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 beta1 and alpha3 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, beta1 and alpha3 integrin receptors co-localize with invading type 1-piliated bacteria and F-actin. FimH-mediated bacterial invasion of host bladder cells is inhibited by beta1 and alpha3 integrin-specific antibodies and by disruption of the beta1 integrin gene in the GD25 fibroblast cell line. Phosphorylation site mutations within the cytoplasmic tail of beta1 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 beta1 and alpha3 integrins are functionally important receptors for type 1 pili-expressing bacteria within the urinary tract and possibly at other sites within the host.

Figure 1. Identification of Potential FimH Receptors

Membrane-associated proteins isolated from 5637 bladder epithelial cells were resolved by SDS-PAGE and transferred to PVDF membrane. Protein bands bound by purified recombinant FimC_6XHisFLAG alone (left) or FimC_6XHisFLAG–FimH complexes in the presence (middle) and absence (right) of 2.5% D-mannose were detected by chemiluminescence in overlay assays using anti-FLAG antibody. The two most prominent bands bound by the FimC_6XHisFLAG–FimH complexes were found by mass spectroscopy analysis to be comprised of α2, β1, α3, α6, and β4 integrins. Molecular weight standards are indicated on the left.

Figure 2. Anti–β1 and Anti–α3 Integrin Antibodies Inhibit Host Cell Invasion by Type 1–Piliated E. coli

5637 bladder epithelial cell monolayers were treated with 1.5 μg/well of the indicated monoclonal antibodies for 30 min prior to infection with the UPEC cystitis isolate UTI89 or AAEC185/pSH2, a type 1 pili–expressing recombinant K-12 strain. Shown are (A and C) intracellular bacterial titers, determined using gentamicin protection assays, and (B and D) total cell-associated bacteria (including both intra- and extracellular bacteria). Data are expressed as the means ± standard error of the mean of at least three independent experiments carried out in triplicate. * p < 0.001, versus untreated control (Ctrl) values, as determined by Student's t-test.

Figure 3. Localization of β1 and α3 Integrins with Type 1 Pili–Expressing E. coli

5637 bladder cells were infected for 30 min with (A–D) UTI89 or (E–G) AAEC185/pSH2 prior to fixation and processing for immunofluorescent confocal microscopy. Samples were stained using antibodies specific for the following individual integrin subunits: (A and E) β1, (B and F) α3, or (C, D and G) heterodimeric α3β1 integrin complexes (green in the merged color images). F-actin (red) was visualized using Alexa568-conjugated phalloidin, while bacteria (blue) were detected using anti–E. coli antibody. For clarity, the merged color image in each row is accompanied by images showing only corresponding single channel signals from integrin or F-actin staining. Arrowheads denote the location of an individual bacterium in each set of images. Based on optical sectioning, the highlighted bacteria in (A–C) and (E–G) were localized at or very near the host cell surface, while the bacteria in (D) have already completely penetrated the target host cell. Scale bar = 10 μm.

Figure 4. Glycosidase Treatment Abrogates In Vitro Binding of FimH to β1 and α3 Integrins

(A) β1 and (B) α3 integrins were immunoprecipitated from 5637 cells that had been transiently transfected with plasmids for overexpression of recombinant human β1 or α3 integrins. The immunoprecipitated proteins were treated ± glycosidases (endoglycosidase Hf [EndoHf] or Peptide: N-glycosidase F [PNGase]) prior to SDS-PAGE and transfer to PVDF membranes. Blots were probed with either (A) anti–β1 or (B) anti–α3 integrin antibodies (Westerns), revealing expected shifts in the electrophoretic mobility of both integrin subunits following glycosidase treatments. For each set of samples, duplicate blots were overlaid with purified recombinant FimC_6XHisFLAG–FimH complexes and probed with anti-FLAG tag antibody (Far Westerns). As additional controls, blots containing untreated β1 or α3 integrins were also incubated with either FimC_6XHisFLAG–FimH complexes plus 2.5% D-mannose or with FimC_6XHisFLAG alone. Molecular weight standards are indicated on the left.

Figure 5. β1 Integrin Cytoplasmic Tail Mutants Differentially Affect FimH-Mediated Bacterial Invasion of Host Cells

β1 integrin–null GD25 cells, as well as GD25 derivatives that constitutively express wild-type or the indicated mutant forms of β1 integrin, were infected with (A and B) UTI89 or (C and D) AAEC185/pSH2. Levels of intracellular bacteria (A and C) were normalized among the different host cell lines by dividing the numbers of intracellular, gentamicin-protected bacteria by the number of total cell-associated bacteria (B and D). Data are expressed relative to results from GD25-β1A control cells and represent the means ± standard error of the mean of at least five independent experiments performed in triplicate. * p < 0.001, versus values from control GD25-β1A cells, as determined by Student's t-test.

Figure 6. FimH-Mediated Bacterial Invasion of Host Cells Requires Src Family Kinases and FAK

(A and B) 5637 cells were treated with 20 μg/ml of the Src family kinase inhibitors PP1 or PP2 or with an inactive analog, PP3, for 1 h prior to infection with UTI89. Levels of intracellular bacteria as determined by gentamicin protection assays (A) or total cell-associated bacteria (B) are expressed relative to results from cells treated with carrier (DMSO) alone. (C and D) Alternately, 5637 cells were transfected with either scrambled control siRNA or siRNA with specificity against FAK. Cells were infected with UTI89 at 72 h after siRNA transfection and intracellular (C) and total cell-associated (D) bacterial counts were determined. FAK knockdown was verified by Western blot analysis using antibody specific for total FAK ([C], inset). Blots were also probed using anti-actin antibody as a protein loading control. (E and F) Intracellular (E) and total cell-associated (F) bacteria were also quantified in assays using FAK−/− and control FAK+/+ mouse embryo fibroblasts. Intracellular bacterial levels were normalized in all assays by dividing the numbers of intracellular, gentamicin-protected bacteria by the number of total cell-associated bacteria. Data are expressed relative to results from the indicated control samples and represent the means ± standard error of the mean of three independent experiments performed in triplicate. * p < 0.001, versus values from control samples, as determined by Student's t-test.