◽  
Stephanie D. Himpsl ◽  
Sara N. Smith ◽  
Harry L. T. Mobley
Keyword(s):  
Ex Vivo ◽  
Cell Adherence ◽  
Upec Strain ◽  
E Coli ◽  
Bladder Cell ◽  
Bladder Cells ◽  
Strain Cft073 ◽  
The Individual ◽  
Tract Infections

Uropathogenic Escherichia coli (UPEC) causes the majority of uncomplicated urinary tract infections (UTI), which affect nearly half of women worldwide. Many UPEC strains encode an annotated intimin-like adhesin ( ila ) locus in their genome related to a well-characterized virulence factor in diarrheagenic E. coli pathotypes. Its role in UPEC uropathogenesis, however, remains unknown. In prototype UPEC strain CFT073, there is an ila locus that encodes three predicted intimin-like genes sinH , sinI , and ratA . We used in silico approaches to determine the phylogeny and genomic distribution of this locus among uropathogens. We found that the currently annotated intimin-encoding proteins in CFT073 are more closely related to invasin proteins found in Salmonella . Deletion of the individual sinH , sinI , and ratA genes did not result in measurable effects on growth, biofilm formation, or motility in vitro . On average, sinH was more highly expressed in clinical strains during active human UTI than in human urine ex vivo . Unexpectedly, we found that strains lacking this ila locus had increased adherence to bladder cells in vitro , coupled with a decrease in bladder cell invasion and death. The sinH mutant displayed a significant fitness defect in the murine model of ascending UTI including reduced inflammation in the bladder. These data confirmed an inhibitory role in bladder cell adherence to facilitate invasion and inflammation; therefore, the ila locus should be termed invasin-like, rather than intimin-like. Collectively, our data suggest that loss of this locus mediates measurable interactions with bladder cells in vitro and contributes to fitness during UTI.

scholarly journals Serogroups and virulence genes of Escherichia coli isolated from psittacine birds

2011 ◽  
Vol 31 (10) ◽  
pp. 916-921 ◽  
Author(s):  
Terezinha Knöbl ◽  
André B.S Saidenberg ◽  
Andrea M Moreno ◽  
Tânia A.T Gomes ◽  
Mônica A.M Vieira ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Virulence Factors ◽  
Virulence Genes ◽  
Serum Resistance ◽  
Temperature Sensitive ◽  
E Coli ◽  
Cytotoxic Necrotizing Factor ◽  
Heat Stable ◽  
Genes Encoding ◽  
Cytotoxic Necrotizing Factor 1

Escherichia coli isolates from 24 sick psittacine birds were serogrouped and investigated for the presence of genes encoding the following virulence factors: attaching and effacing (eae), enteropathogenic E. coli EAF plasmid (EAF), pili associated with pyelonephritis (pap), S fimbriae (sfa), afimbrial adhesin (afa), capsule K1 (neu), curli (crl, csgA), temperature-sensitive hemagglutinin (tsh), enteroaggregative heat-stable enterotoxin-1 (astA), heat-stable enterotoxin -1 heat labile (LT) and heat stable (STa and STb) enterotoxins, Shiga-like toxins (stx1 and stx2), cytotoxic necrotizing factor 1 (cnf1), haemolysin (hly), aerobactin production (iuc) and serum resistance (iss). The results showed that the isolates belonged to 12 serogroups: O7; O15; O21; O23; O54; O64; O76; O84; O88; O128; O152 and O166. The virulence genes found were: crl in all isolates, pap in 10 isolates, iss in seven isolates, csgA in five isolates, iuc and tsh in three isolates and eae in two isolates. The combination of virulence genes revealed 11 different genotypic patterns. All strains were negative for genes encoding for EAF, EAEC, K1, sfa, afa, hly, cnf, LT, STa, STb, stx1 and stx2. Our findings showed that some E. coli isolated from psittacine birds present the same virulence factors as avian pathogenic E. coli (APEC), uropathogenic E. coli (UPEC) and Enteropathogenic E. coli (EPEC) pathotypes.

