Serogroups of Escherichia coli strains producing cytotoxic necrotizing factors CNF1 and CNF2

The serogroups of 396 necrotizing Escherichia coli of human and bovine origin isolated in Spain between 1979 and 1991 have been determined. The 270 cytotoxic necrotizing factor strains belonged to 22 different O serogroups; however, 84% (226 of 270) were of one of seven serogroups (O2, O4, O6, O14, O22, O75 and O83). Although necrotizing E. coli producing cytotoxic necrotizing factor 2 belonged to 28 different serogroups, only six of them (O1, O3, O15, O55, O88 and O123) accounted for 60% (76 of 126) of cytotoxic necrotizing factor 2 strains. Furthermore, only 3% (4 of 126) of cytotoxic necrotizing factor 2 strains belonged to serogroups most common among strains producing cytotoxic necrotizing factor 1. The majority of necrotizing E. coli producing cytotoxic necrotizing factor 1 were obtained from human extraintestinal infections, whereas cytotoxic necrotizing factor 2 strains were isolated from stools of healthy and diarrhoeic calves.     

Molecular localization of the Escherichia coli cytotoxic necrotizing factor CNF1 cell-binding and catalytic domains

Cytotoxic necrotizing factor type 1 (CNF1) induces, in epithelial cells, the development of stress fibres via the GTPase Rho pathway. We showed that CNF1 is able to modify Rho both in vitro and in vivo. Recombinant N-terminal 33 kDa (CNF1Nter) and C-terminal 14.8–31.5 kDa (CNF1Cter) regions of the CNF1 protein allowed us to demonstrate that the N-terminal region contains the cell-binding domain of the toxin and that the C-terminal region is responsible for its catalytic activity. CNF1Nter lowered the activity of CNF1 when provided to cells before the toxin whereas CNF1Cter had no effect on CNF1 cell toxicity. CNF1Cter was sufficient to induce a typical CNF1 phenotype when microinjected into African green monkey kidney cells (Vero cells), and was able to modify Rho as previously reported for CNF1. The C-terminal domain lost its catalytic activity when deleted of various subdomains, suggesting a scattered distribution of catalytic-site amino acids. Elucidation of the CNF1 functional organization and analysis of amino acid homologies between CNFs (CNF1, CNF2), Pasteurella multocida toxin (PMT) and dermonecrotic toxin of Bordetella pertussis (DNT) allowed us to postulate that CNFs and DNT act on Rho via the same enzymatic activity located in their C-terminus, and that CNFs and PMT probably bind to analogous cell receptors.     

Identification of the region of rho involved in substrate recognition by Escherichia coli cytotoxic necrotizing factor 1 (CNF1).

The Escherichia coli cytotoxic necrotizing factor 1 (CNF1) and the Bordetella dermonecrotic toxin (DNT) activate Rho GTPases by deamidation of Gln(63) of RhoA (Gln(61) of Cdc42 and Rac). In addition, both toxins possess in vitro transglutaminase activity in the presence of primary amines. Here we characterized the region of Rho essential for substrate recognition by the toxins using Rho/Ras chimeras as protein substrates. The chimeric protein Ras55Rho was deamidated or transglutaminated by CNF1. Rat pheochromocytoma PC12 cells microinjected with Ras55Rho developed formation of neurite-like structures after treatment with the CNF1 holotoxin indicating activation of the Ha-Ras chimera and Ras-like effects in intact cells. The Ras59Rho78Ras chimera protein contained the minimal Rho sequence allowing deamidation or transglutamination by CNF1. A peptide covering mainly the switch II region and consisting of amino acid residues Asp(59) through Asp(78) of RhoA was substrate for CNF1. Changes of amino acid residues Arg(68) or Leu(72) of RhoA into the corresponding residues of Ras (R68ARhoA and L72QRhoA) inhibited deamidation and transglutamination of the mutants by CNF1. In contrast to CNF1, DNT did not modify Rho/Ras chimeras or the switch II peptide (Asp(59) through Asp(78)). Glucosylation of RhoA at Thr(37) blocked deamidation by DNT but not by CNF. The data indicate that CNF1 recognizes Rho GTPases exclusively in the switch II region, whereas the substrate recognition by DNT is characterized by additional structural requirements.     

