Cytotoxic Necrotizing Factor Type Research Articles

Context-Dependent Requirements for FimH and Other Canonical Virulence Factors in Gut Colonization by Extraintestinal Pathogenic Escherichia coli.

ABSTRACTExtraintestinal pathogenic Escherichia coli (ExPEC) acts as a commensal within the mammalian gut but can induce pathology upon dissemination to other host environments such as the urinary tract and bloodstream. ExPEC genomes are likely shaped by evolutionary forces encountered within the gut, where the bacteria spend much of their time, provoking the question of how their extraintestinal virulence traits arose. The principle of coincidental evolution, in which a gene that evolved in one niche happens to be advantageous in another, has been used to argue that ExPEC virulence factors originated in response to selective pressures within the gut ecosystem. As a test of this hypothesis, the fitness of ExPEC mutants lacking canonical virulence factors was assessed within the intact murine gut in the absence of antibiotic treatment. We found that most of the tested factors, including cytotoxic necrotizing factor type 1 (CNF1), Usp, colibactin, flagella, and plasmid pUTI89, were dispensable for gut colonization. The deletion of genes encoding the adhesin PapG or the toxin HlyA had transient effects but did not interfere with longer-term persistence. In contrast, a mutant missing the type 1 pilus-associated adhesin FimH displayed somewhat reduced persistence within the gut. However, this phenotype varied dependent on the presence of specific competing strains and was partially attributable to aberrant flagellin expression in the absence of fimH. These data indicate that FimH and other key ExPEC-associated factors are not strictly required for gut colonization, suggesting that the development of extraintestinal virulence traits is not driven solely by selective pressures within the gut.

Distribution of Uropathogenic Virulence Genes in Escherichia coli Isolated from Children with Urinary Tract Infection in Sanandaj, Iran

Background and Objectives: Uropathogenic Escherichia coli (UPEC) strains are the most common cause of urinary tract infections (UTIs) in children. UPEC isolates express a range of virulence traits promoting effective colonization of urinary tract. The aim of this study was to determine antibiotic susceptibility and virulence determinants of UPEC isolated from children. Methods: This cross-sectional study was performed on 32 E. coli strains recovered from urine samples of children with UTI aged 0 to 12 years in spring 2015 (between April and June) in Sanandaj, Iran. The isolates were examined by PCR for the presence of virulence genes encoding haemolysin (hly), cytotoxic necrotizing factor type 1 (cnf1), P-fimbriae (Pap), and afimbrial adhesin (afa). Sensitivity to antibiotics was determined using the disk diffusion method. Results: The prevalence of genes encoding adhesins was 25% for pap, and 15.6% for afa. The hly and cnf genes encoding toxins were amplified in 15.6% and 25% of isolates, respectively. The strains isolated from hospitalized patients displayed a greater number of virulence genes compared to the isolates from outpatients. Different patterns of virulence genes were identified. Nitrofurantoin and trimethoprim/sulfamethoxazole were the most and least effective antibiotics with susceptibility rates of 96.9% and 21.9%, respectively. Conclusions: These data show the need for monitoring of drug resistance and its consideration in the treatment of E. coli infections. Investigation of bacterial pathogenicity associated with UTI may help have better medical intervention and management of UTI.

Secretion of Alpha-Hemolysin by Escherichia coli Disrupts Tight Junctions in Ulcerative Colitis Patients.

Objectives:The potential of Escherichia coli (E. coli) isolated from inflammatory bowel disease (IBD) patients to damage the integrity of the intestinal epithelium was investigated.Methods:E. coli strains isolated from patients with ulcerative colitis (UC) and healthy controls were tested for virulence capacity by molecular techniques and cytotoxic assays and transepithelial electric resistance (TER). E. coli isolate p19A was selected, and deletion mutants were created for alpha-hemolysin (α-hemolysin) (hly) clusters and cytotoxic necrotizing factor type 1 (cnf1). Probiotic E. coli Nissle and pathogenic E. coli LF82 were used as controls.Results:E. coli strains from patients with active UC completely disrupted epithelial cell tight junctions shortly after inoculation. These strains belong to phylogenetic group B2 and are all α-hemolysin positive. In contrast, probiotic E. coli Nissle, pathogenic E. coli LF82, four E. coli from patients with inactive UC and three E. coli strains from healthy controls did not disrupt tight junctions. E. coli p19A WT as well as cnf1, and single loci of hly mutants from cluster I and II were all able to damage Caco-2 (Heterogeneous human epithelial colorectal adenocarcinoma) cell tight junctions. However, this phenotype was lost in a mutant with knockout (Δ) of both hly loci (P<0.001).Conclusions:UC-associated E. coli producing α-hemolysin can cause rapid loss of tight junction integrity in differentiated Caco-2 cell monolayers. This effect was abolished in a mutant unable to express α-hemolysin. These results suggest that high Hly expression may be a mechanism by which specific strains of E. coli pathobionts can contribute to epithelial barrier dysfunction and pathophysiology of disease in IBD.

