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, 74 (9), 5106-13

Uropathogenic Escherichia Coli Induces Extrinsic and Intrinsic Cascades to Initiate Urothelial Apoptosis

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Uropathogenic Escherichia Coli Induces Extrinsic and Intrinsic Cascades to Initiate Urothelial Apoptosis

David J Klumpp et al. Infect Immun.

Abstract

A murine model of urinary tract infection identified urothelial apoptosis as a key event in the pathogenesis mediated by uropathogenic Escherichia coli (UPEC), yet the mechanism of this important host response is not well characterized. We employed a culture model of UPEC-urothelium interactions to examine the biochemical events associated with urothelial apoptosis induced by the UPEC strain NU14. NU14 induced DNA cleavage within 5 h that was inhibited by the broad caspase inhibitor ZVAD, and urothelial caspase 3 activity was induced within 3 h of exposure to type 1 piliated NU14 and was dependent upon interactions mediated by the type 1 pilus adhesin FimH. Flow cytometry experiments using chloromethyl-X-rosamine and Indo-1 revealed FimH-dependent mitochondrial membrane depolarization and elevated [Ca(2+)](in), respectively, indicating activation of the intrinsic apoptotic pathway. Consistent with this possibility, overexpression of Bcl(XL) inhibited NU14 activation of caspase 3. Immunoblotting, caspase inhibitors, and caspase activity assays implicated both caspase 2 and caspase 8 in apoptosis, suggesting the involvement of the intrinsic and extrinsic apoptotic cascades. To reconcile the apparent activation of both extrinsic and intrinsic pathways, we examined Bid-green fluorescent protein localization and observed translocation from the cytosol to mitochondria in response to either NU14 or purified FimH. These data suggest that FimH acts as a tethered toxin of UPEC that activates caspase-dependent urothelial apoptosis via direct induction of the extrinsic pathway and that the intrinsic pathway is activated indirectly as a result of coupling by caspase 8-mediated Bid cleavage.

