Integrin-Mediated Host Cell Invasion by Type 1–Piliated Uropathogenic Escherichia coli

Abstract

Uropathogenic Escherichia coli (UPEC), the primary causative agent of urinary tract infections, typically express filamentous adhesive organelles called type 1 pili that mediate both bacterial attachment to and invasion of bladder urothelial cells. Several host proteins have previously been identified as receptors for type 1 pili, but none have been conclusively shown to promote UPEC entry into host bladder cells. Using overlay assays with FimH, the purified type 1 pilus adhesin, and mass spectroscopy, we have identified β1 and α3 integrins as key host receptors for UPEC. FimH recognizes N-linked oligosaccharides on these receptors, which are expressed throughout the urothelium. In a bladder cell culture system, β1 and α3 integrin receptors co-localize with invading type 1–piliated bacteria and F-actin. FimH-mediated bacterial invasion of host bladder cells is inhibited by β1 and α3 integrin–specific antibodies and by disruption of the β1 integrin gene in the GD25 fibroblast cell line. Phosphorylation site mutations within the cytoplasmic tail of β1 integrin that alter integrin signaling also variably affect UPEC entry into host cells, by either attenuating or boosting invasion frequencies. Furthermore, focal adhesion and Src family kinases, which propagate integrin-linked signaling and downstream cytoskeletal rearrangements, are shown to be required for FimH-dependent bacterial invasion of target host cells. Cumulatively, these results indicate that β1 and α3 integrins are functionally important receptors for type 1 pili–expressing bacteria within the urinary tract and possibly at other sites within the host.

Uropathogenic Escherichia coli Invades Host Cells via an HDAC6-modulated Microtubule-dependent Pathway

“…Human bladder epithelial cells (ATCC 5637 cells) were incubated with increasing concentrations of each drug for 60 min prior to infection with the well-characterized type 1-piliated human cystitis isolate UTI89 (9,27). Host cell invasion by UTI89 requires expression of type 1 pili, and specifically the FimH adhesin (2,18). As determined by using standard gentamicin protection-based invasion assays, all three drugs were found to inhibit UTI89 invasion in a dose-dependent fashion (Fig.…”

“…Protein Analysis-Western blots were performed and visualized using either enhanced chemiluminescence or an Odyssey Infrared Imaging System (LI-COR Biosciences) as previously described (18). Primary antibodies used include rabbit anti-HA (Bethyl Laboratories), rabbit anti-␣-tubulin (Rockland), mouse anti-acetylated tubulin (clone 6-11B-1), and anti-FLAG M2 (Sigma-Aldrich), rabbit anti-cortactin (H222), anti-Aurora A (1G4), and anti-GEF-H1 (55B6, Cell Signaling), rabbit anti-HDAC6 (Santa Cruz Biotechnology), mouse anti-pan-actin, and anti-␤-actin antibodies (Abcam).…”

“…Bacterial Invasion and Cell Association Assays-Gentamicin protection bacterial invasion and cell association assays were performed as described (18). 5637 cells were seeded into 24-well tissue culture plates and grown for 18 -24 h to confluency.…”

“…The FimH adhesin binds mannose-containing glycoprotein receptors and is sufficient to stimulate bacterial uptake by bladder epithelial cells (2). Interactions between FimH and host receptors like ␣3␤1 integrin activate a signaling cascade involving focal adhesion, Src, and phosphoinositide 3-kinases, as well as Rho-family GTPases and the actin bundling and adaptor proteins ␣-actinin and vinculin (2,17,18). The coordinated activation of these host factors drives localized rearrangements of the actin cytoskeleton, leading to the envelopment and internalization of bound UPEC.…”

Comprehensive mutagenesis of thefimSpromoter regulatory switch reveals novel regulation of type 1 pili in uropathogenicEscherichia coli

“…They are caused mainly by uropathogenic Escherichia coli (UPEC), which rely on several virulence factors to cause disease (2); type 1 pili (T1P) are perhaps the most important of these (3). T1P present a tip adhesin, FimH, that binds specifically to mannose, which is found on several glycosylated surface proteins, such as uroplakins and α1-and β3-integrins (4,5), in the mammalian bladder. Binding to mannosylated proteins initiates a cascade of events including UPEC invasion of host tissues (2), formation of intracellular structures (6), and activation of the host immune response (7).…”