Risk factors causing the development of infection and inflammation of the lower urinary tract

Persistence as well as high survival rate and resistance of microorganisms belong to the key reasons promoting symptoms of lower urinary tract diseases. They can stay active for a long time and can substantially deteriorate the life quality of patients suffering from chronic infections and inflammations in the wall of the urinary bladder even after the eradication of the pathogen. Recent studies in the field of clinical microbiology reflect serious qualitative and quantitative changes in the infection incidence structure, which is mainly related to changes in the bacterial pathogenicity. Pathogenicity island genes control the synthesis of different adhesins, invasins, hemolysins and toxins (hemolysin, cytotoxic necrotizing factor type 1, etc.) as well as iron ion absorption system, which are essential for the pathogen propagation and activity in tissues. The determination of genetic determinants will provide a new insight into the bacterial evolution process and will help to understand the reason of prompt formation and propagation of virulent strains as well as fast development of the infection and inflammation in the lower urinary tract, which may explain different severity of the pathology and symptoms of the lower urinary tract.

Antibodies against Hemolysin and Cytotoxic Necrotizing Factor Type 1 (CNF1) Reduce Bladder Inflammation in a Mouse Model of Urinary Tract Infection with Toxigenic Uropathogenic Escherichia coli

Uropathogenic Escherichia coli (UPEC) is the leading cause of cystitis. Cytotoxic necrotizing factor 1 (CNF1) and hemolysin (Hly) are toxins made by approximately 50% of UPEC isolates. CNF1 and Hly contribute to the robust inflammatory response in the bladders of mice challenged with UPEC strain CP9. We hypothesized that antibodies against CNF1 and/or Hly would reduce cystitis caused by CP9. To test this theory, we immunized female C3H/HeOuJ mice subcutaneously with a genetically derived Hly toxoid or genetically derived CNF1 toxoid plus sublethal doses of CNF1. We collected serum and observed increasing titers of specific and neutralizing antibodies against Hly or CNF1 over time. We challenged the mice intraurethrally with CP9 and euthanized them 24 h later. We observed 10-fold lower bacterial titers in the urine of Hly-immunized mice than in that of sham-immunized mice but no difference in kidney bacterial titers. Immunized mice also exhibited significantly less cystitis than sham-immunized mice. In CNF1-vaccinated mice, we detected neither a difference in urine or kidney bacterial titers nor a reduction in the severity of cystitis versus that of sham-immunized mice. We then passively administered an anti-CNF1 monoclonal antibody intraperitoneally to female C3H/HeOuJ mice prior to intraurethral challenge with CP9. Upon challenge, we noted no difference in colonization of the urine or kidney; however, cystitis was reduced significantly in mice treated with the anti-CNF1 antibody versus that in the bladders of mice given an isotype control antibody. Taken together, our data demonstrate that antibodies against CNF1 or Hly reduce the bladder pathology caused by UPEC.

Tu1760 Functional Evidence for a Causative Role of the Intestinal Microbiota in Crohn's Disease-Like Ileitis Using Antibiotic-Treated and Germ-Free TNFDeltaare/+ Mice

