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. ...

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.

LasA protease is a 20-kDa elastolytic and staphylolytic enzyme secreted by Pseudomonas aeruginosa. LasA is synthesized as a preproenzyme that undergoes proteolysis to remove a 22-kDa amino-terminal propeptide. Like the propeptides of other bacterial proteases, the LasA propeptide may act as an intramolecular chaperone that correctly folds the mature domain into an active protease. To locate regions of functional importance within proLasA, linker-scanning insertional mutagenesis was employed using a plasmid containing lasA as the target. Among the 5 missense insertions found in the mature domain of proLasA, all abolished enzymatic activity but not secretion. In general, the propeptide domain was more tolerant to insertions. However, insertions within a 9-amino-acid region in the propeptide caused dramatic reductions in LasA enzymatic activity. All mutant proLasA proteins were still secreted, but extracellular stability was low due to clustered insertions within the propeptide. The codons of 16 residues within and surrounding the identified 9-amino-acid region were subjected to site-directed mutagenesis. Among the alanine substitutions in the propeptide that had a major effect on extracellular LasA activity, two (L92A and W95A) resulted in highly unstable proteins that were susceptible to proteolytic degradation and three (H94A, I101A, and N102A) were moderately unstable and allowed the production of a LasA protein with low enzymatic activity. These data suggest that these clustered residues in the propeptide may play an important role in promoting the correct protein conformation of the mature LasA protease domain.

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.Cytotoxic necrotizing factor type 1 (CNF1), a toxin made by many uropathogenic and other extraintestinal isolates of Escherichia coli, is a 115-kDa member of the Rho family of GTPaseactivating toxins (for reviews, see references 2, 3, 6, 19, and 26). CNF1 deamidates glutamine 63 (Q63) of RhoA and glutamine 61 (Q61) of Rac1 and Cdc42; these modifications lead to constitutive activation of the target GTPases (1), which in turn results in downstream effects on the mammalian cell phenotype and cell cycle. The typical phenotype associated with CNF1 intoxication in cell cultures is multinucleation, as has been reported previously for HEp-2 cells (14, 15), HeLa cells (13), and Swiss 3T3 cells (23). In addition, this toxin can be cytotoxic to cell lines such as 5637 human bladder cells (25). CNF1-intoxicated cells may also display formation of stress fibers, focal adhesions, lamellipodia, and filopodia and rearrangement of the actin cytoskeleton (16,20,30). In vivo, CNF1 evokes necrosis when it is injected intradermally into rabbits (9). The contribution of CNF1 to uropathogenic E. coli virulence has also been demonstrated using a rat model of acute prostatitis (28), as well as a mouse model of ascending urinary tract infection (11,29,33). In the latter model, intraurethral infection with a CNF1-positive strain leads to an increase...