Neosynthesis and activation of Rho by Escherichia coli cytotoxic necrotizing factor (CNF1) reverse cytopathic effects of ADP-ribosylated Rho.

Clostridium botulinum exoenzyme C3 inactivates the small GTPase Rho by ADP-ribosylation. We used a C3 fusion toxin (C2IN-C3) with high cell accessibility to study the kinetics of Rho inactivation by ADP-ribosylation. In primary cultures of rat astroglial cells and Chinese hamster ovary cells, C2IN-C3 induced the complete ADP-ribosylation of RhoA and concomitantly the disassembly of stress fibers within 3 h. Removal of C2IN-C3 from the medium caused the recovery of stress fibers and normal cell morphology within 4 h. The regeneration was preceded by the appearance of non-ADP-ribosylated RhoA. Recovery of cell morphology was blocked by the proteasome inhibitor lactacystin and by the translation inhibitors cycloheximide and puromycin, indicating that intracellular degradation of the C3 fusion toxin and the neosynthesis of Rho were required for reversal of cell morphology. Escherichia coli cytotoxic necrotizing factor CNF1, which activates Rho by deamidation of Gln(63), caused reconstitution of stress fibers and cell morphology in C2IN-C3-treated cells within 30-60 min. The effect of CNF1 was independent of RhoA neosynthesis and occurred in the presence of completely ADP-ribosylated RhoA. The data show three novel findings; 1) the cytopathic effects of ADP-ribosylation of Rho are rapidly reversed by neosynthesis of Rho, 2) CNF1-induced deamidation activates ADP-ribosylated Rho, and 3) inhibition of Rho activation but not inhibition of Rho-effector interaction is a major mechanism underlying inhibition of cellular functions of Rho by ADP-ribosylation.     

Comparative genomics of Escherichia coli strains causing urinary tract infections

The virulence determinants of uropathogenic Escherichia coli have been studied extensively over the years, but relatively little is known about what differentiates isolates causing various types of urinary tract infections. In this study, we compared the genomic profiles of 45 strains from a range of different clinical backgrounds, i.e., urosepsis, pyelonephritis, cystitis, and asymptomatic bacteriuria (ABU), using comparative genomic hybridization analysis. A microarray based on 31 complete E. coli sequences was used. It emerged that there is little correlation between the genotypes of the strains and their disease categories but strong correlation between the genotype and the phylogenetic group association. Also, very few genetic differences may exist between isolates causing symptomatic and asymptomatic infections. Only relatively few genes that could potentially differentiate between the individual disease categories were identified. Among these were two genomic islands, namely, pathogenicity island (PAI)-CFT073-serU and PAI-CFT073-pheU, which were significantly more associated with the pyelonephritis and urosepsis isolates than with the ABU and cystitis isolates. These two islands harbor genes encoding virulence factors, such as P fimbriae (pyelonephritis-associated fimbriae) and an important immunomodulatory protein, TcpC. It seems that both urovirulence and growth fitness can be attributed to an assortment of genes rather than to a specific gene set. Taken together, urovirulence and fitness are the results of the interplay of a mixture of factors taken from a rich menu of genes.     

Structure of the Rho-activating domain of Escherichia coli cytotoxic necrotizing factor 1.

Certain uropathogenic and neonatal meningitis-causing strains of Escherichia coli express a 114 kDa protein toxin called cytotoxic necrotizing factor 1 (CNF1). The toxin causes alteration of the host cell actin cytoskeleton and promotes bacterial invasion of blood-brain barrier endothelial cells. CNF1 belongs to a unique group of large cytotoxins that cause constitutive activation of Rho guanosine triphosphatases (GTPases), which are key regulators of the actin cytoskeleton. This group also includes E. coli cytotoxic necrotizing factor 2 (CNF2, 114 kDa) and dermonecrotic toxins (DNT, 159 kDa) of Bordetella spp. with related sequences occurring in Yersinia spp. Here we show that the catalytic region of CNF1 exhibits a novel protein fold as determined by its 1.83 A resolution crystal structure. The structure reveals that CNF1 has a Cys-His-main chain oxygen catalytic triad reminiscent of enzymes belonging to the catalytic triad superfamily. The position of the catalytic Cys residue at the base of a deep pocket restricts access to potential substrates and helps explain the high specificity of this and related toxins.     