BACKGROUND: An increased prevalence of phylogenetic B2 group Escherichia coli (E. coli) has been associated with active disease in IBD patients (IBDEC). Many of the B2 group strains harbor virulence genes present in extraintestinal pathogenic E.coli including hemolysin (hly) and cytotoxic necrotizing factor type 1 (CNF1) the loci of which is often linked. Here we investigated the potential of B2 strains of E.coli isolated from IBD patients to damage the integrity of the intestinal epithelium and the potential contribution of Hly and CNF1. METHODS: E coli strains isolated from 35 patients with IBD were tested for virulence (Petersen et al 2009) and of those isolate C691-04A showed high cytotoxicity on dendritic cells ( submitted) and a rapidly decrease of the trans-epithelial electric resistance (TEER) of a monolayer of intestinal epithelial cell lines. Genome sequencing of C691-04A confirmed the presence of hly cluster. It is known that some E coli have two hly-clusters. In order to investigate the role of hly and CNF1 in disruption of the epithelial integrity the following mutants were created in strain C691-04A; 1: CAS965, hly cluster II upstream of CNF1; 2: CAS954, hly cluster I mutant; 3: CAS956 is a knock out of both loci of hly; 4: CAS964, CNF1 mutant; 5: CAS966, clbA mutant. Caco-2 cells were grown to confluence in 24-well trans-well devices for 14 days to form a polarized cell monolayer and TER measured constantly during exposure to E. coli strains at MOI 10. The wild type and commensal E. coli were included as controls. RESULTS: E. coli C691-04A WT as well as CNF1, clbA and single loci of hly mutants from cluster I and II were all able to damage Caco-2 cell tight junctions in less than 6 hrs co-culture (see figure). Confocal microscopy of occludin stained trans-well sections revealed loss of occludin from the tight junction within 3 hours. However, this phenotype was lost in a mutant with knock out of both hly loci (p<0.001). Commensal E. coli isolated from healthy controls also did not cause loss of TER in 6 h. CONCLUSION: IBD-associated E. coli such as C691-04A can cause rapid loss of tight junction integrity in differentiated Caco-2 cell monolayers. This effect is abolished in a mutant with knock out of both loci of the hly genes and diminished in one of the single knockout mutants. These results suggest that high Hly expression may be a mechanism by which specific strains of E. coli pathobionts can contribute to epithelial barrier dysfunction and pathophysiology of disease in IBD.

Toxins of Extraintestinal Escherichia coli Isolated from Blood Culture

Extraintestinal pathogenic Escherichia coli (ExPEC) is one of the main etiological agents of bloodstream infections caused by Gram-negative bacilli. ExPEC pathogenicity is due to the presence of genes, located on plasmids or chromosomes that encode virulence factors. E. coli virulence factors such as adhesins, toxins, invasins are able to modify the metabolism of host cell, contributing to disease. In this study, 80 strains of E. coli were isolated by hemoculture from septicemic patients and examinated by polymerase chain reaction to identify the virulence factors genes encoding toxins and to determine the phylogenetic group. We characterized genes encoding 3 different types of toxins: α-hemolysin (hlyA), cytotoxic necrotizing factor type 1 (cnf1) and five subtypes of cytolethal distending toxins (cdt-I to cdt-V). 23.75% of E. coli strains contained cnf1 gene and 22.5% hlyA. Genes cdt-I and cdt-IV were detected in 1 of 80 strains. Phylogenetic classification showed that E. coli strains fall into 4 groups (A, B1, B2, D) based on PCR detection (chuA and yjaA genes and DNA fragment TSPE4.C2). Virulent ExPEC belong mostly to groups B2 and D. Our results confirmed this fact: 56% of E. coli strains belonged to group B2 and 24% to group D.

Virulence characteristics and antimicrobial susceptibility of uropathogenic Escherichia coli strains

Eight virulence factors associated with uropathogenic Escherichia coli (UPEC) were investigated in 204 clinical isolates of E. coli recovered from urine cultures at counts ≥10(5). The bacteria were classified into two groups according to the number of leukocytes in urine samples from which they were isolated: group I ≤8 leukocytes/hpf, 104 strains; group II >8 leukocytes/hpf, 100 strains. Two multiplex PCR systems were used to detect genes encoding adhesin P (pap), adhesin S (sfa), afimbrial adhesin I (afa), siderophore aerobactin (aer), alpha-hemolysin (hly), cytotoxic necrotizing factor type 1 (cnf1), and traT associated with serum resistance. The PAI marker for the virulence island identified in strains CFT072 and CVD432, a marker of enteroaggregative E. coli, was also investigated using PCR. The susceptibility profile of E. coli strains was determined by disk diffusion method. Ninety percent UPEC showed at least one of the virulence genes, the prevalence being traT (76%), aer (41%), PAI (32%), sfa (26%), pap (25%), cnf1 (18%), afa (6%), and hly (5%). There was no significant difference in the distribution of virulence genes between groups I and II. A significantly higher degree of virulence was detected in UPEC group II. The CVD432 gene was not detected in any of the UPECs. Fifty-nine percent of the strains were resistant to at least one of the antimicrobials that we tested; the most common being resistance to ampicillin (51%) and trimethoprim-sulfamethoxazole (44%).

