Identification and Analysis of the Regulatory RegionR&R* with thecnf1Gene Encoding the Cytotoxic Necrotizing Factor Type 1 That Closely Links to theluxRegulon ofVibrio fischeri

1998 ◽  
Vol 250 (2) ◽  
pp. 462-465 ◽  
Author(s):  
Juey-Wen Lin ◽  
Li-Ming Chen ◽  
Hui-Yi Chen ◽  
Shu-Fen Weng
Keyword(s):  
Cytotoxic Necrotizing Factor ◽  
Factor Type

scholarly journals Cytotoxic Necrotizing Factor Type 1 Delivered by Outer Membrane Vesicles of Uropathogenic Escherichia coli Attenuates Polymorphonuclear Leukocyte Antimicrobial Activity and Chemotaxis

2006 ◽  
Vol 74 (8) ◽  
pp. 4401-4408 ◽  
Author(s):  
Jon M. Davis ◽  
Humberto M. Carvalho ◽  
Susan B. Rasmussen ◽  
Alison D. O'Brien
Keyword(s):  
Escherichia Coli ◽  
Outer Membrane ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Biologically Active ◽  
Single Amino Acid ◽  
Upec Strain ◽  
Cytotoxic Necrotizing Factor ◽  
Factor Type

ABSTRACT Cytotoxic necrotizing factor type 1 (CNF1), a toxin produced by many strains of uropathogenic Escherichia coli (UPEC), constitutively activates small GTPases of the Rho family by deamidating a single amino acid within these target proteins. Such activated GTPases not only stimulate actin polymerization within affected cells but also, as we previously reported, decrease membrane fluidity on mouse polymorphonuclear leukocytes (PMNs). In that same investigation we found that this diminished membrane movement impedes the clustering of the complement receptor CD11b/CD18 on PMNs and, in turn, decreases PMN phagocytic capacity and microbicidal activity on PMNs in direct contact with CNF1-expressing UPEC as well as on those in proximity to wild-type UPEC. The latter observation suggested to us that CNF1 is released from neighboring bacteria, although at the time of initiation of the study described here, no specific mechanism for export of CNF1 from UPEC had been described. Here we present evidence that CNF1 is released from the CNF1-expressing UPEC strain CP9 (serotype O4/H5/K54) in a complex with outer membrane vesicles (OMVs) and that these CNF1-bearing vesicles transfer biologically active CNF1 to PMNs and attenuate phagocyte function. Furthermore, we show that CNF1-bearing vesicles act in a dose-dependent fashion on PMNs to inhibit their chemotactic response to formyl-Met-Leu-Phe, while purified CNF1 does not. We conclude that OMVs provide a means for delivery of CNF1 from a UPEC strain to PMNs and thus negatively affect the efficacy of the acute inflammatory response to these organisms.

scholarly journals 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

2008 ◽  
Vol 77 (1) ◽  
pp. 170-179 ◽  
Author(s):  
Kerian K. Grande ◽  
Karen C. Meysick ◽  
Susan B. Rasmussen ◽  
Alison D. O'Brien
Keyword(s):  
Amino Acids ◽  
Monoclonal Antibody ◽  
Dependent Manner ◽  
Triple Mutant ◽  
Potent Inducer ◽  
Cytotoxic Necrotizing Factor ◽  
Toxin Binding ◽  
Neutralizing Monoclonal Antibody ◽  
Factor Type

ABSTRACTCytotoxic necrotizing factor type 1 (CNF1) and CNF2 are toxins of pathogenicEscherichia colithat 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 (CNF1D591E F593L) and the CNF1H661Qsingle mutant displayed drastically reduced reactivity with MAb NG8. A reverse chimeric triple mutant, CNF1E591D L593F Q661H, imparted MAb NG8 reactivity to CNF2. MAb NG8 neutralized CNF2E591D L593F Q661Hactivity 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.

scholarly journals Two Domains of Cytotoxic Necrotizing Factor Type 1 Bind the Cellular Receptor, Laminin Receptor Precursor Protein

