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Isolation and nucleotide sequence of the gene encoding cytotoxic necrotizing factor 1 of Escherichia coli.

ABSTRACT: Cytotoxic necrotizing factors (CNFs) are dermonecrotic protein toxins produced by human and animal clinical isolates of Escherichia coli. In this study, the CNF1 determinant was isolated and sequenced, showing that expression of biologically active toxin is governed by a unique open reading frame encoding a protein of 1,014 amino acids with a predicted molecular mass of 113.7 kDa. Nucleotide and protein data base searches showed significant homology between CNF1 and the dermonecrotic toxin of Pasteurella multocida. In particular, the two toxins were found to share a hydrophobic region of about 220 amino acids which is a potential membrane-spanning domain.

SUBMITTER: Falbo V

PROVIDER: S-EPMC281255 | biostudies-other | 1993 Nov

REPOSITORIES: biostudies-other

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Isolation and nucleotide sequence of the gene encoding cytotoxic necrotizing factor 1 of Escherichia coli.

Falbo V V Pace T T Picci L L Pizzi E E Caprioli A A

Infection and immunity 19931101 11

Cytotoxic necrotizing factors (CNFs) are dermonecrotic protein toxins produced by human and animal clinical isolates of Escherichia coli. In this study, the CNF1 determinant was isolated and sequenced, showing that expression of biologically active toxin is governed by a unique open reading frame encoding a protein of 1,014 amino acids with a predicted molecular mass of 113.7 kDa. Nucleotide and protein data base searches showed significant homology between CNF1 and the dermonecrotic toxin of Pa ...[more]

PMID: 8406895

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Project description:Background:Many Escherichia coli strains are considered to be a component of the normal flora found in the human and animal intestinal tracts. While most E. coli strains are commensal, some strains encode virulence factors that enable the bacteria to cause intestinal and extra-intestinal clinically-relevant infections. Colibactin, encoded by a genomic island (pks island), and cytotoxic necrotizing factor (CNF), encoded by the cnf gene, are genotoxic and can modulate cellular differentiation, apoptosis and proliferation. Some commensal and pathogenic pks+ and cnf+ E. coli strains have been associated with inflammation and cancer in humans and animals. Results:In the present study, E. coli strains encoding colibactin and CNF were identified in macaque samples. We performed bacterial cultures utilizing rectal swabs and extra-intestinal samples from clinically normal macaques. A total of 239 E. coli strains were isolated from 266 macaques. The strains were identified biochemically and selected isolates were serotyped as O88:H4, O25:H4, O7:H7, OM:H14, and OM:H16. Specific PCR for pks and cnf1 gene amplification, and phylogenetic group identification were performed on all E. coli strains. Among the 239 isolates, 41 (17.2%) were pks+/cnf1-, 19 (7.9%) were pks-/cnf1+, and 31 (13.0%) were pks+/cnf1+. One hundred forty-eight (61.9%) E. coli isolates were negative for both genes (pks-/cnf1-). In total, 72 (30.1%) were positive for pks genes, and 50 (20.9%) were positive for cnf1. No cnf2+ isolates were detected. Both pks+ and cnf1+ E. coli strains belonged mainly to phylogenetic group B2, including B21. Colibactin and CNF cytotoxic activities were observed using a HeLa cell cytotoxicity assay in representative isolates. Whole genome sequencing of 10 representative E. coli strains confirmed the presence of virulence factors and antibiotic resistance genes in rhesus macaque E. coli isolates. Conclusions:Our findings indicate that colibactin- and CNF-encoding E. coli colonize laboratory macaques and can potentially cause clinical and subclinical diseases that impact macaque models.

Project description:Although many Escherichia coli strains are considered commensals in mammals, strains encoding the cyclomodulin genotoxins are associated with clinical and subclinical disease in the urogenital and gastrointestinal tracts, meningitis, and inflammatory disorders. These genotoxins include the polyketide synthase (pks) pathogenicity island, cytolethal distending toxin (cdt), and hemolysin-associated cytotoxic necrotizing factor (cnf). E. coli strains are not excluded from rodents housed under SPF conditions in academic or vendor facilities. This study isolated and characterized genotoxin-encoding E. coli from laboratory rats obtained from 4 academic institutions and 3 vendors. A total of 69 distinct E. coli isolates were cultured from feces, rectal swab, nares, or vaginal swab of 52 rats and characterized biochemically. PCR analysis for cyclomodulin genes and phylogroup was performed on all 69 isolates. Of the 69 isolates, 45 (65%) were positive for pks, 20/69 (29%) were positive for cdt, and 4 (6%) were positive for cnf. Colibactin was the sole genotoxin identified in 21 of 45 pks+ isolates (47%), whereas cdt or cnf was also present in the remaining 24 isolates (53%); cdt and cnf were never present together or without pks. All genotoxin-associated strains were members of pathogen-associated phylogroup B2. Fisher exact and χ² tests demonstrated significant differences in genotoxin prevalence and API code distribution with regard to vendor. Select E. coli isolates were characterized by HeLa cell in vitro cytotoxicity assays, serotyped, and whole-genome sequenced. All isolates encoding cyclomodulins induced megalocytosis. Serotypes corresponded with vendor origin and cyclomodulin composition, with the cnf+ serotype representing a known human uropathogen. Whole-genome sequencing confirmed the presence of complete pks, cdt, and hemolysin-cnf pathogenicity islands. These findings indicate that genotoxin-encoding E. coli colonize laboratory rats from multiple commercial vendors and academic institutions and suggest the potential to contribute to clinical disease and introduce confounding variables into experimental rat models.

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Isolation and nucleotide sequence of the gene encoding cytotoxic necrotizing factor 1 of Escherichia coli.

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Isolation and nucleotide sequence of the gene encoding cytotoxic necrotizing factor 1 of Escherichia coli.

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