Cytotoxic Necrotizing Factor Type Research Articles

Gene clusters encoding the cytotoxic necrotizing factor type 1, Prs-fimbriae and alpha-hemolysin form the pathogenicity island II of the uropathogenic Escherichia coli strain J96.

The uropathogenic Escherichia coli strain J96 (04:K6) is able to produce four adherence factors [P-fimbriae (pap and prs), F1C-fimbriae (foc) and Type 1-fimbriae (fim)], two alpha-hemolysins (hlyI and II) and the cytotoxic necrotizing factor type 1 (cnf1). Using phenotypic test systems and genotypic analysis, it has been shown that the mutant strain J96-M1 has lost the hlyII, prs and cnf1 genes. The three virulence associated determinants are linked on one particular region on the chromosome, which is termed 'pathogenicity island II' (Pai II).

Serotypes of CNF1-producing Escherichia coli strains that cause extraintestinal infections in humans.

The O:K:H serotypes of 137 necrotoxigenic Escherichia coli (NTEC) producing the cytotoxic necrotizing factor type 1 (CNF1) isolated from human extraintestinal infection were determined. Although NTEC producing CNF1 belonged to 58 different serotypes, only 10 of them accounted for 54% of strains. The most common serotypes, in order of frequency, were: O4:K?:H5, O6:K13:H1, O83:K1:H31, O75:K95:H5, O2:K1:H6, O2:K7:H-, O75:K1:H7, O2:K?:H1, O4:K12:H1 and O22:K13:H1. CNF1 strains of serotypes O2:K7:H- and O4:K12:H1 express P-fimbriae, whereas CNF1 strains of serotypes O2:K?:H1, O2:K1:H6 and O75:K95:H5 possess the adhesin responsible for MRHA type III. Among CNF1 strains of serotype O4:K?:H5 there exist some that express P-fimbriae and others that possess MRHA type III. Lastly, the majority of CNF1 strains of serotypes O6:K13:H1, O22:K13:H1, O75:K1:H7 and O83:K1:H31 do not express P-fimbriae nor the adhesin responsible to MRHA type III. Our results show that extraintestinal infections are caused by a limited number of virulent clones, as suggested by the theory of special pathogenicity.

F17-like fimbriae from an invasive Escherichia coli strain producing cytotoxic necrotizing factor type 2 toxin.

The F17b fimbriae encoded by the transmissible virulence plasmid Vir, also coding for cytotoxic necrotizing factor type 2, were characterized. A 5.7-kb region of Vir mediates in vitro N-acetylglucosamine-sensitive adhesion to calf intestinal villi. Sequence analysis revealed that this region codes for a structural subunit and an adhesin closely related to the F17-A and F17-G proteins encoded by the F17 fimbrial gene cluster. The F17b-A gene presents an open reading frame of 540 bp encoding a polypeptide of 180 amino acids with a putative signal peptide of 21 residues. The mature protein shows an identity of 74% with the F17-A structural subunit. This 20-kDa protein is recognized by antiserum directed against F17 fimbriae. The F17b-G gene shows an open reading frame of 1,029 bp encoding a polypeptide of 343 amino acids with a putative signal peptide of 22 residues. The F17b-G polypeptide exhibits 95% identity with the F17-G adhesin. The functional homology of the gene products was further confirmed by demonstrating that mutants in the F17-A gene can be complemented by the F17b-A gene and vice versa. These results prove that fimbriae belonging to the F17 family are also found on pathogenic Escherichia coli strains other than enterotoxigenic isolates producing heat-labile or heat-stable enterotoxin.

Detection of Escherichia coli strains producing cytotoxic necrotizing factor type two (CNF2) by enzyme-linked immunosorbent assay

Sheep and rabbit antisera were produced against lysates of E. coli strain 711 (pVir). This K-12 strain carries the Vir plasmid which codes for Cytotoxic Necrotizing Factor type 2 (CNF2). Immunoglobulin G (IgG) fractions of both immune sera were subsequently purified by a two-step precipitation method. To increase the specificity for CNF2, the sheep IgG preparation was extensively adsorbed against both a sonicated extract of isogenic K-12 strain 711 and intact phenol-treated cells of vaccine strain 711 (pVir). An enzyme-linked immunosorbent assay (ELISA) was developed to detect clinical isolates of E. coli producing CNF2, using the final preparations of rabbit and sheep IgG in a double sandwich technique. The results obtained with this CNF2-ELISA were compared to those obtained with the conventional HeLa cell cytotoxicity assay. The testing of 133 E. coli strains (49 CNF2 positive strains and 84 negative strains) resulted in no false-negative and no false-positive. Therefore, the CNF2-ELISA offers a good alternative to the HeLa cell culture assay for the detection of CNF2-producing strains where facilities for and experience with cell cultures is lacking.