Cytotoxic Necrotizing Factor Type 1-Neutralizing Monoclonal Antibody NG8 Recognizes Three Amino Acids in a C-Terminal Region of the Toxin and Reduces Toxin Binding to HEp-2 Cells

Cytotoxic necrotizing factor type 1 (CNF1) and CNF2 are toxins of pathogenic Escherichia coli that share 85% identity over 1,014 amino acids. Although both of these toxins modify GTPases, CNF1 is a more potent inducer of multinucleation in HEp-2 cells, binds more efficiently to HEp-2 cells, and, despite the conservation of amino acids (C866 and H881) required for enzymatic activity of the toxins, deamidates RhoA and Cdc42 better than CNF2. Here we exploited the differences between CNF1 and CNF2 to define the epitope on CNF1 to which the CNF1-specific neutralizing monoclonal antibody (MAb) (MAb NG8) binds and to determine the mechanism by which MAb NG8 neutralizes CNF1 activity on HEp-2 cells. For these purposes, we generated a panel of 21 site-directed mutants in which amino acids in CNF1 were exchanged for the amino acids in CNF2 between amino acids 546 and 869 and vice versa. This region of CNF1 not only is recognized by MAb NG8 but also is involved in binding of this toxin to HEp-2 cells. All the mutants retained the capacity to induce multinucleation of HEp-2 cells. However, the CNF1 double mutant with D591E and F593L mutations (CNF1(D591E F593L)) and the CNF1(H661Q) single mutant displayed drastically reduced reactivity with MAb NG8. A reverse chimeric triple mutant, CNF1(E591D L593F Q661H), imparted MAb NG8 reactivity to CNF2. MAb NG8 neutralized CNF2(E591D L593F Q661H) activity in a dose-dependent manner and reduced the binding of this chimeric toxin to HEp-2 cells. Taken together, these results pinpoint three amino acids in CNF1 that are key amino acids for recognition by neutralizing MAb NG8 and further help define a region in CNF1 that is critical for full toxin binding to HEp-2 cells.

Genotypic characterization of virulence factors in Escherichia coli strains from patients with cystitis

Adhesins (P-fimbriae, S-fimbriae, type 1 fimbriae and afimbrial adhesin), toxins (alpha-hemolysin and cytotoxic necrotizing factor type 1), iron acquisition systems (aerobactin) and host defense avoidance mechanisms (capsule or lipopolysaccharide) have been shown to be prevalent in Escherichia coli strains associated with urinary tract infections. In this work, 162 Uropathogenic Escherichia coli (UPEC) strains from patients with cystitis were genotypically characterized by polymerase chain reaction (PCR) assay. We developed three multiplex PCR assays for virulence-related genes papC, papE/F, papG alleles, fimH, sfa/foc, afaE, hly, cnf-1, usp, cdtB, iucD, and kpsMTII, all of them previously identified in UPEC strains. The PCR assay results identified 158 fimH (97.5%), 86 kpsMTII (53.1%), 53 papC/papEF/papG (32.7%), 45 sfa (27.8%), 42 iucD (25.9%), 41 hly (25.3%), 36 usp (22.2%), 30 cnf-1(18.5%) and 10 afa (6.2%) strains. No strain was positive for cdtB. In this work, we also demonstrated that adhesins may be multiple within a single strain and that several virulence genes can occur combined in association.