scholarly journals Suppression of Bladder Epithelial Cytokine Responses by Uropathogenic Escherichia coli

2005 ◽  
Vol 73 (7) ◽  
pp. 3999-4006 ◽  
Author(s):  
David A. Hunstad ◽  
Sheryl S. Justice ◽  
Chia S. Hung ◽  
Scott R. Lauer ◽  
Scott J. Hultgren
Keyword(s):  
Escherichia Coli ◽  
Epithelial Cells ◽  
Outer Membrane Proteins ◽  
Cytokine Signaling ◽  
E Coli ◽  
Cytokine Responses ◽  
Genes Encoding ◽  
Vitro Model ◽  
Tract Infections

ABSTRACT Urinary tract infections are most commonly caused by uropathogenic strains of Escherichia coli (UPEC), which invade superficial bladder epithelial cells via a type 1 pilus-dependent mechanism. Inside these epithelial cells, UPEC organisms multiply to high numbers to form intracellular bacterial communities, allowing them to avoid immune detection. Bladder epithelial cells produce interleukin-6 (IL-6) and IL-8 in response to laboratory strains of E. coli in vitro. We investigated the ability of UPEC to alter epithelial cytokine signaling by examining the in vitro responses of bladder epithelial cell lines to the cystitis strains UTI89 and NU14. The cystitis strains induced significantly less IL-6 than did the laboratory E. coli strain MG1655 from 5637 and T24 bladder epithelial cells. The cystitis strains also suppressed epithelial cytokine responses to exogenous lipopolysaccharide (LPS) and to laboratory E. coli. We found that insertional mutations in the rfa and rfb operons and in the surA gene all abolished the ability of UTI89 to suppress cytokine induction. The rfa and rfb operons encode LPS biosynthetic genes, while surA encodes a periplasmic cis-trans prolyl isomerase important in the biogenesis of outer membrane proteins. We conclude that, in this in vitro model system, cystitis strains of UPEC have genes encoding factors that suppress proinflammatory cytokine production by bladder epithelial cells.

scholarly journals Cytotoxic Necrotizing Factor Type 1 of Uropathogenic Escherichia coli Kills Cultured Human Uroepithelial 5637 Cells by an Apoptotic Mechanism

2000 ◽  
Vol 68 (10) ◽  
pp. 5869-5880 ◽  
Author(s):  
Melody Mills ◽  
Karen C. Meysick ◽  
Alison D. O'Brien
Keyword(s):  
Escherichia Coli ◽  
Cell Line ◽  
Cell Lines ◽  
Human Bladder ◽  
E Coli ◽  
Cytotoxic Necrotizing Factor ◽  
Bladder Cell ◽  
Factor Type ◽  
Tract Infections

ABSTRACT Pathogenic Escherichia coli associated with urinary tract infections (UTIs) in otherwise healthy individuals frequently produce cytotoxic necrotizing factor type 1 (CNF1), a member of the family of bacterial toxins that target the Rho family of small GTP-binding proteins. To gain insight into the function of CNF1 in the development of E. coli-mediated UTIs, we examined the effects of CNF1 intoxication on a panel of human cell lines derived from physiologically relevant sites (bladder, ureters, and kidneys). We identified one uroepithelial cell line that exhibited a distinctly different CNF1 intoxication phenotype from the prototypic one of multinucleation without cell death that is seen when HEp-2 or other epithelial cells are treated with CNF1. The 5637 bladder cell line detached from the growth surface within 72 h of CNF1 intoxication, a finding that suggested frank cytotoxicity. To determine the basis for the unexpected toxic effect of CNF1 on 5637 cells, we compared the degree of toxin binding, actin fiber formation, and Rho modification with those CNF1-induced events in HEp-2 cells. We found no apparent difference in the amount of CNF1 bound to 5637 cells and HEp-2 cells. Moreover, CNF1 modified Rho, in vivo and in vitro, in both cell types. In contrast, one of the classic responses to CNF1 in HEp-2 and other epithelial cell lines, the formation of actin stress fibers, was markedly absent in 5637 cells. Indeed, actin stress fiber induction by CNF1 did not occur in any of the other human bladder cell lines that we tested (J82, SV-HUC-1, or T24). Furthermore, the appearance of lamellipodia and filopodia in 5637 cells suggested that CNF1 activated the Cdc42 and Rac proteins. Finally, apoptosis was observed in CNF1-intoxicated 5637 cells. If our results with 5637 cells reflect the interaction of CNF1 with the transitional uroepithelium in the human bladder, then CNF1 may be involved in the exfoliative process that occurs in that organ after infection with uropathogenic E. coli.

