Bacteria-Induced Uroplakin Signaling Mediates Bladder Response to Infection

Urinary tract infections are the second most common infectious disease in humans and are predominantly caused by uropathogenic E. coli (UPEC). A majority of UPEC isolates express the type 1 pilus adhesin, FimH, and cell culture and murine studies demonstrate that FimH is involved in invasion and apoptosis of urothelial cells. FimH initiates bladder pathology by binding to the uroplakin receptor complex, but the subsequent events mediating pathogenesis have not been fully characterized. We report a hitherto undiscovered signaling role for the UPIIIa protein, the only major uroplakin with a potential cytoplasmic signaling domain, in bacterial invasion and apoptosis. In response to FimH adhesin binding, the UPIIIa cytoplasmic tail undergoes phosphorylation on a specific threonine residue by casein kinase II, followed by an elevation of intracellular calcium. Pharmacological inhibition of these signaling events abrogates bacterial invasion and urothelial apoptosis in vitro and in vivo. Our studies suggest that bacteria-induced UPIIIa signaling is a critical mediator of bladder responses to insult by uropathogenic E. coli

Characterization of Mannheimia (Pasteurella) haemolytica leukotoxin interaction with bovine alveolar macrophage β\beta2 integrins

Mannheimia (Pasteurella) haemolytica, the etiologic agent of bovine pneumonic mannheimiosis, produces an exotoxic leukotoxin. The leukotoxin (LktA) is a member of the RTX (repeats in toxin) family of bacterial cytolysins and is distinguished from other toxins by its unique target cell specificity to ruminant leukocytes occurring through binding to a specific receptor. We have demonstrated previously that the $\beta$2 integrin LFA-1 is a receptor for LktA in bovine leukocytes and is involved in leukotoxin-induced biological effects. However the subunits within LFA-1 involved in binding to LktA, and post-binding signaling leading to cellular activation have not been well characterized. The purpose of our study was to pinpoint these precise subunits on bovine alveolar macrophages and to characterize their interaction with LktA. The results in this study indicate that although LktA can efficiently bind to the CD18 subunit of both LFA-1 and Mac-1, post-binding signaling events including elevation of intracellular calcium and CD18 tail phosphorylation are only observed through LFA-1. Furthermore, LktA also binds to the CD11a subunit of LFA-1. LktA binding to CD11a could be inhibited by a small molecule inhibitor of the I(inserted)-domain, the major ligand binding interface on CD11a. I-domain inhibition significantly blunts LktA-induced intracellular calcium elevation and tyrosine phosphorylation of the CD18 tail. Based on our results we suggest that LFA-1 serves as the functional leukotoxin receptor on bovine alveolar macrophages

Uropathogenic Escherichia coli Induces Extrinsic and Intrinsic Cascades To Initiate Urothelial Apoptosis

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