Cytotoxic necrotizing factor type 2 produced by virulent Escherichia coli modifies the small GTP-binding proteins Rho involved in assembly of actin stress fibers.

Cytotoxic necrotizing factor type 2 (CNF2) produced by Escherichia coli strains isolated from intestinal and extraintestinal infections is a dermonecrotic toxin of 110 kDa. We cloned the CNF2 gene from a large plasmid carried by an Escherichia coli strain isolated from a lamb with septicemia. Hydropathy analysis of the deduced amino acid sequence revealed a largely hydrophilic protein with two potential hydrophobic transmembrane domains. The N-terminal half of CNF2 showed striking homology (27% identity and 80% conserved residues) to the N-terminal portion of Pasteurella multocida toxin. Methylamine protection experiments and immunofluorescence studies suggested that CNF2 enters the cytosol of the target cell through an acidic compartment and induces the reorganization of actin into stress fibers. Since the formation of stress fibers in eukaryotic cells involves Rho proteins, we radiolabeled these small GTP-binding proteins from CNF2-treated and control cells with a Rho-specific ADP-ribosyltransferase. The [32P]ADP-ribosylated Rho proteins from CNF2-treated cells migrated slightly more slowly in SDS/PAGE than did the labeled proteins from the control cells. This shift in mobility of Rho proteins in SDS/PAGE was also observed when CNF2 and the RhoA protein were coexpressed in E. coli. We propose that Rho proteins are the targets of CNF2 in mammalian cells.

Serotypes of bovine Escherichia coli producing cytotoxic necrotizing factor type 2 (CNF2)

The serotypes of 101 faecal bovine necrotoxigenic Escherichia coli (NTEC) producing the cytotoxic necrotizing factor type 2 (CNF2) were determined. Although, NTEC producing CNF2 belonged to 48 different O:K:H serotypes, only eleven of them accounted for 54% of strains. The most common serotypes in order of frequency were: O123:K-:H16, O3:K-:H21, O88:K-:H8, O15:K14:H21, O1:K-:H12, O1:K1:H46, O2:K1:H5, O55:H21, O88:K?:H25, O117:K?:H21 and O123:K-:H-. The serotypes of CNF2 NTEC were different from those found in NTEC producing CNF1 and in enterotoxigenic, verotoxigenic, enteropathogenic, enteroinvasive and enteroadherent E. coli strains that cause infections in humans and animals.

Haemolytic Escherichia coli strains isolated from stools of healthy cats produce cytotoxic necrotizing factor type 1 (CNF1)

A total of 159 Escherichia coli colonies isolated from the stools of 23 healthy cats were studied for production of α-haemolysin (Hly), enterohaemolysin (EntHly), cytotoxic necrotizing factors (CNF1 and CNF2), verotoxins (VT) and heat-labile enterotoxin (LT). Hly +CNF1 +, Hly +CNF2 +, Hly +VT + and Hly + E. coli colonies were isolated from 12 (48%), 1 (4%) and 2 (8%) respectively of the cats sampled. None of the 159 E. coli colonies produced LT or EntHly. Nine of 12 Hly +CNF1 + strains from the cats belonged to serogroup O6 and eleven to serotypes (O4:K?:H5 or H-, O6:K13:H1, O6:K53:H-, O6:K53:H1, O6:K53:H7 and O6:K14:H31) found among Hly +CNF1 + E. coli that cause urinary tract infections and sepsis in humans. Furthermore, 10 Hly +CNF1 + strains from the cats expressed the mannose-resistant haemagglutination (MRHA) type III. By contrast, the majority of nontoxigenic E. coli strains were MRHA negative and belonged to different O groups. We conclude that cats are a important reservoir of Hly +CNF1 + E. coli strains that possess similar characteristics to strains that can cause extraintestinal infections in humans and that Hly + E. coli from cats usually do not produce shiga-like toxins with cytotoxic activity on Vero cells.

Induction of phagocytic behaviour in human epithelial cells by Escherichia coli cytotoxic necrotizing factor type 1.