scholarly journals Lack of a Role of Cytotoxic Necrotizing Factor 1 Toxin from Escherichia coli in Bacterial Pathogenicity and Host Cytokine Response in Infected Germfree Piglets

2000 ◽  
Vol 68 (2) ◽  
pp. 839-847 ◽  
Author(s):  
S. Fournout ◽  
C. M. Dozois ◽  
M. Odin ◽  
C. Desautels ◽  
S. Pérès ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Bacterial Colonization ◽  
Cytokine Expression ◽  
Cytokine Response ◽  
Mrna Levels ◽  
Mesenteric Lymph Nodes ◽  
E Coli ◽  
Cytotoxic Necrotizing Factor ◽  
Cytotoxic Necrotizing Factor 1

ABSTRACT Some Escherichia coli strains isolated from intestinal or extraintestinal infections in pigs produce cytotoxic necrotizing factor 1 (CNF1). In order to analyze the role of CNF1 in the pathogenesis of porcine colibacillosis, newborn colostrum-deprived germfree piglets were orally inoculated with a wild-type CNF1-producing strain (M623) or with an isogenic cnf1 mutant (M623ΔCNF1). The two isogenic strains induced a high mortality with similar lung and serosal inflammatory lesions, indicating that both strains were pathogenic in these piglets. Bacterial counts in various organs of inoculated piglets revealed an intestinal predisposition of M623 and M623ΔCNF1 strains for the cecum and colon. Extraintestinal organs (lungs, liver, spleen, and kidney) were also colonized by both strains. Similar colonization of intestinal and extraintestinal tissues in animals inoculated with either strain was observed, except in the ileum, where M623 showed a higher colonization than M623ΔCNF1. Intestinal (ileum and colon), extraintestinal (lung and kidney), and immune (mesenteric lymph nodes and spleen) tissues were sampled at 1 day postinoculation and analyzed for cytokine expression by a reverse transcriptase PCR technique. Inoculation with E. coli M623 induced an enhanced expression of inflammatory cytokines (interleukin-1α [IL-1α], tumor necrosis factor α, and IL-12p40) in the intestinal organs compared to uninoculated piglets or piglets inoculated with nonpathogenic intestinal E. coli 862B, which is also able to colonize the intestinal tract. There was little difference in cytokine transcript levels in the intestinal and extraintestinal organs in piglets inoculated with E. colistrains M623 or M623ΔCNF1, except in the ileum, where IL-1α and IL-8 mRNA levels correlated with bacterial colonization. Expression of regulatory cytokines (gamma interferon and IL-4) was weak in immune tissues from piglets inoculated with M623 or M623ΔCNF1. Taken together, our data indicate that the CNF1-producing strain, M623, is pathogenic and induces inflammatory cytokine expression in germfree, colostrum-deprived piglets. Nevertheless, in this model, the CNF1 toxin does not appear to be a major factor for pathogenicity or cytokine response, as demonstrated by the use of an isogenic cnf1mutant.

scholarly journals Deamidation of Cdc42 and Rac by Escherichia coliCytotoxic Necrotizing Factor 1: Activation of c-Jun N-Terminal Kinase in HeLa Cells

