Comparative evaluation of three tests for the detection of Escherichia coli cytotoxic necrotizing factors (CNF1 and CNF2) using filtrates of cultures treated with mitomycin C

Blanco, Jorge; Blanco, M; González, Enrique A.; Alonso, Marı́a Pilar; Garabal, J.I.

doi:10.1016/0378-1097(90)90086-6

Cited by 20 publications

(17 citation statements)

References 21 publications

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“…These two toxins share a high degree of amino acid identity and have similar catalytic activities (14,17,31,34,37), and yet CNF1 is a more potent inducer of multinucleation in vitro, while CNF2 appears to produce more necrosis in both mouse footpad assays and rabbit skin tests (5,10). By exchanging the CNF1-specific or crossreactive epitopes between the two toxins, new information may be revealed concerning the substrate and receptor specificities of CNF1 and CNF2.…”

Section: Discussionmentioning

confidence: 99%

Epitope Mapping of Monoclonal Antibodies Capable of Neutralizing Cytotoxic Necrotizing Factor Type 1 of Uropathogenic Escherichia coli

2001

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Cytotoxic necrotizing factor type 1 (CNF1) of uropathogenic Escherichia coli belongs to a family of bacterial toxins that target the small GTP-binding Rho proteins that regulate the actin cytoskeleton. Members of this toxin family typically inactivate Rho; however, CNF1 and the highly related CNF2 activate Rho by deamidation. Other investigators have reported that the first 190 amino acids of CNF1 constitute the cellular binding domain and that the CNF1 enzymatic domain lies within a 300-amino-acid stretch in the C terminus of the toxin. Amino acids 53 to 75 appear to be critical for cell receptor recognition, while amino acids Cys866 and His881 are considered essential for deamidation activity. To delineate further the functional domains of CNF1, we generated 16 monoclonal antibodies (MAbs) against the toxin and used them for epitope mapping studies. Based on Western blot immunoreactivity patterns obtained from a series of truncated CNF1 proteins, this panel of MAbs mapped to epitopes located throughout the toxin, including the binding and enzymatic domains. All MAbs showed reactivity to CNF1 by Western and dot blot analyses. However, only 7 of the 16 MAbs exhibited crossreactivity with CNF2. Furthermore, only three MAbs demonstrated the capacity to neutralize toxin in either HEp-2 cell assays (inhibition of multinucleation) or 5637 bladder cell assays (inhibition of cytotoxicity). Since CNF1 epitopes recognized by neutralizing MAbs are likely to represent domains or regions necessary for the biological activities of the toxin, the epitopes recognized by these three MAbs, designated JC4 (immunoglobulin G2a [IgG2a]), BF8 (IgA), and NG8 (IgG2a), were more precisely defined. MAbs JC4 and BF8 reacted with epitopes that were common to CNF1 and CNF2 and located within the putative CNF1 binding domain. MAb JC4 recognized an epitope spanning amino acids 169 to 191, whereas MAb BF8 mapped to an epitope between amino acids 135 and 164. Despite the capacity of both MAbs to recognize CNF2 in Western blot analyses, only MAb BF8 neutralized CNF2. MAb NG8 showed reactivity to a CNF1-specific epitope located between amino acids 683 and 730, a region that includes a very small portion of the putative enzymatic domain. Taken together, these findings identify three new regions of the toxin that appear to be critical for the biological activity of CNF1.

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Section: Discussionmentioning

confidence: 99%

Epitope Mapping of Monoclonal Antibodies Capable of Neutralizing Cytotoxic Necrotizing Factor Type 1 of Uropathogenic Escherichia coli

2001

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“…Strains that produced a clear zone of lysis after incubation for 24 hours at 37 °C were considered to be Hly ÷. For detection of CNF1 the filtrates of cultures treated with mitomycin C were assayed on Veto and HeLa cells as previously described [21]. Seroneutralization assays with CNF1 antiserum were carried out to confirm the production of CNF1 [21,22].…”

Section: Methodsmentioning

confidence: 99%

Virulence factors and O groups of Escherichia coli isolates from patients with acute pyelonephritis, cystitis and asymptomatic bacteriuria

