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, CP9⌬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 CP9⌬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 CP9⌬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.Urinary tract infections (UTIs) include infections of the bladder (cystitis) and/or kidney (pyelonephritis) and account for more than 7 million office visits each year by otherwise healthy women (23). Extraintestinal pathogenic Escherichia coli (ExPEC) is the causative agent of at least 80% of all uncomplicated UTIs. ExPEC strains that cause a UTI are called uropathogenic E. coli (UPEC). UPEC strains typically are members of phylogenetic group B2 or D and often exhibit specific O:K:H serotypes, as well as various combinations of virulence factors, including, among others, adhesins or fimbriae, siderophore systems, and toxins. The virulence-associated genes in UPEC are frequently clustered together in "pathogenicity islands" (PAIs) (25), and many UPEC isolates harbor more than one PAI. For instance, the prototypic UPEC clinical strain J96 (O4:K6) carries two PAIs, PAI I J96 and PAI II J96 (58). PAI I J96 is over 170 kb long and contains operons encoding alpha-hemolysin (hly) and Pap fimbriae (pap). PAI II J96 is 110 kb long and also contains an hly operon in addition to genes encoding Prs fimbriae (prs) and cytotoxic necrotizing factor type 1 (CNF1) (cnf 1 ) (6). Here, we hypothesized that the link between PAI II J96 and the urovirulence of E. coli strains may largely reflect the fact that these strains produce both alpha-hemolysin (referred to as hemolysin [Hly] here) and CNF1. ...

Bacillus collagen-like protein of anthracis (BclA) is the immunodominant glycoprotein on the exosporium of Bacillus anthracis spores. Here, we sought to assess the impact of BclA on spore germination in vitro and in vivo, surface charge, and interaction with host matrix proteins. For that purpose, we constructed a markerless bclA null mutant in B. anthracis Sterne strain 34F2. The growth and sporulation rates of the ⌬bclA and parent strains were nearly indistinguishable, but germination of mutant spores occurred more rapidly than that of wild-type spores in vitro and was more complete by 60 min. Additionally, the mean time to death of A/J mice inoculated subcutaneously or intranasally with mutant spores was lower than that for the wild-type spores even though the 50% lethal doses of the two strains were similar. We speculated that these in vitro and in vivo differences between mutant and wild-type spores might reflect the ease of access of germinants to their receptors in the absence of BclA. We also compared the hydrophobic and adhesive properties of ⌬bclA and wild-type spores. The ⌬bclA spores were markedly less water repellent than wild-type spores, and, probably as a consequence, the extracellular matrix proteins laminin and fibronectin bound significantly better to mutant than to wild-type spores. These studies suggest that BclA acts as a shield to not only reduce the ease with which spores germinate but also change the surface properties of the spore, which, in turn, may impede the interaction of the spore with host matrix substances.Bacillus anthracis is a gram-positive, spore-forming bacillus that can cause anthrax (15). The spore is the form of the organism found in its natural habitat, the soil, and is also the infectious form for herbivores, the typical vertebrate host for the bacterium, and humans (15). The B. anthracis spore is covered by a loose balloon-like membranous structure called the exosporium (8). BclA (for bacillus collagen-like protein of anthracis) was first described by Sylvestre et al. (23), who constructed an insertional bclA mutant and compared it to its wild-type parent. These investigators and, subsequently, others found that BclA is a glycoprotein and a major component of the hair-like projections that cover the exosporium (16,22,23,25). BclA is also an immunodominant marker on the outside of the spore (22). The finding that BclA does not play a significant role in the virulence of a Sterne-like strain for mice was first reported by Sylvestre et al. (23). Sterne strains contain pXO1 but not pX02 and are attenuated in humans and many other animals except certain mouse strains (26). In support of the findings of Sylvestre and colleagues, Bozue and coworkers recently constructed a bclA mutant of the fully virulent B. anthracis Ames strain and showed that the absence of BclA had no impact on the lethality of that strain for guinea pigs or mice (5). Whether BclA, the substance on the spore with which the host cells probably first interact, plays a more subtle role in B. anthracis patho...

Human bladder 5637 cells cultivated under microgravity conditions formed organoids that displayed characteristics of in vivo tissue-specific differentiation. Uropathogenic Escherichia coli (UPEC) strain CP9 colonized and penetrated the organoids and induced ␣-hemolysin-mediated exfoliation of uroepithelial cells. We propose these uro-organoids as models that simulate the interactions between UPEC and terminally differentiated human urothelium.