The involvement of SelB in the expression of cytotoxic necrotizing factor 1 in Escherichia coli

Cytotoxic necrotizing factor 1 (CNF1) plays an important role in meningitis-causing Escherichia coli. Mini-Tn5 mutagenesis of meningitis-causing E. coli revealed that transposon mutants of selA and selB genes failed to express CNF1. We subsequently showed that SelB and selenocysteine, however, are not essential for the expression of CNF1, but the deletion of 47 amino acids of SelB at its C terminus has a dominant negative effect on CNF1 expression at the translational level. Bioinformatic analysis of the mRNA of cnf1 predicted two putative selenocysteine incorporation sequence (SECIS) elements, but we failed to detect any selenocysteine in CNF1 protein. These findings suggest that SelB is involved in translational regulation of CNF1 expression but without incorporation of selenocysteine in CNF1 protein.     

Frequency of Escherichia coli serotypes in urinary infections]

Among the large O-groups of E.coli isolated from urinary-tract infections, a few groups appear more frequent. The AA report about this frequency in the district of L'Aquilla. Of 147 stocks of E.coli, the most frequent O-groups are the O6 (28,5%), the O75 (20,4%), the O2 (10,2%), the O18 (6,1%), the O5 (4,1%), the O39 (2%).     

Necrotoxigenic Escherichia coli from sheep and goats produce a new type of cytotoxic necrotizing factor (CNF3) associated with the eae and ehxA genes.

Fecal samples from sheep and goats were screened by tissue-culture assays and PCR for the presence of necrotoxigenic Escherichia coli (NTEC) producing cytotoxic necrotizing factors (CNFs). Of the 18 NTEC strains assayed, four were positive for the cnf1 gene while 14 strains were negative for the cnf1 and cnf2 genes. All of the NTEC strains had the eae gene and most of them also carried the ehxA gene. Moreover, all the cnf1- cnf2- NTEC strains were negative for several virulence markers associated with CNF1+ or CNF2+ strains. The cnf gene present in one of these strains was sequenced and analysis of the gene product revealed a new type of CNF, which was named CNF3 (and the coding gene cnf3). Oligonucleotide primers were designed to PCR-amplify a fragment of cnf3. The results showed that all strains examined in this study, except one cnf1+strain, were cnf3+. The association of cnf3 with eae and ehxA suggests that cnf3+ NTEC strains might be pathogenic for humans.     

Pathogenic characteristics of Escherichia coli strains in cases of asymptomatic bacteriuria and symptomatic urinary tract infections

The aim of this study was to examine if E. coli isolated from asymptomatic bacteriuria differed in pathogenic features from strains isolated from symptomatic infections of urinary tract. In this study 130 strains of E. coli isolated from women having asymptomatic bacteriuria and 112 strains isolated from patients with symptoms of urinary tract infection were examined. It was shown that E. coli isolated from patients with symptomatic urinary tract infection showed the more frequently ability to cause mannose-resistant haemagglutination of human erythrocytes, resistance to bactericidal activity of serum and haemolytic properties than those isolated from asymptomatic bacteriuria. These strains showed also the higher ability to adhere to Vero cells in tissue culture. Among E. coli strains isolated from persons with asymptomatic bacteriuria the pathogenic features were most frequently found in strains from healthy women and the most rarely in isolated from diabetic women.     

