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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Cytotoxic necrotizing factor type 1 (CNF1) and CNF2 are highly homologous toxins that are produced by certain pathogenic strains of Escherichia 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, ⌬N63, ⌬N545, and ⌬C469, 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.Cytotoxic necrotizing factor type 1 (CNF1) is produced by many strains of uropathogenic Escherichia coli (UPEC), which are agents that are responsible for the majority of uncomplicated urinary tract infections (9). CNF1 is a 115-kDa cytoplasmic protein that is a member of a family of toxins that target small GTPases. Specifically, CNF1 deamidates glutamine 63 of RhoA and glutamine 61 of Rac1 and Cdc42, modifications that result in constitutive activation of these small GTPases (1). This activation leads to the formation of stress fibers and focal adhesions (RhoA), lamellipodia (Rac1), and filopodia (Cdc42) in CNF1-intoxicated cells and ultimately results in rearrangement of the cytoskeleton (12,18,28). Phenotypically, CNF1 causes multinucleation of various tissue culture cells (8, 11) but can also be cytotoxic against certain cell lines, including Swiss 3T3 and 5637 bladder cells (19,22). In vivo, CNF1 evokes necrosis when it is injected intradermally into rabbit skin (4). Moreover, members of our laboratory, in collaboration with colleagues, demonstrated that in two animal systems CNF1 expression contributes to the virulence of UPEC strains. In a rat model of acute prostatitis, we found that intraurethral infection with a CNF1-positive strain leads to a significantly enhanced inflammatory response compared to that elicited by an isogenic, CNF1-negative mutant, even when the bacterial counts are equivalent (26). Similarly, in a mouse model of urinary tract infection, the production of CNF1 by UPEC strains results in higher bacterial counts and increased inflammation compared to the results for cnf1 isogenic mutants, in part due to...

To study the role of vaginal flora and pH in the pathogenesis of Trichomonas vaginalis, an intravaginal mouse model of infection was established. By employing this model, the vaginal flora and pH of mice could be monitored for changes caused by the parasite. As a baseline, the endemic vaginal flora of BALB/c mice was examined first and found to consist mainly of Staphylococcus aureus and Enterococcus species (32-76%). Lactobacilli and enteric bacilli were moderate (16-32%) in their frequency of isolation, and the prevalence of both anaerobic species and coagulase-negative staphylococci was low (4-16%). Vaginal pH was recorded at 6.5 +/- 0.3. Estrogenization, which was required for a sustained T. vaginalis infection, did not significantly alter vaginal flora; however, a slight rise in the number of bacterial species isolated per mouse and a drop in vaginal pH (6.2 +/- 0.5) were observed. Trichomonas vaginalis-infected mice did not appear to show significant changes in vaginal flora although vaginal pH was slightly increased. This mouse model could have applications in both immunologic and pathogenic studies of T. vaginalis and, with further modifications, aid in the study of protist-bacterial interactions.

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.