Hemolysin of Uropathogenic Escherichia coli Evokes Extensive Shedding of the Uroepithelium and Hemorrhage in Bladder Tissue within the First 24 Hours after Intraurethral Inoculation of Mice

Many uropathogenic Escherichia coli (UPEC) strains produce both hemolysin (Hly) and cytotoxic necrotizing factor type 1 (CNF1), and the loci for these toxins are often linked. The conclusion that Hly and CNF1 contribute to urovirulence is supported by the results of epidemiological studies associating the severity of urinary tract infections (UTIs) with toxin production by UPEC isolates. Additionally, we previously reported that mouse bladders and rat prostates infected with UPEC strain CP9 exhibit a more profound inflammatory response than the organs from animals challenged with CP9cnf(1) and that CNF1 decreases the antimicrobial activities of polymorphonuclear leukocytes. More recently, we created an Hly mutant, CP9Delta hlyA(1)::cat, and showed that it was less hemolytic and destructive for cultured bladder cells than CP9 was. Here we evaluated the relative effects of mutations in hlyA(1) or cnf(1) alone or together on the pathogenicity of CP9 in a mouse model of ascending UTI. To do this, we constructed an hlyA(1)-complemented clone of CP9Delta hlyA(1)::cat and an hlyA(1) cnf(1) CP9 double mutant. We found that Hly had no influence on bacterial colonization of the bladder or kidneys in single or mixed infections with the wild type and CP9Delta hlyA(1)::cat but that it did provoke sloughing of the uroepithelium and bladder hemorrhage within the first 24 h after challenge. Finally, we confirmed that CNF1 expression induces bladder inflammation and, in particular, as shown in this study, submucosal edema. From these data, we speculate that Hly and CNF1 may be largely responsible for the signs and symptoms of cystitis in humans infected with toxigenic UPEC.

A single amino acid substitution in the enzymatic domain of cytotoxic necrotizing factor type 1 of Escherichia coli alters the tissue culture phenotype to that of the dermonecrotic toxin of Bordetella spp.

Cytotoxic necrotizing factor type 1 (CNF1) and dermonecrotic toxin (DNT) share homology within their catalytic domains and possess deamidase and transglutaminase activities. Although each toxin has a preferred enzymatic activity (i.e. deamidation for CNF1 and transglutamination for DNT) as well as target substrates, both modify a specific glutamine residue in RhoA, Rac1 and Cdc42, which renders these GTPases constitutively active. Here we show that despite their similar mechanisms of action CNF1 and DNT induced unique phenotypes on HEp-2 and Swiss 3T3 cells. CNF1 induced multinucleation of HEp-2 cells and was cytotoxic for Swiss 3T3 cells (with binucleation of the few surviving cells) while DNT showed no morphological effects on HEp-2 cells but did induce binucleation of Swiss 3T3 cells. To determine if the enzymatic domain of each toxin dictated the induced phenotype, we constructed enzymatically active chimeric toxins and mutant toxins that contained single amino acid substitutions within the catalytic site and tested these molecules in tissue culture and enzymatic assays. Moreover, both site-directed mutant toxins showed reduced time to maximum transglutamination of RhoA compared with the parent toxins. Nevertheless, the substitution of threonine for Lys(1310) in the DNT-based mutant, while affecting transglutamination efficiency of the toxin, did not abrogate that enzymatic activity.

Cytotoxic necrotizing factor type 1 production by uropathogenic Escherichia coli modulates polymorphonuclear leukocyte function.

Many strains of uropathogenic Escherichia coli (UPEC) produce cytotoxic necrotizing factor type 1 (CNF1), a toxin that constitutively activates the Rho GTPases RhoA, Rac1, and Cdc42. We previously showed that CNF1 contributes to the virulence of UPEC in a mouse model of ascending urinary tract infection and a rat model of acute prostatitis and that a striking feature of the histopathology of the mouse bladders and rat prostates infected with CNF1-positive strains is an elevation in levels of polymorphonuclear leukocytes (PMNs). We also found that CNF1 synthesis leads to prolonged survival of UPEC in association with human neutrophils. Here, we tested the hypothesis that CNF1 production by UPEC diminishes the antimicrobial capacity of mouse PMNs by affecting phagocyte function through targeting Rho family GTPases that are critical to phagocytosis and the generation of reactive oxygen species. We found that, as with human neutrophils, CNF1 synthesis provided a survival advantage to UPEC incubated with mouse PMNs. We also observed that CNF1-positive UPEC down-regulated phagocytosis, altered the distribution of the complement receptor CR3 (CD11b/CD18), enhanced the intracellular respiratory burst, and increased levels of Rac2 activation in PMNs. From these results, we conclude that modulation of PMN function by CNF1 facilitates UPEC survival during the acute inflammatory response.

