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

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.

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Distribution of Lactoferrin Is Related with Dynamics of Neutrophils in Bacterial Infected Mice Intestine

Molecules ◽

10.3390/molecules25071496 ◽

2020 ◽

Vol 25 (7) ◽

pp. 1496 ◽

Cited By ~ 1

Author(s):

Li Liang ◽

Zhen-Jie Wang ◽

Guang Ye ◽

Xue-You Tang ◽

Yuan-Yuan Zhang ◽

...

Keyword(s):

Escherichia Coli ◽

Antimicrobial Activity ◽

Mucosal Immunity ◽

Mrna Levels ◽

Iron Binding ◽

E Coli ◽

New Knowledge ◽

Activated Neutrophils ◽

Binding Glycoprotein

Lactoferrin (Lf) is a conserved iron-binding glycoprotein with antimicrobial activity, which is present in secretions that recover mucosal sites regarded as portals of invaded pathogens. Although numerous studies have focused on exogenous Lf, little is known about its expression of endogenous Lf upon bacterial infection. In this study, we investigated the distribution of Lf in mice intestine during Escherichia coli (E. coli) K88 infection. PCR and immunohistology staining showed that mRNA levels of Lf significantly increased in duodenum, ileum and colon, but extremely decreased in jejunum at 8 h and 24 h after infection. Meanwhile, endogenous Lf was mostly located in the lamina propria of intestine villi, while Lf receptor (LfR) was in the crypts. It suggested that endogenous Lf-LfR interaction might not be implicated in the antibacterial process. In addition, it was interesting to find that the infiltration of neutrophils into intestine tissues was changed similarly to Lf expression. It indicated that the variations of Lf expression were rather due to an equilibrium between the recruitment of neutrophils and degranulation of activated neutrophils. Thus, this new knowledge will pave the way to a more effective understanding of the role of Lf in intestinal mucosal immunity.

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

Infection and Immunity ◽

10.1128/iai.67.7.3657-3661.1999 ◽

1999 ◽

Vol 67 (7) ◽

pp. 3657-3661 ◽

Cited By ~ 8

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.

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Characterization of the Roles of Hemolysin and Other Toxins in Enteropathy Caused by Alpha-Hemolytic Escherichia coli Linked to Human Diarrhea

Infection and Immunity ◽

10.1128/iai.66.5.2040-2051.1998 ◽

1998 ◽

Vol 66 (5) ◽

pp. 2040-2051 ◽

Cited By ~ 40

Author(s):

Simon J. Elliott ◽

S. Srinivas ◽

M. John Albert ◽

Khorshed Alam ◽

Roy M. Robins-Browne ◽

...

Keyword(s):

Escherichia Coli ◽

Virulence Factors ◽

Virulence Factor ◽

Adult Rabbit ◽

Cell Hyperplasia ◽

E Coli ◽

Alpha Hemolysin ◽

A Cell ◽

Cytotoxic Necrotizing Factor 1

ABSTRACT Escherichia coli strains producing alpha-hemolysin have been associated with diarrhea in several studies, but it has not been clearly demonstrated that these strains are enteropathogens or that alpha-hemolysin is an enteric virulence factor. Such strains are generally regarded as avirulent commensals. We examined a collection of diarrhea-associated hemolytic E. coli (DHEC) strains for virulence factors. No strain produced classic enterotoxins, but they all produced an alpha-hemolysin that was indistinguishable from that of uropathogenic E. coli strains. DHEC strains also produced other toxins including cytotoxic necrotizing factor 1 (CNF1) and novel toxins, including a cell-detaching cytotoxin and a toxin that causes HeLa cell elongation. DHEC strains were enteropathogenic in the RITARD (reversible intestinal tie adult rabbit diarrhea) model of diarrhea, causing characteristic enteropathies, including inflammation, necrosis, and colonic cell hyperplasia in both small and large intestines. Alpha-hemolysin appeared to be a major virulence factor in this model since it conferred virulence to nonpathogenic E. colistrains. Other virulence factors also appear to be contributing to virulence. These findings support the epidemiologic link to diarrhea and suggest that further research into the role of DHEC and alpha-hemolysin in enteric disease is warranted.

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Serogroups and virulence genes of Escherichia coli isolated from psittacine birds

Pesquisa Veterinária Brasileira ◽

10.1590/s0100-736x2011001000013 ◽

2011 ◽

Vol 31 (10) ◽

pp. 916-921 ◽

Cited By ~ 9

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.

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

Infection and Immunity ◽

10.1128/iai.66.7.3384-3389.1998 ◽

1998 ◽

Vol 66 (7) ◽

pp. 3384-3389 ◽

Cited By ~ 20

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.

