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, 19 (12), 2803-12

Type 1 Pilus-Mediated Bacterial Invasion of Bladder Epithelial Cells

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Type 1 Pilus-Mediated Bacterial Invasion of Bladder Epithelial Cells

J J Martinez et al. EMBO J.

Abstract

Most strains of uropathogenic Escherichia coli (UPEC) encode filamentous adhesive organelles called type 1 pili. We have determined that the type 1 pilus adhesin, FimH, mediates not only bacterial adherence, but also invasion of human bladder epithelial cells. In contrast, adherence mediated by another pilus adhesin, PapG, did not initiate bacterial internalization. FimH-mediated invasion required localized host actin reorganization, phosphoinositide 3-kinase (PI 3-kinase) activation and host protein tyrosine phosphorylation, but not activation of Src-family tyrosine kinases. Phosphorylation of focal adhesin kinase (FAK) at Tyr397 and the formation of complexes between FAK and PI 3-kinase and between alpha-actinin and vinculin were found to correlate with type 1 pilus-mediated bacterial invasion. Inhibitors that prevented bacterial invasion also blocked the formation of these complexes. Our results demonstrate that UPEC strains are not strictly extracellular pathogens and that the type 1 pilus adhesin FimH can directly trigger host cell signaling cascades that lead to bacterial internalization.

Figures

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Fig. 1. Invasion of bladder epithelial cells by type 1-piliated E.coli. 5637 bladder cells were examined by (A) SEM or (B and C) TEM 1–2 h after infection with NU14. Adherent bacteria were often observed being enveloped by bladder epithelial cells by SEM and could be detected within membrane-bound vacuoles by TEM. The scale bars indicate 1 µm.
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Fig. 2. Type 1 pili mediate bacterial invasion of bladder cells. (A) Gentamicin protection assays indicate that NU14 can invade 5637 cells, while the isogenic fimH mutant, NU14-1, was non-invasive. Addition of 2.5% d-mannose to the cell culture medium inhibited NU14 invasion. The plasmid pHJ20 (encoding FimH), but not pHJ19 (encoding FimH in the incorrect orientation), complements the invasion defect of NU14-1. (B) The non-invasive K-12 strain AAEC185 (type 1), when transformed with the plasmid pSH2 (type 1+, FimH+), could also invade 5637 cells. AAEC185 strains transformed with pUT2002 (type 1+, FimH) or pPIL110-35 (P pili+, with or without the PapG adhesin) were non-invasive. (C) Adherence assays indicate that AAEC185 expressing either wild-type type 1 pili (fimH+) or wild-type P pili (papG+) are able to adhere well to 5637 cells (46.2 ± 5.9% versus 16.5 ± 0.6%, respectively) in comparison with strains expressing type 1 or P pili lacking the FimH and PapG adhesins, respectively. (D) Calculated invasion indices (c.f.u. invaded/c.f.u. adherent) demonstrate that AAEC185 type 1 pili (FimH+) are ∼98% more efficient at mediating bacterial invasion than bacteria expressing P pili (PapG+). All data are representative of three or more independent assays, each performed in triplicate. Between experiments, the invasion frequencies of the type 1-piliated E.coli strains varied from 0.5 to 10%. This variability appears to be related to the passage number and status of the host cells and to the degree of bacterial piliation on the day of the experiment.
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Fig. 3. Internalization of FimCH-coated beads. 5637 cells were incubated with FimCH- or BSA-coated beads for 30–60 min and then processed for SEM (A–C) or TEM (D–G). (A and B) FimCH-coated beads were internalized efficiently by 5637 cells, while the few adherent BSA-coated beads (C) did not induce any appreciable alterations in the 5637 cell membrane. (DG) The host cell membrane appears to zipper around and eventually envelope FimCH-coated beads during the internalization process. Scale bars represent 5.0 µm (A and E), 0.5 µm (B–D) and 1.0 µm (F and G).
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Fig. 4. FimH is necessary and seemingly sufficient to mediate internalization. (A) 5637 cells, grown on sterile coverslips, were incubated with equal amounts of FimCH-, FimC- or BSA-coated beads ±2.5% d-mannose for 30–60 min. FimCH-coated beads were internalized in a d-mannose-inhibitable manner. In contrast, few control FimC- or BSA-coated beads were internalized and their uptake was not inhibited by d-mannose. Numbers of intracellular beads were determined as described in Materials and methods. Data are presented as an invasion index (total number of internalized beads divided by the total number of host cell-associated beads) and are representative of at least four independent assays. (B and C) Representative images of the internalization assay demonstrating the internalization of FimCH-coated beads into 5637 cells. (C) Fluorescent images (TRITC filter setting) were superimposed onto the corresponding phase contrast images such that extracellular beads appear red (arrow). The scale bar represents 5 µm.
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Fig. 5. Host cell actin polymerization plays an important role in the invasion process. (A) 5637 cells were pre-treated with cytochalasin D for 30 min prior to infection with AAEC185/pSH2. Gentamicin protection assays demonstrate that cytochalasin D (1 µg/ml) can completely abolish invasion. The effects of cytochalasin D are reversible if the host cells are washed with PBS prior to infection in fresh medium without drug. Similar results were obtained using NU14. (B) Cytochalasin D also inhibits the internalization of FimCH-coated beads.
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Fig. 6. Actin rearrangements leading to internalization of type 1-piliated E.coli. (A) Texas red–phalloidin staining of uninfected 5637 cells highlighting cortical actin (red). (B) Localized actin polymerization is induced at points of contact (arrows) with AAEC185/pSH2/pcomGFP (type 1+, GFP+). (C and D) Images from (B) (arrows) and (E) are enlarged to show detail. The scale bar represents 2 µm.
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Fig. 7. Inhibition of host protein tyrosine phosphorylation and PI 3-kinase activation diminishes invasion. (A) Gentamicin protection assays show that genistein prevents FimH-mediated bacterial internalization into 5637 cells in a concentration-dependent manner (>90% inhibition at 250 µM). (B) PP1, a Src-family tyrosine kinase inhibitor, had no effect on the internalization process. 5637 cells were treated with PP1 at concentrations that are known to inhibit Src-family kinase activity effectively (Gerwein et al., 1999). (C and D) Wortmannin and LY294002, two compounds that inhibit PI 3-kinase activity, can block FimH-mediated bacterial invasion of 5637 cells (∼90% inhibition at 100 nM and 50 µM, respectively). (E) Internalization of FimCH-coated beads into 5637 cells is also inhibited by wortmannin (∼80% inhibition at 100 nM).
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Fig. 8. Type 1-piliated E.coli induce complex formation between FAK and PI 3-kinase and between vinculin and α-actinin. (A) Shortly after infection of 5637 cells with AAEC185/pSH2 (FimH+), blots probed with a phospho-specific anti-FAK antibody show that FAK becomes phosphorylated at Y397. Infection with AAEC185/pUT2002 (FimH) did not induce phosphorylation of FAK Y397. (B) PI 3-kinase (the p85α subunit) co-precipitates with FAK within 15 min after infection with AAEC185/pSH2, but not after infection with AAEC185/pUT2002. (D) α-actinin, but not tensin or talin, transiently co-precipitates with vinculin beginning within 15 min after infection with AAEC185/pSH2, but not after infection with AAEC185/pUT2002. Control lysates containing tensin, talin and α-actinin were used to ensure that the different antibodies were functional in these assays. (C and E) Inhibitors of type 1 pilus-mediated bacterial invasion block complex formation between FAK and PI 3-kinase and between vinculin and α-actinin. The Src-family kinase inhibitor, PP1, had no effect on complex formation.

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