Detection of cytotoxic necrotizing factor types 1 and 2 among fecal Escherichia coli isolates from Brazilian children with and without diarrhea.

The enteropathogenic role of cytotoxic necrotizing factor (CNF)-producing Escherichia coli was investigated by searching cnf genes among 2074 isolates from 200 children with and 200 without acute diarrhea in Brazil. Fourteen (7%) cases versus 10 (5%) control children carried at least one cnf positive isolate (P = 0.50) and most isolates expressed CNF type 1. DNA sequences of virulence factors of extraintestinal pathogenic E. coli (ExPEC) were detected in 78.6% of CNF1-producing isolates. Besides not being associated with human acute diarrhea, the CNF1-producing isolates here identified may represent potential ExPEC transitorily composing the normal intestinal flora.

Patterns of variations in Escherichia coli strains that produce cytolethal distending toxin.

A collection of 20 Escherichia coli strains that produce cytolethal distending toxin (CDT) were analyzed for their virulence-associated genes. All of these strains were serotyped, and multiplex PCR analysis was used to ascertain the presence of genes encoding other virulence factors, including Shiga toxin, intimin, enterohemolysin, cytotoxic necrotizing factor type 1 (CNF1) and CNF2, heat-stable toxin, and heat-labile toxin. These CDT-producing strains possessed various combinations of known virulence genes, some of which have not been noted before. Partial cdtB sequences were obtained from 10 of these strains, and their predicted CdtB sequences were compared to known E. coli CdtB sequences; some of the sequences were identical to known CdtB sequences, but two were not. PCR primers based on sequence differences between the known cdt sequences were tested for their ability to detect CDT producers and to determine CDT type. Correlations between the type of CDT produced, the presence of other virulence properties, and overall strain relatedness revealed that the CDT producers studied here can be divided into three general groups, with distinct differences in CDT type and in their complement of virulence-associated genes.

Cytotoxic necrotizing factor 1 enhances reactive oxygen species-dependent transcription and secretion of proinflammatory cytokines in human uroepithelial cells.

Uropathogenic Escherichia coli strains frequently produce a Rho-activating protein toxin named cytotoxic necrotizing factor type 1 (CNF1). We herein report that CNF1 promotes transcription and release of tumor necrosis factor alpha, gamma interferon, interleukin-6 (IL-6), and IL-8 proinflammatory cytokines and increases the production of reactive oxygen species (ROS) in uroepithelial T24 cells. The antioxidant N-acetyl-L-cysteine counteracts these phenomena, a fact which suggests a role for ROS-mediated signaling in CNF1-induced proinflammatory cytokine production.

Response from Emmanuel Lemichez: A new turn in Rho GTPase activation by Escherichia coli cytotoxic necrotizing factors