Figures

FIG. 1.
FIG. 1.
Strain NU14 induces caspase-dependent urothelial apoptosis. TEU-1 cells were infected with strain NU14 at an initial MOI of 250 in the presence or absence of the broad caspase inhibitor ZVAD-FMK (ZVAD) for 5 h. Following incubation, apoptotic cells were identified by fluorescent TUNEL assay for DNA degradation. Strain NU14 induced DNA degradation in urothelial cells (lower left panel), but similar DNA degradation was blocked in cells infected with strain NU14 in the presence of ZVAD-FMK (lower right panel). Magnification, ×40. Data are representative of triplicate experiments.
FIG. 2.
FIG. 2.
Strain NU14 induces FimH-dependent caspase 3 activity. (A) TEU-1 cells were infected with strain NU14, with the ΔfimH mutant NU14-1, or with NU14 in the presence of 25 mM methyl α-d-mannopyranoside (Mann). All infections were performed at an initial MOI of 250, and urothelial caspase 3 enzymatic activity levels were then determined for cell extracts by cleavage of the fluorogenic substrate Ac-DEVD-AFC and compared with the activity level for extracts of untreated cultures (−). (B) The caspase 3 activity level was also determined in a similar experiment using cultures of SR22A bladder urothelial cells. Caspase 3 activity was induced in both TEU-1 and SR22A cultures by strain NU14 but was blocked by the competitive inhibitor methyl α-d-mannopyranoside or a mutation in the gene encoding FimH. Error represents the standard deviation for triplicate infections.
FIG. 3.
FIG. 3.
Strain NU14 induces FimH-dependent mitochondrial membrane depolarization and increased cytosolic calcium. TEU-2 cultures were infected with strain NU14 or strain NU14-1 at an initial MOI of 250 for 5 h and then analyzed by flow cytometry for mitochondrial membrane potential (ΔΨmito; left panels) or cytosolic calcium ([Ca2+]in; right panels) by use of CMXRos and Indo-1, respectively. Control cultures (upper panels; −CTL) had high mitochondrial membrane potential and low cytosolic calcium, but NU14-treated cultures (middle panels) exhibited decreased mitochondrial membrane potential and increased cytosolic calcium. Cultures infected with strain NU14-1 (lower panels) were largely unaffected. Vertical dashed lines indicate the population mode of control cultures and facilitate comparison of panels within the columns. Data are representative of triplicate experiments.
FIG. 4.
FIG. 4.
Strain NU14 induces cytochrome c release, and caspase 3 activity is blocked by BclXL. (A) TEU-1 cultures were infected with strain NU14 at an initial MOI of 250 for 2.5 h in the presence or absence of 25 mM methyl α-d-mannopyranoside (Mann). S-100 extracts were prepared, and supernatants were analyzed for cytosolic cytochrome c by ELISA. Strain NU14 induced significant accumulation of cytosolic cytochrome c (P < 0.01) that was blocked by methyl α-d-mannopyranoside (P < 0.01 relative to NU14 treatment alone) and not observed for untreated cultures. Immunoblotting detected VDAC protein in S-100 pellets (P) but not in S-100 soluble fractions (S). Data are representative of duplicate experiments. (B) TEU-1 cultures were infected with adenoviruses encoding luciferase (AdLuc) or BclXL (AdBclXL). The following day, cultures were infected with strain NU14, and then caspase 3 activity was quantified by DEVD-AFC cleavage. Strain NU14 induced caspase 3 activity that was not induced by the adenoviruses. Adenovirus encoding BclXL inhibited NU14-induced caspase 3 activity, whereas adenovirus encoding luciferase did not. Data are representative of triplicate experiments.
FIG. 5.
FIG. 5.
Strain NU14 induces caspase 2 and caspase 8. (A) TEU-1 cultures were infected with strain NU14 at an initial MOI of 250 in the presence of specific inhibitors of caspase 2 and caspase 8 (iC-2 [z-VDVAD-FMK] and iC-8 [z-IETD-FMK], respectively). NU14-induced caspase 3 activation was largely blocked in cultures treated with the caspase 2 or caspase 8 inhibitors. (B) TEU-1 cultures were infected with strain NU14 for 1.5 h and assayed for caspase 2 (C-2) activity or caspase 8 (C-8) activity by the cleavage of the fluorogenic caspase 2 substrate Ac-VDVAD-AFC or the caspase 8 substrate Ac-IETD-AFC, respectively. NU14 induced both caspase 2 and 8 activities. (C) TEU-1 cultures were infected with strain NU14 at an initial MOI of 250. After infection for 0 to 3.0 h, whole-cell extracts were prepared and analyzed for accumulation of cleaved caspases by immunoblotting (30 μg/lane). Blotted membranes were then stripped and reprobed with an antibody specific for GAPDH as a loading control. Cleaved caspases were most abundant at 1.5 h and at 1.0 to 2.0 h (caspases 8 and 2, respectively).
FIG. 6.
FIG. 6.
Strain NU14 induces Bid translocation to mitochondria. TEU-1 cultures (A, C, and E) and SR22A cultures were transfected with BD4EGFP-Bid. After 24 h, cultures were infected with strain NU14 at an initial MOI of 500, and GFP-Bid localization was monitored via epifluorescence at 0 min (A and B), 90 min (C and D), and 180 min (E and F). Bid underwent a shift in localization within from diffuse in untreated cells (A and B) to concentrated at perinuclear sites (arrows in C to F). (G) SR22A cells were stained with MitoTracker after 180 min of exposure to NU14. Mitochondria were localized to perinuclear sites where Bid-GFP was concentrated (arrows). BD4EGFP-Bid-transfected TEU-1 cells were also exposed to 10 μg/ml FimC · H, and translocation was evident at 90 min (I) compared with the diffuse fluorescence of resting cells at 0 min (H). Scale bars, 15 μm (shown in panel A for all panels except panel G) and 10 μm (panel G).

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