Bacillus collagen-like protein of anthracis (BclA) is an immunodominant glycoprotein located on the exosporium of Bacillus anthracis. We hypothesized that antibodies to this spore surface antigen are largely responsible for the augmented immunity to anthrax that has been reported for animals vaccinated with inactivated spores and protective antigen (PA) compared to vaccination with PA alone. To test this theory, we first evaluated the capacity of recombinant, histidine-tagged, nonglycosylated BclA (rBclA) given with adjuvant to protect A/J mice against 10 times the 50% lethal dose of Sterne strain spores introduced subcutaneously. Although the animals elicited anti-rBclA antibodies and showed a slight but statistically significant prolongation in the mean time to death (MTD), none of the mice survived. Similarly, rabbit anti-rBclA immunoglobulin G (IgG) administered intraperitoneally to mice before spore inoculation increased the MTD statistically significantly but afforded protection to only 1 of 10 animals. However, all mice that received suboptimal amounts of recombinant PA and that then received rBclA 2 weeks later survived spore challenge. Additionally, anti-rBclA IgG, compared to anti-PA IgG, promoted a sevenfold-greater uptake of opsonized spores by mouse macrophages and markedly decreased intramacrophage spore germination. Since BclA has some sequence similarity to human collagen, we also tested the extent of binding of anti-rBclA antibodies to human collagen types I, III, and V and found no discernible cross-reactivity. Taken together, these results support the concept of rBclA as being a safe and effective boost for a PA-primed individual against anthrax and further suggest that such rBclA-enhanced protection occurs by the induction of spore-opsonizing and germination-inhibiting antibodies.Spores of Bacillus anthracis, the causative agent of anthrax, are the infectious form of the organism and can persist in soil in a dormant stage for decades (25). Although herbivores are the primary reservoir of anthrax, humans can contract anthrax, albeit rarely, if inoculated with spores cutaneously, orally, or inhalationally (8). Although anthrax is typically seen only in individuals involved in certain occupations, the potential for infection of larger numbers of people by the aerosol route is of public health concern because of the misuse of B. anthracis spores that occurred in the United States in 2001 (9).One way to protect vulnerable individuals and populations against anthrax is through a strategy of prophylactic immunization. Currently, the anthrax vaccine adsorbed (AVA) preparation is the only licensed anthrax vaccine for use in the United States. AVA is comprised of a formalin-treated, aluminum salt-adsorbed, cell-free culture filtrate from an attenuated strain of B. anthracis (3). Although AVA is considered to be safe and effective, the utility of the vaccine is limited by its availability, reactogenicity, requirement for the administration of multiple doses (3), and the generally adverse publicity that ...

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.

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.Urinary tract infections rank high among the most common types of symptomatic infections in humans (10, 31). Indeed, more than 6 million cases of cystitis, i.e., urinary tract infections that are restricted to the bladder, occur annually in the United States with an estimated health care cost of over $1 billion (31). Uropathogenic Escherichia coli (UPEC) are responsible for the majority of urinary tract infections and account for 85% to 95% of all cystitis isolates (31). Nearly twice as many women as men are seen by physicians for symptomatic urinary tract infection, and 40 to 50% of women experience at least one urinary tract infection in their lifetime (10). Longterm complications of urinary tract infection are rare; however, an ascending urinary tract infection can result in pyelonephritis and septicemia (16,21).The pathogenesis of E. coli-mediated urinary tract infection is not completely understood but likely involves the expression by the uropathogen of an array of virulence factors that facilitate colonization and evasion of the immune response. Type 1 fimbriae are established as UPEC virulence factors (8), while expression of other adhesins, such as P-pili and S-fimbriae, is strongly linked to UPEC pathogenicity (11). The precise role that ␣-hemolysin, a secreted toxin made by many UPEC isolates, plays in urovirulence is not understood. However, one possible explanation is that ␣-hemolysin changes host responses, a theory based on the finding that ␣-hemolysin modulates Ca 2ϩ signaling in cultured primary renal epithelial cells (21). Cytotoxic necrotizing factor type 1 (CNF1) is often coproduced with ␣-hemolysin by UPEC strains (9) a...

Int6/eIF3-p48 was ®rst identi®ed as a common integration site for MMTV in mouse mammary tumors. In all cases, the MMTV integration event resulted in an interruption of the normal Int6 transcript from one allele leaving the second allele intact and operative. We hypothesize that insertion of MMTV into Int6 results in a mutated allele that encodes a shortened Int6 mRNA and protein (Int6sh), which either modi®es normal Int6 function or possesses a new independent function. To con®rm the transforming potential of the mutation and its dominant function, we transfected two mammary epithelial cell lines, MCF10A (human), and HC11 (mouse), with Int6sh under the control of the elongation factor-1a (eEF1A) promoter. Expression of Int6sh in MCF10A and HC11 mammary epithelial cells leads to anchorage-independent growth in soft agar indicative of a transformed phenotype. Colonies selected from agar exhibited high levels of mutated Int6sh and wild type Int6 RNA transcripts by RT ± PCR and Northern blot analysis. In addition, Int6sh transformed MCF10A and HC11 cells formed nodular growths, in vivo, in immune compromised hosts. NIH3T3 cells, mouse embryo ®broblasts, were also transformed to anchorage-independent growth in vitro by Int6sh expression. These observations provide direct evidence that the Int6 mutations observed in MMTV-induced tumors and hyperplasia contribute to the malignant transformation of the mammary epithelial cells. Oncogene (2001) 20, 5291 ± 5301.

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...