Blanco

Alonso

1996

Eur J Epidemiol

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The relationship between the presence of bacterial virulence factors and the severity of urinary tract infection (UTI) was analized in this study. The production of alpha-hemolysin (Hly), the expression of P-fimbriae and the mannose-resistant hemagglutination (MRHA) type IVa (associated with the presence of P-fimbriae), were all detected more frequently in Escherichia coli strains from acute pyelonephritis than in strains isolated from cystitis and asymptomatic bacteriuria. In contrast, the production of cytotoxic necrotizing factor type 1 (CNF1) and the expression of MRHA types III and IVb were distributed uniformly between strains causing different clinical categories of UTI. Thus 88% of the E. coli strains from acute pyelonephritis showed some of the virulence factors investigated in this study, whereas only 60% (p < 0.01) and 56% (p < 0.01) repectively of the strains isolated from cystitis and asymptomatic bacteriuria possessed virulence factors. There were no significant differences in the prevalence of virulence properties between strains isolated from patients with or without complicating factors. Only 16% (p < 0.001) of the fecal isolates from healthy individuals showed virulence factors. The virulence factors were concentrated in strains belonging to 10 (O1, O2, O4, O6, O7, O14, O18, O22, O75 and O83) of the 12 serogroups most frequently detected in uropathogenic E. coli strains. The majority of uropathogenic O4, O6, O14, O22, O75 and O83 E.coli strains were Hly+CNF1+ and expressed P-fimbriae or MRHA type III, whereas the strains of serogroup O18 were Hly+CNF1- and P-fimbriated. Among O1 and O7 strains we found Hly-CNF1-strains that expressed P-fimbriae. Among O2 strains we found Hly+CNF1+ strains that expressed P-fimbriae or MRHA type III and other Hly-CNF1-strains that possessed P-fimbriae. We conclude that E.coli strains isolated from pyelonephritis show virulence factors more frequently than those from cystitis and asymptomatic bacteriuria, and that strains that cause urinary tract infections in Spain belong to the same serogroups as uropathogenic E.coli isolated in other areas of the world. Our results support the special pathogenicity theory and suggest that many cases of serious urogenital disease may be caused by a limited number of P-fimbriated E.coli strains that usually produce alpha-hemolysin.

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“…The supernatants were filtered through 0.22 µm membrane filters (Millipore Corp., Bedford, USA) and stored at -20ºC. CNF production by E. coli strains was assessed in the presence of mytomycin C (1 µg/mL) as described by Blanco et al (7). The culture supernatants of all strains were tested in duplicate for detection of STx, CNF and LT toxins on Vero (African Green Monkey Kidney) cells supplied by Fort Dodge Laboratories, Inc. (Campinas, Brazil).…”

Section: Toxin Productionmentioning

confidence: 99%

Virulence factors of Escherichia coli isolated from calves with diarrhea in Brazil

Salvadori¹,

Valadares²,

Leite³

et al. 2003

Braz. J. Microbiol.

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Two hundred and five Escherichia coli strains isolated from calves with diarrhea from mid-western Brazil were screened for the presence of virulence factors associated with bovine colibacillosis. One hundred and two (49.8%) of the E. coli strains produced toxins: Shiga toxins 1 (9.7%) and 2 (6.3%), α-hemolysin (9.7%), enterohemolysin (6.8%), Cytotoxic Necrotizing Factors type 1 (0.5%), and type 2 (4.4%), enterotoxins LT-II (8.3%) and STa (3.9%). No strain produced enterotoxin LT-I. Fimbrial adhesins F5 and F17 were produced by 7.3% and 4.8% of the strains, respectivly, and none expressed F41. Seven strains (3.4%) possessed the gene eae and belonged to serotypes O26:H-; O111:H-and O118:H16. These results suggest that calves in Brazil may be an important source of pathogenic E. coli for animals and humans.

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Comparative evaluation of three tests for the detection of Escherichia coli cytotoxic necrotizing factors (CNF1 and CNF2) using filtrates of cultures treated with mitomycin C

Cited by 20 publications

References 21 publications

Epitope Mapping of Monoclonal Antibodies Capable of Neutralizing Cytotoxic Necrotizing Factor Type 1 of Uropathogenic Escherichia coli

Epitope Mapping of Monoclonal Antibodies Capable of Neutralizing Cytotoxic Necrotizing Factor Type 1 of Uropathogenic Escherichia coli

Virulence factors and O groups of Escherichia coli isolates from patients with acute pyelonephritis, cystitis and asymptomatic bacteriuria

Virulence factors of Escherichia coli isolated from calves with diarrhea in Brazil

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