2007 ◽  
Vol 75 (11) ◽  
pp. 5095-5104 ◽  
Author(s):  
Beth A. McNichol ◽  
Susan B. Rasmussen ◽  
Humberto M. Carvalho ◽  
Karen C. Meysick ◽  
Alison D. O'Brien
Keyword(s):  
Amino Acids ◽  
Catalytic Domain ◽  
Precursor Protein ◽  
Laminin Receptor ◽  
Cellular Receptor ◽  
Cytotoxic Necrotizing Factor ◽  
C Terminus ◽  
Factor Type

ABSTRACTCytotoxic necrotizing factor type 1 (CNF1) and CNF2 are highly homologous toxins that are produced by certain pathogenic strains ofEscherichia coli. These 1,014-amino-acid toxins catalyze the deamidation of a specific glutamine residue in RhoA, Rac1, and Cdc42 and consist of a putative N-terminal binding domain, a transmembrane region, and a C-terminal catalytic domain. To define the regions of CNF1 that are responsible for binding of the toxin to its cellular receptor, the laminin receptor precursor protein (LRP), a series of CNF1 truncated toxins were characterized and assessed for toxin binding. In particular, three truncated toxins, ΔN63, ΔN545, and ΔC469, retained conformational integrity and in vitro enzymatic activity and were immunologically reactive against a panel of anti-CNF1 monoclonal antibodies (MAbs). Based on a comparison of these truncated toxins with wild-type CNF1 and CNF2 in LRP and HEp-2 cell binding assays and in MAb and LRP competitive binding inhibition assays and based on the results of confocal microscopy, we concluded that CNF1 contains two major binding regions: one located within the N terminus, which contained amino acids 135 to 164, and one which resided in the C terminus and included amino acids 683 to 730. The data further indicate that CNF1 can bind to an additional receptor(s) on HEp-2 cells and that LRP can also serve as a cellular receptor for CNF2.

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

1993 ◽  
Vol 38 (1-2) ◽  
pp. 157-165 ◽  
Author(s):  
Jorge Blanco ◽  
Miguel Blanco ◽  
Idania Wong ◽  
Jesús E. Blanco
Keyword(s):  
Escherichia Coli ◽  
Cytotoxic Necrotizing Factor ◽  
Factor Type

scholarly journals Epitope Mapping of Monoclonal Antibodies Capable of Neutralizing Cytotoxic Necrotizing Factor Type 1 of UropathogenicEscherichia coli

2001 ◽  
Vol 69 (4) ◽  
pp. 2066-2074 ◽  
Author(s):  
K. C. Meysick ◽  
M. Mills ◽  
A. D. O'Brien
Keyword(s):  
Amino Acids ◽  
Monoclonal Antibodies ◽  
Western Blot ◽  
Epitope Mapping ◽  
Biological Activities ◽  
Binding Domain ◽  
Cytotoxic Necrotizing Factor ◽  
Cell Assays ◽  
Factor Type

ABSTRACT Cytotoxic necrotizing factor type 1 (CNF1) of uropathogenicEscherichia 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 cross-reactivity 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.

scholarly journals Mutation of the Gene Encoding Cytotoxic Necrotizing Factor Type 1 (cnf1) Attenuates the Virulence of Uropathogenic Escherichia coli

2001 ◽  
Vol 69 (6) ◽  
pp. 3954-3964 ◽  
Author(s):  
Karen E. Rippere-Lampe ◽  
Alison D. O'Brien ◽  
Richard Conran ◽  
Hank A. Lockman
Keyword(s):  
Escherichia Coli ◽  
Urinary Tract ◽  
Human Neutrophils ◽  
Wild Type ◽  
Single Strain ◽  
E Coli ◽  
Cytotoxic Necrotizing Factor ◽  
Positive Strain ◽  
Factor Type