The high frequency and high cost of urinary tract infections (UTIs) make these pathologies a major public health problem in developed countries. Uropathogenic Escherichia coli (UPEC), the major causative agent of UTIs, and its frequently associated cytotoxic necrotizing factor-1 (CNF1) toxin, have therefore attracted much attention. Investigations carried out in Aktories and Boquet's laboratories, together with Horiguchi's laboratory working on the CNF1-related dermonecrotic toxin of Bordetella, have clarified the molecular mechanism of action of this toxin family towards Rho GTPase proteins [1xBacterial toxins that modify the actin cytoskeleton. Barbieri, J.T. et al. Annu. Rev. Cell Dev. Biol. 2002; 18: 315–344Crossref | PubMed | Scopus (107)See all References, 2xSee all References].In vitro, these toxins cause permanent activation of Rho GTPases by modifying the glutamine residue responsible for GTP hydrolysis. Recent observations have revealed that in fact, CNF1 has a dual mechanism of action, comprising activation followed by the proteasome-dependent degradation of RhoA, Rac and Cdc42, thereby resulting in long-term, low-grade stimulation of the Rho GTPases [3xCNF1 exploits the ubiquitin-proteasome machinery to restrict Rho GTPase activation for bacterial host cell invasion. Doye, A. et al. Cell. 2002; 111: 553–564Abstract | Full Text | Full Text PDF | PubMed | Scopus (163)See all References, 4xProteasomal degradation of cytotoxic necrotizing factor 1-activated Rac. Lerm, M. et al. Infect. Immun. 2002; 70: 4053–4058Crossref | PubMed | Scopus (69)See all References]. This dual mechanism has been hidden until now owing to the lower capacity of some carcinoma cell lines used for CNF1 studies to efficiently carry out ubiquitination and proteasomal degradation of permanently activated Rho GTPases [3xCNF1 exploits the ubiquitin-proteasome machinery to restrict Rho GTPase activation for bacterial host cell invasion. Doye, A. et al. Cell. 2002; 111: 553–564Abstract | Full Text | Full Text PDF | PubMed | Scopus (163)See all References[3].Previous work has highlighted a role for CNF1 as an invasive factor [5xInduction of phagocytic behaviour in human epithelial cells by Escherichia coli cytotoxic necrotizing factor type 1. Falzano, L. et al. Mol. Microbiol. 1993; 9: 1247–1254Crossref | PubMedSee all References[5]. In addition, we could demonstrate that reaching a low level of Rac activation is required for efficient bacterial entry into non-professional phagocytic cells, together with associated cellular motility and cellular junction dynamic [3xCNF1 exploits the ubiquitin-proteasome machinery to restrict Rho GTPase activation for bacterial host cell invasion. Doye, A. et al. Cell. 2002; 111: 553–564Abstract | Full Text | Full Text PDF | PubMed | Scopus (163)See all References[3]. Both cellular motility and bacterial invasion thus appeared tightly coupled, requiring cell polarization [6xQuo vadis: polarized membrane recycling in motility and phagocytosis. Mellman, I. J. Cell Biol. 2000; 149: 529–530Crossref | PubMed | Scopus (24)See all References[6]. Our observations also raised an alternative hypothesis that pathogenic bacteria have developed antagonist virulence factors toward Rho GTPases to protect host cells from toxicity arising from massive activation of Rho GTPases. We suggest that instead, these virulence factors might cooperate for efficient bacterial invasion. Consistent with this model, different pathogenic bacteria appear to have developed different strategies to achieve a local polymerization of the actin cytoskeleton for cell penetration. For example, Salmonella injects both SopE/E2 and SptP virulence factors for the respective activation and inhibition of Rac and Cdc42 [7xStriking a balance: modulation of the actin cytoskeleton by Salmonella. Galan, J.E. and Zhou, D. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8754–8761Crossref | PubMed | Scopus (179)See all References[7]. Likewise, Shigella injects IpaC for Rac and Cdc42 activation, and IpaA, which produces indirect disassembly of local actin filaments, both factors acting together to produce efficient bacterial entry into cells [8xBacterial signals and cell responses during Shigella entry into epithelial cells. Tran Van Nhieu, G. et al. Cell Microbiol. 2000; 2: 187–193Crossref | PubMed | Scopus (99)See all References[8].The crosstalk between bacterial virulence factors and the ubiquitin/proteasome machineries is a major point of interest for future research. Consistent with this, the YopJ/P virulence factor of Yersinia has been reported to cleave SUMO-1 ubiquitin-like polypeptide, and SigD/SopB inositol phosphatase of Salmonella appeared regulated by ubiquitylation [9xDisruption of signaling by Yersinia effector YopJ, a ubiquitin-like protein protease. Orth, K. et al. Science. 2000; 290: 1594–1597Crossref | PubMedSee all References, 10xSalmonella enterica serovar Typhimurium effector SigD/SopB is membrane-associated and ubiquitinated inside host cells. Marcus, S.L. et al. Cell Microbiol. 2002; 4: 435–446Crossref | PubMed | Scopus (70)See all References].Finally, in addition to the demonstration that CNF1 is a bona fide invasive factor, we have evidence that an oncogenic protein, exemplified by Rac, is rapidly withdrawn upon permanent activation by the ubiquitin-proteasome machinery, pointing out the proteasome to be a guardian of the cellular homeostasis.