Cytotoxic necrotizing factor type 1 (CNF1) from strains of pathogenic Escherichia coli induces in human epithelial HEp-2 cells, a profound reorganization of the actin cytoskeleton into prominent stress fibres and membrane ruffles. We report here that this process is associated with induction of phagocytic-like activity. CNF1-treated cells acquired the ability to ingest latex beads as well as non-invasive bacteria such as Listeria innocua, which were taken as a model system. Uptake of bacteria was similar to pathogen-induced phagocytosis, since L. innocua transformed with DNA coding for the pore-forming toxin listeriolysin O behaved, with respect to intracellular growth, like the invasive, pathogenic species L. monocytogenes. Our results raise the possibility that, in vivo, pathogenic CNF1-producing E. coli may invade epithelia by this novel induced phagocytic-like mechanism.

Interaction ofEscherichia coli cytotoxic necrotizing factor type 1 (CNF1) with cultured cells

The cytotoxic necrotizing factor type 1 (CNF1) fromE. coli causes necrosis in rabbit skin and multinucleation in cultured cells. Cells exposed to CNF1 were characterized by changes in actin organization, mainly consisting in the presence of well-developed stress fibers and membrane ruffles. The interaction of CNF1 with the cell cytoskeleton probably promotes the cell spreading and interferes with the cytokinesis, leading to the formation of giant multinucleated cells.

Establishment of three categories of P-fimbriated Escherichia coli strains that show different toxic phenotypes and belong to particular O serogroups.

Eight hundred and nineteen strains of Escherichia coli isolated in Spain between 1986 and 1991 from extraintestinal infections and feces of healthy controls were investigated for expression of P-fimbriae using a particle agglutination test. Among strains causing urinary tract infections, sepsis and other extraintestinal infections, P-fimbriae were found in 31% (130/420) (P < 0.001), 25% (30/118) (P < 0.001) and 12% (11/92) (P < 0.5) respectively. In contrast, only 7% (14/189) of faecal isolates from healthy individuals carried P-fimbriae. According to two more common toxic markers detected in this study (alpha-haemolysin and cytotoxic necrotizing factor type 1), P-fimbriated E. coli strains were grouped into three categories: haemolysin+cytotoxic necrosing factor+ (Hly+CNF1+) (68/185; 37%), haemolysin+cytotoxic necrosing factor- (Hly+CNF1-) (61/185; 33%) and Hly-CNF1- (56/185; 30%). The 185 P-fimbriated strains belonged to 17 different O serogroups. However, 148 (80%) were of one of six serogroups (O1, O2, O4, O6, O7 and O18). The most frequent serogroups determined in the Hly+CNF1+ strains were the O4 and O6 (53/68; 78%), in the Hly+CNF1- strains it was the O18 (27/61; 44%) and in the Hly-CNF1- strains the O1, O2 and O7 (41/56; 73%). The majority (160/185; 86%) of P-fimbriated E. coli expressed the mannose-resistant haemagglutinin type IVa.

Establishment of three categories of P-fimbriatedEscherichia colistrains that show different toxic phenotypes and belong to particular O serogroups

Eight hundred and nineteen strains of Escherichia coli isolated in Spain between 1986 and 1991 from extraintestinal infections and feces of healthy controls were investigated for expression of P-fimbriae using a particle agglutination test. Among strains causing urinary tract infections, sepsis and other extraintestinal infections, P-fimbriae were found in 31% (130/420) (P < 0.001), 25% (30/118) (P < 0.001) and 12% (11/92) (P < 0.5) respectively. In contrast, only 7% (14/189) of faecal isolates from healthy individuals carried P-fimbriae. According to two more common toxic markers detected in this study (alpha-haemolysin and cytotoxic necrotizing factor type 1), P-fimbriated E. coli strains were grouped into three categories: haemolysin+cytotoxic necrosing factor+ (Hly+CNF1+) (68/185; 37%), haemolysin+cytotoxic necrosing factor- (Hly+CNF1-) (61/185; 33%) and Hly-CNF1- (56/185; 30%). The 185 P-fimbriated strains belonged to 17 different O serogroups. However, 148 (80%) were of one of six serogroups (O1, O2, O4, O6, O7 and O18). The most frequent serogroups determined in the Hly+CNF1+ strains were the O4 and O6 (53/68; 78%), in the Hly+CNF1- strains it was the O18 (27/61; 44%) and in the Hly-CNF1- strains the O1, O2 and O7 (41/56; 73%). The majority (160/185; 86%) of P-fimbriated E. coli expressed the mannose-resistant haemagglutinin type IVa.