ABSTRACT Cytotoxic necrotizing factor type 1 (CNF1) is a 115-kDa toxin that activates Rho GTPases and is produced by uropathogenicEscherichia coli (UPEC). While both epidemiological studies that link CNF1 production by E. coli with urinary tract disease and the cytopathic effects of CNF1 on cultured urinary tract cells are suggestive of a role for the toxin as a UPEC virulence factor, few in vivo studies to test this possibility have been reported. Therefore, in this investigation, we evaluated the importance of CNF1 in a murine model of urinary tract infection (UTI) by comparing the degree of colonization and damage induced by three different CNF1-producing E. coli strains with isogenic CNF1-deficient derivatives. The data from single-strain challenge experiments with C3H/HeOuJ mice indicated a trend toward higher counts of the wild-type strains in the urine and bladders of these animals up to 3 days after challenge in two of three strain pairs. Furthermore, this difference was statistically significant at day 2 of infection with one strain pair, C189 and C189cnf 1. To control for the animal-to-animal variability inherent in this model, we infected C3H/HeOuJ mice with a mixture of CNF1-positive and -negative isogenic derivatives of CP9. The CNF1-positive strain was recovered in higher numbers than the CNF1-negative strain in the urine, bladders, and kidneys of the mice up to 9 days postinfection. These striking coinfection findings, taken with the trends observed in single-strain infections, led us to conclude that CNF1-negative strains were generally attenuated compared to the wild type in the C3H/HeOuJ mouse model of UTI. Furthermore, histopathological examination of bladder specimens from mice infected with CNF1-positive strains consistently showed deeper, more extensive inflammation than in those infected with the isogenic mutants. Lastly, we found that CNF1-positive strain CP9 was better able to resist killing by fresh human neutrophils than were CP9cnf 1 bacteria. From these data in aggregate, we propose that CNF1 production increases the capacity of UPEC strains to resist killing by neutrophils, which in turn permits these bacteria to gain access to deeper tissue and persist better in the lower urinary tract.

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

1992 ◽  
Vol 143 (9) ◽  
pp. 869-878 ◽  
Author(s):  
J Blanco ◽  
M Blanco ◽  
M.P Alonso ◽  
J.E Blanco ◽  
E.A González ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cytotoxic Necrotizing Factor ◽  
Factor Type

scholarly journals Cytotoxic Necrotizing Factor Type 1 of Uropathogenic Escherichia coli Kills Cultured Human Uroepithelial 5637 Cells by an Apoptotic Mechanism

2000 ◽  
Vol 68 (10) ◽  
pp. 5869-5880 ◽  
Author(s):  
Melody Mills ◽  
Karen C. Meysick ◽  
Alison D. O'Brien
Keyword(s):  
Escherichia Coli ◽  
Cell Line ◽  
Cell Lines ◽  
Human Bladder ◽  
E Coli ◽  
Cytotoxic Necrotizing Factor ◽  
Bladder Cell ◽  
Factor Type ◽  
Tract Infections

ABSTRACT Pathogenic Escherichia coli associated with urinary tract infections (UTIs) in otherwise healthy individuals frequently produce cytotoxic necrotizing factor type 1 (CNF1), a member of the family of bacterial toxins that target the Rho family of small GTP-binding proteins. To gain insight into the function of CNF1 in the development of E. coli-mediated UTIs, we examined the effects of CNF1 intoxication on a panel of human cell lines derived from physiologically relevant sites (bladder, ureters, and kidneys). We identified one uroepithelial cell line that exhibited a distinctly different CNF1 intoxication phenotype from the prototypic one of multinucleation without cell death that is seen when HEp-2 or other epithelial cells are treated with CNF1. The 5637 bladder cell line detached from the growth surface within 72 h of CNF1 intoxication, a finding that suggested frank cytotoxicity. To determine the basis for the unexpected toxic effect of CNF1 on 5637 cells, we compared the degree of toxin binding, actin fiber formation, and Rho modification with those CNF1-induced events in HEp-2 cells. We found no apparent difference in the amount of CNF1 bound to 5637 cells and HEp-2 cells. Moreover, CNF1 modified Rho, in vivo and in vitro, in both cell types. In contrast, one of the classic responses to CNF1 in HEp-2 and other epithelial cell lines, the formation of actin stress fibers, was markedly absent in 5637 cells. Indeed, actin stress fiber induction by CNF1 did not occur in any of the other human bladder cell lines that we tested (J82, SV-HUC-1, or T24). Furthermore, the appearance of lamellipodia and filopodia in 5637 cells suggested that CNF1 activated the Cdc42 and Rac proteins. Finally, apoptosis was observed in CNF1-intoxicated 5637 cells. If our results with 5637 cells reflect the interaction of CNF1 with the transitional uroepithelium in the human bladder, then CNF1 may be involved in the exfoliative process that occurs in that organ after infection with uropathogenic E. coli.

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