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Understanding the Role of Agricultural Practices in the Potential Colonization and Contamination by Escherichia coli in the Rhizospheres of Fresh Produce

Journal of Food Protection ◽

10.4315/0362-028x-73.11.2001 ◽

2010 ◽

Vol 73 (11) ◽

pp. 2001-2009 ◽

Cited By ~ 13

Author(s):

MUSSIE Y. HABTESELASSIE ◽

MARIANNE BISCHOFF ◽

BRUCE APPLEGATE ◽

BRADLEY REUHS ◽

RONALD F. TURCO

Keyword(s):

Escherichia Coli ◽

Irrigation Water ◽

Bacterial Colonization ◽

Agricultural Practices ◽

E Coli ◽

Lettuce Seeds ◽

Growth System ◽

Low Levels

To better protect consumers from exposure to produce contaminated with Escherichia coli, the potential transfer of E. coli from manure or irrigation water to plants must be better understood. We used E. coli strains expressing bioluminescence (E. coli O157:H7 lux) or multiantibiotic resistance (E. coli2+) in this study. These marked strains enabled us to visualize in situ rhizosphere colonization and metabolic activity and to track the occurrence and survival of E. coli in soil, rhizosphere, and phyllosphere. When radish and lettuce seeds were treated with E. coli O157:H7 lux and grown in an agar-based growth system, rapid bacterial colonization of the germinating seedlings and high levels of microbial activity were seen. Introduction of E. coli2+ to soil via manure or via manure in irrigation water showed that E. coli could establish itself in the lettuce rhizosphere. Regardless of introduction method, 15 days subsequent to its establishment in the rhizosphere, E. coli2+ was detected on the phyllosphere of lettuce at an average number of 2.5 log CFU/g. When E. coli2+ was introduced 17 and 32 days postseeding to untreated soil (rather than the plant surface) via irrigation, it was detected at low levels (1.4 log CFU/g) on the lettuce phyllosphere 10 days later. While E. coli2+ persisted in the bulk and rhizosphere soil throughout the study period (day 41), it was not detected on the external portions of the phyllosphere after 27 days. Overall, we find that E. coli is mobile in the plant system and responds to the rhizosphere like other bacteria.

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

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.50.2.649-653.2006 ◽

2006 ◽

Vol 50 (2) ◽

pp. 649-653 ◽

Cited By ~ 60

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.

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Ferredoxin Is Involved in Secretion of Cytotoxic Necrotizing Factor 1 across the Cytoplasmic Membrane in Escherichia coli K1

Infection and Immunity ◽

10.1128/iai.00674-09 ◽

2009 ◽

Vol 78 (2) ◽

pp. 838-844 ◽

Cited By ~ 15

Author(s):

Hao Yu ◽

Kwang Sik Kim

Keyword(s):

Escherichia Coli ◽

Deletion Mutant ◽

Cytoplasmic Membrane ◽

Genetic Determinants ◽

E Coli ◽

Outer Membranes ◽

Cytotoxic Necrotizing Factor ◽

Cytotoxic Necrotizing Factor 1 ◽

First Time ◽

Escherichia Coli K1

ABSTRACT We previously showed that cytotoxic necrotizing factor 1 (CNF1) contributes to Escherichia coli K1 invasion of human brain microvascular endothelial cells (HBMEC) and interacts with the receptor on the surface of HBMEC. CNF1 is the cytoplasmic protein, and it remains incompletely understood how CNF1 is secreted across the inner and outer membranes in E. coli K1. In order to investigate the genetic determinants for secretion of CNF1 in E. coli K1, we performed Tn5 mutagenesis screening by applying β-lactamase as a reporter to monitor secretion of CNF1. We identified a Tn5 mutant that exhibited no β-lactamase activity in the culture supernatant and in which the mutated gene encodes a ferredoxin gene (fdx). In the fdx deletion mutant, there was no evidence of translocation of CNF1 into HBMEC. Western blot analysis of the fdx deletion mutant revealed that ferredoxin is involved in translocation of CNF1 across the cytoplasmic membrane. The fdx mutant exhibited significantly decreased invasion of HBMEC, similar to the decreased HBMEC invasion observed with the CNF1 mutant. The failures to secrete CNF1 and invade HBMEC of the fdx mutant were restored to the levels of the parent strain by complementation with fdx. These findings demonstrate for the first time that ferredoxin is involved in secretion of CNF1 across the inner membrane in meningitis-causing E. coli K1.

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

Journal of Medical Microbiology ◽

10.1099/jmm.0.012088-0 ◽

2009 ◽

Vol 58 (10) ◽

pp. 1354-1358 ◽

Cited By ~ 6

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.

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