Characteristics of haemolytic Escherichia coli with particular reference to production of cytotoxic necrotizing factor type 1 (CNF1)

A total of 1,106 Escherichia coli strains isolated in Spain between 1986 and 1991 from extraintestinal infections and faeces of healthy controls were examined for production of α-haemolysin (Hly). Among strains causing urinary tract infections, sepsis and other extraintestinal infections, Hly production was detected in 51 % (P < 0.001), 32 % (P < 0.001) and 18 % (P < 0.02), respectively. In contrast, only 9 % of faecal isolates from healthy individuals synthesized Hly. The 356 haemolytic E. coli strains characterized in this study belonged to 28 different serogroups. However, 284 (80 %) were of one of eight serogroups (02, 04, 06, 08, 018, 022, 075 and 083); 40 % and 31 % of haemolytic strains expressed P fimbriae and mannose-resistant haemagglutination (MRHA) type III, respectively. We have found that haemolytic isolates of E. coli may clearly be divided into two categories on the basis of the ability to produce cytotoxic necrotizing factor type 1 (CNF1). The serogroups and adhesins determined in Hly +CNF1 + strains were generally different from those found in Hly +CNF1 − strains. Thus, serogroups 02, 06 and 075 were associated with haemolytic E. coli producing CNF1 +, whereas serogroups 01, 08, 018, 028 and 086 were established more frequently among in Hly +CNF1 − strains. While expression of P fimbriae was more frequently detected in Hly +CNF1 − strains (70 versus 29 %, (P < 0.001), MRHA type III was usually identified in Hly +CNF1 + E. coli (42 versus 1 %, P < 0.001). Furthermore, the sonic extracts of Hly +CNF1 + strains caused necrosis in rabbit skin (96 versus 25 %, (P < 0.001) and death in intraperitoneally injected mice (73 versus 11 %, P < 0. 001) more frequently than sonic extracts of Hly +CNF1 − strains.

Virulence factors associated with cytotoxic necrotizing factor type two in bovine diarrheic and septicemic strains of Escherichia coli

Forty-three bovine isolates of Escherichia coli producing a second type of cytotoxic necrotizing factor (CNF2) and three K-12 strains carrying different Vir plasmids coding for CNF2 were tested for the presence of several virulence factors. Most of the strains were serum resistant (79%), produced an aerobactin (70%), and adhered to calf villi (53%); some of them produced a colicin (32%) and a hemolysin (9%). These strains were also tested by a colony hybridization assay with gene probes for six toxins (classical heat-stable [STaP and STb] and heat-labile [LT-I and LT-IIa] enterotoxins and Shiga-like toxins [SLT-I and SLT-II]) and five adhesion factors (K99, K88, 987P, F17, and F41). Only two gene probes, LT-IIa (9%) and F17A (53%), hybridized with the CNF2 strains. However, antibodies raised against F17 fimbriae did not agglutinate the strains hybridizing with the F17A probe. In contrast, all except one of these strains adhered to calf villi. Interestingly, these two properties, F17A positivity and adherence to calf villi, were the only ones expressed by the K-12 strains carrying different Vir plasmids. In conclusion, this study confirmed that CNF2-producing strains are unrelated to previously described toxigenic E. coli strains and also demonstrated that in half of the strains the production of CNF2 was associated with an adhesion factor genetically related to, but different from, F17, which is more than likely encoded by Vir plasmids.

Characteristics of α-hemolytic strains of Escherichia coli isolated from dogs with gastroenteritis

Twenty-four hemolysin producing (Hly +) strains of Escherichia coli isolated from dogs with gastroenteritis were investigated for their virulence markers and their phenotypic properties. The strains were distributed over eleven known E. coli O-serogroups and most of them were heterogeneous for their phenotypes. All strains were found to produce α-hemolysin which was detected by Southern hybridization and colony immunoblotting using a specific gene probe and a monoclonal antibody. Eight strains were carrying plasmids encoding α-hemolysin sequences ( hly-plasmids) and 16 strains carried chromosomal hly-determinants. Twelve of the strains showed enterotoxic activities which were tested for in different assays. Among these, three O42:H37 and two O7O:H- strains carrying hyl-plasmids were found to harbour other plasmids encoding the heat-stable enterotoxin ST A1. The other seven strains showing enterotoxicity in the ileal loop or the suckling mouse assay were negative for ST A1, ST A2, or LT. None of the 24 strains were positive for invasiveness or for production of Vero (Shiga-like) toxins. The production of α-hemolysin was closely associated with the production of cytotoxic necrotizing factor (CNF), which was detected in 17 of 24 strains. Of these, 16 elaborated CNF1 and one strain produced an unknown CNF type. Surprisingly, all strains carrying ST-plasmids and six of eight strains carrying hly-plasmids were negative for CNF. Thus, in canine E. coli strains CNF production seems to be closely associated with production of chromosomally encoded α-hemolysin whereas hly-plasmids are more often associated with ST-producing, CNF negative isolates.