Role of Rac and Cdc42 in lysophosphatidic acid-mediated cyclo-oxygenase-2 gene expression

The role of Rho proteins in lysophosphatidic acid (LPA)-mediated induction of cyclo-oxygenase-2 (Cox-2) was investigated in renal mesangial cells. Previous studies had shown that toxin B, an inhibitor of Rho, Rac and Cdc42, suppressed Cox-2 induction. A role for RhoA in pertussis toxin-sensitive LPA signalling was excluded with C3 transferase from Clostridium limosum, used as the fusion toxin C2IN—C3 (where C2IN is part of the C2I toxin of C. botulinum). Incubation of the cells with C2IN—C3 disrupted cytosolic actin stress fibres, but had no effect on Cox-2 induction. Similarly, activation of p42/44 mitogen-activated protein kinase (MAP kinase), an upstream step in Cox-2 induction, was inhibited by toxin B, but not affected by C2IN—C3. Upon treatment with toxin B, focal adhesion kinase and paxillin were dephosphorylated at tyrosine residues and the actin cytoskeleton was completely destroyed. An intact cytoskeleton, however, was not required for p42/44 MAP-kinase activation or Cox-2 induction, as shown by the actin-depolymerizing agent cytochalasin D. Toxin B did not influence functionality of LPA receptors, because Gi-mediated Ca2+ release from intracellular stores remained unchanged. Within 1h, toxin B inactivated and translocated RhoA and Cdc42 to the cellular membranes. Within the same time frame, monoglucosylated Rac1 was degraded. Direct stimulation of Rho proteins by cytotoxic necrotizing factor type 1 (CNF1) induced Cox-2 expression, which was sensitive to inhibition of the MAP-kinase pathway by PD98059, but not to an inhibitor of RhoA kinase. By exclusion of RhoA and non-specific cytoskeletal effects, the results in the present study indicate an important role for Rac and/or Cdc42 in pertussis toxin-sensitive LPA-mediated Cox-2 induction.

Role of Rac and Cdc42 in lysophosphatidic acid-mediated cyclo-oxygenase-2 gene expression

The role of Rho proteins in lysophosphatidic acid (LPA)-mediated induction of cyclo-oxygenase-2 (Cox-2) was investigated in renal mesangial cells. Previous studies had shown that toxin B, an inhibitor of Rho, Rac and Cdc42, suppressed Cox-2 induction. A role for RhoA in pertussis toxin-sensitive LPA signalling was excluded with C3 transferase from Clostridium limosum, used as the fusion toxin C2IN-C3 (where C2IN is part of the C2I toxin of C. botulinum). Incubation of the cells with C2IN-C3 disrupted cytosolic actin stress fibres, but had no effect on Cox-2 induction. Similarly, activation of p42/44 mitogen-activated protein kinase (MAP kinase), an upstream step in Cox-2 induction, was inhibited by toxin B, but not affected by C2IN-C3. Upon treatment with toxin B, focal adhesion kinase and paxillin were dephosphorylated at tyrosine residues and the actin cytoskeleton was completely destroyed. An intact cytoskeleton, however, was not required for p42/44 MAP-kinase activation or Cox-2 induction, as shown by the actin-depolymerizing agent cytochalasin D. Toxin B did not influence functionality of LPA receptors, because G(i)-mediated Ca(2+) release from intracellular stores remained unchanged. Within 1 h, toxin B inactivated and translocated RhoA and Cdc42 to the cellular membranes. Within the same time frame, monoglucosylated Rac1 was degraded. Direct stimulation of Rho proteins by cytotoxic necrotizing factor type 1 (CNF1) induced Cox-2 expression, which was sensitive to inhibition of the MAP-kinase pathway by PD98059, but not to an inhibitor of RhoA kinase. By exclusion of RhoA and non-specific cytoskeletal effects, the results in the present study indicate an important role for Rac and/or Cdc42 in pertussis toxin-sensitive LPA-mediated Cox-2 induction.

Occurrence and genetic association of selected virulence factors in clinical Escherichia coli isolates.

Occurrence of cnf1+ E. coli pathogenic strains among extraintestinal E. coli isolates was evaluated to explain an impact of cytotoxic necrotizing factor type 1 (CNF1) in human infections. A total of 120 E. coli isolates were characterized for presence of virulence factors cnf1- and pap--specific sequences by PCR, and the production of alpha-hemolysin using blood agar-plate test. Different association patterns among the detected virulence factors were obtained by comparison of various groups of clinical E. coli isolates. These differences probably reflect a potential impact of CNF1 in the colonization of vaginal environment.