1999 ◽  
Vol 67 (2) ◽  
pp. 496-503 ◽  
Author(s):  
M. Lerm ◽  
J. Selzer ◽  
A. Hoffmeyer ◽  
U. R. Rapp ◽  
K. Aktories ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Molecular Mass ◽  
Hela Cells ◽  
Fold Increase ◽  
Gtpase Activating Protein ◽  
Intact Cells ◽  
Tryptic Peptides ◽  
Cytotoxic Necrotizing Factor ◽  
Cytotoxic Necrotizing Factor 1

ABSTRACT Recently, Escherichia coli cytotoxic necrotizing factor 1 (CNF1) was shown to activate the low-molecular-mass GTPase RhoA by deamidation of Gln63, thereby inhibiting intrinsic and GTPase-activating protein (GAP)-stimulated GTPase activities (G. Schmidt, P. Sehr, M. Wilm, J. Selzer, M. Mann, and K. Aktories, Nature 387:725–729, 1997; G. Flatau, E. Lemichez, M. Gauthier, P. Chardin, S. Paris, C. Fiorentini, and P. Boquet, Nature 387:729–733, 1997). Here we report that in addition to RhoA, Cdc42 and Rac also are targets for CNF1 in vitro and in intact cells. Treatment of HeLa cells with CNF1 induced a transient formation of microspikes and formation of membrane ruffles. CNF1 caused a transient 10- to 50-fold increase in the activity of the c-Jun N-terminal kinase. Tryptic peptides of Cdc42 obtained from CNF1-treated cells by immunoprecipitation exhibited an increase in mass of 1 Da compared to control peptides, indicating the deamidation of glutamine 61 by the toxin. The same increase in mass was observed with the respective peptides obtained from CNF1-modified recombinant Cdc42 and Rac1. Modification of recombinant Cdc42 and Rac1 by CNF1 inhibited intrinsic and GAP-stimulated GTPase activities and retarded binding of 2′(3′)- O-(N -methylanthraniloyl)GDP. The data suggest that recombinant as well as cellular Cdc42 and Rac are substrates for CNF1.

In Vitro Activities of Cephalosporins and Quinolones against Escherichia coli Strains Isolated from Diarrheic Dairy Calves

1999 ◽  
Vol 43 (3) ◽  
pp. 510-513 ◽  
Author(s):  
José Antonio Orden ◽  
José Antonio Ruiz-Santa-Quiteria ◽  
Silvia García ◽  
Dolores Cid ◽  
Ricardo de la Fuente
Keyword(s):  
Escherichia Coli ◽  
Nalidixic Acid ◽  
Virulence Factors ◽  
Dairy Calves ◽  
Neonatal Diarrhea ◽  
E Coli ◽  
Cytotoxic Necrotizing Factor ◽  
Highly Effective

ABSTRACT The in vitro activities of several cephalosporins and quinolones against 195 strains of Escherichia coli isolated from dairy calves affected by neonatal diarrhea were determined. One hundred thirty-seven of these strains produced one or more potential virulence factors (F5, F41, F17, cytotoxic necrotizing factor, verotoxin, and theeae gene), but the remaining 58 strains did not produce any of these factors. From 11 to 18% of the E. coli strains were resistant to cephalothin, nalidixic acid, enoxacin, and enrofloxacin. However, cefuroxime, cefotaxime, and cefquinome were highly effective against the E. coli isolates tested. Some significant differences (P < 0.05) in resistance to quinolones between the strains producing potential virulence factors and nonfimbriated, nontoxigenic, eae-negative strains were found. Thus, eae-positive, necrotoxigenic, and verotoxigenic (except for nalidixic acid) E. coli strains were significantly more sensitive to nalidixic acid, enoxacin, and enrofloxacin than nonfimbriated, nontoxigenic, eae-negative strains. Moreover, eae-positive strains were significantly more sensitive to enoxacin and enrofloxacin than F5-positive strains. Thus, the results of this study suggest that the bovine E. coli strains that produce some potential virulence factors are more sensitive to quinolones than those that do not express these factors.

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