Aerobactin production and dulcitol fermentation in clonal groups of Escherichia coli O1: K1 strains from urinary tract infections

Abstract 70 urinary Escherichia coli O1:K1 strains were characterized for O1 antigen factors, mannose-resistant hemagglutination of human erythrocytes, flagellar and fimbrial antigens, dulcitol fermentation and aerobactin production. On the basis of their O1 and H antigens the strains could be assigned to 6 distinct groups. The most prevalent groups were: O1abcd: H⁻ :F9 (33 strains; pattern II), O1abc: H⁻ :F11 (9 strains; pattern IV), and O1abc: H7: F11 (19 strains; pattern V). Strains with patterns IV and V, both expressing fimbrial antigen F11, fermented dulcitol and produced aerobactin, whereas strains with pattern II were negative for both characteristics.     

Urinary tract infections of Escherichia coli strains of chaperone-usher system

Urinary tract infections are a very serious health and economic problem affecting millions of people each year worldwide. The most common etiologic agent of this type of bacterial infections, involving the upper and lower urinary tract, are E. coli strains representing approximately 80% of cases. Uropathogenic E. coli strains produce several urovirulence factors which can be divided into two main types, surface virulence factors and exported virulence factors. Surface-exposed structures include mainly extracellular adhesive organelles such as fimbriae/pili necessary in adhesion, invasion, biofilm formation and cytokine induction. Among the surface-exposed polymeric adhesive structures there are three most invasive groups, type 1 pili, type P pili and Dr family of adhesins which are bioassembled via the conserved, among Gram-negative bacteria, chaperone-usher secretion system. Type 1 and P-piliated E. coli cause cystitis and pyelonephritis. The Dr family of adhesins recognizing DAF receptor is responsible for cystitis, pyelonephritis (especially in pregnant women) and diarrhoea (in infants). In addition, Dr-positive E. coli strains carry the risk of recurrent urinary tract infections. Pyelonephritis in pregnant women leads to a series of complications such as bacteremia, urosepsis, acute respiratory distress syndrome and even death. In the era of increasing drug resistance of bacteria, the development of vaccines, drugs termed pilicides and inhibitors of adhesion may be a promising tool in the fight against urogenital infections.     

Proteomic analysis of Escherichia coli associated with urinary tract infections

Escherichia coli is a major cause of urinary tract infections (UTIs) where the initial infection arises from bacteria originating in the bowel. However, significant differences are observed between the genomes of intestinal and urinary E. coli strains with the latter possessing many adaptations that promote growth in the urinary tract. To define further the adaptation of urinary E. coli isolates, the cellular proteomes of 41 E. coli strains, collected from cases of UTIs or random faecal samples, were compared by 2-D gel electrophoresis and principal component analysis. The data indicated that individual patients carried relatively homogenous E. coli populations, as defined by their cellular proteomes, but the populations were distinct between patients. For one patient, E. coli, isolated during two recurrent infections 3 months apart, were indistinguishable, indicating that for this patient the infections were possibly caused by the same bacterial population. To understand the basis of the discrimination of the bacteria, selected protein spots were identified by peptide fragment fingerprinting. The identified proteins were involved in a variety of metabolic and structural roles. The data obtained for these E. coli strains provide a basis from which to target key bacterial proteins for further investigation into E. coli pathogenesis.     

Induction of phagocytic behaviour in human epithelial cells by Escherichia coli cytotoxic necrotizing factor type1

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 listeriolysln 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 of<Emphasis Type="Italic">Escherichia coli</Emphasis> 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.     

Cross-protection phenomenon in Escherichia coli strains harbouring cytotoxic necrotizing factors and cytolethal distending toxins

Micro-organisms which are subjected to non-lethal stress can exhibit significantly greater resistance when both the same or an unrelated stress is subsequently reapplied. This latter phenomenon is termed 'cross-protection'. In experiments using three strains of Escherichia coli harbouring cytotoxic necrotizing factor 1 and/or cytolethal distending toxin all three exhibited significantly greater (P < 0.05) resistance to salt (20% w/v) or heat (56 degrees C for up to 75 min) when prestressed with lactic acid (pH 4). This work indicates that the cross-protection phenomenon should be taken into account when devising food process operations designed to minimize the risk posed by these pathogens.