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First published online December 31, 2008
doi: 10.1242/10.1242/jcs.035600

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Integrin-linked kinase is required for vitronectin-mediated internalization of Streptococcus pneumoniae by host cells

Simone Bergmann^1,2, Anke Lang², Manfred Rohde³, Vaibhav Agarwal^1,*, Claudia Rennemeier^2,, Carsten Grashoff^4,, Klaus T. Preissner⁵ and Sven Hammerschmidt^1,2,¶,**

¹ Max von Pettenkofer-Institute for Hygiene and Medical Microbiology, Ludwig-Maximilians University, Pettenkoferstrasse 9a, 80336 München, Germany
² University of Würzburg, Research Center for Infectious Diseases, Röntgenring 11, 97070 Würzburg, Germany
³ Department of Microbial Pathogenesis, Helmholtz Center for Infection Research, Inhoffenstrasse 7, D-38124 Braunschweig, Germany
⁴ Department of Molecular Medicine, Max Planck Institute of Biochemistry, 82152 Martinsried, Germany
⁵ Institute for Biochemistry, Justus-Liebig-University, Friedrichstrasse 24, 35392 Giessen, Germany

View larger version (27K): [in this window] [in a new window]   Fig. 1. Species-unspecific recruitment of vitronectin by Streptococcus pneumoniae. (A) S. pneumoniae (NCTC10319) binding of vitronectin derived from human plasma was detected by immunoblot analysis using a polyclonal anti-vitronectin antibody. Enolase protein of pneumococci was used as a loading control and detected with an anti-enolase IgG (Bergmann et al., 2003). Human plasma vitronectin was detected as 65- and 75-kDa protein bands. Incubation of bacteria with PBS instead of plasma was used as negative control (PBS). (B) S. pneumoniae (NCTC10319) recruitment of human or mouse plasma-derived vitronectin was monitored by flow cytometry analysis after incubation of pneumococci with various concentrations of plasma. Dose-dependent and species-unspecific recruitment of plasma-derived vitronectin by pneumococci was monitored by using monoclonal antibody VN7. Results are presented as geometric mean fluorescence intensity (GMFI) x percentage of gated events. (C) Documentation of purified native and multimeric (multi) human vitronectin by non-denaturating PAGE. (D) Recruitment of purified human vitronectin (VN) isoforms by S. pneumoniae was analyzed by flow cytometry after incubation of bacteria (NCTC10319: Cps+; serotype 35A) with indicated amounts of multimeric or native vitronectin. Binding data are presented as GMFI x percentage of gated events. (E) Vitronectin that is bound to bacteria is shown as dot plots of a representative flow cytometric analysis. The control dot plot shows the GMFI in the absence of vitronectin but after incubation of the sample with the antibodies. (F) The influence of pneumococcal encapsulation on the binding of purified multimeric vitronectin was analyzed by flow cytometry, and results are expressed as GMFI x percentage of gated events (means ± s.d. of at least three independent experiments, each done in triplicate; *P<0.05).

View larger version (42K): [in this window] [in a new window]   Fig. 2. Host-cell-associated multimeric vitronectin promotes adherence to and invasion of pneumococci into host tissue cells. (A) Pneumococcal (NCTC10319) adherence (at 4 hours) was investigated by immunofluorescence staining after pre-incubation of Detroit 562 cells with 3.0 µg native vitronectin or 3.0 µg of commercially purchased (com) or 3.0 µg self-generated (gen) multimeric vitronectin. The number of attached bacteria per cell in the absence of vitronectin (none) was used as control. Results represent the mean ± s.d. of at least three independent experiments, each done in duplicate. *P<0.005; n.s., non-significant. (B) Multimeric vitronectin promotes the adherence of pneumococci to Detroit 562 cells in a dose-dependent manner. (C) Adherence of pneumococci to the human epithelial cells Detroit 562 or A549, and the endothelial cell line HBMEC after 4 hours of infection was determined by immunofluorescence in the absence (none) or presence of 3.0 µg multimeric vitronectin (VN). Results represent the mean ± s.d. of at least three independent experiments, each done in duplicate. *P<0.05. (D) Immunofluorescence microscopy of pneumococci attached to host cells in the absence (none) or presence of multimeric vitronectin. (E) Invasion and intracellular survival of pneumococci in Detroit 562 epithelial cells and the endothelial cell line HBMEC in the absence (none) or presence of multimeric vitronectin (VN) was determined by the antibiotic protection assay. Results represent the mean ± s.d. of at least three independent experiments, each done in duplicate. *P<0.03.

View larger version (14K): [in this window] [in a new window]   Fig. 3. Influence of heparin on the recruitment of vitronectin and invasion into host cells. (A) The dose-dependent influence of heparin on the binding of multimeric vitronectin to pneumococci was analyzed by flow cytometry using a polyclonal anti-vitronectin antibody and FITC-labelled secondary antibody. Results are presented as the mean ± s.d. of at least three independent experiments. (B) The influence of heparin on the adherence of pneumococci to Detroit 562 cells in the absence (none) or presence of 3.0 µg multimeric vitronectin (VN) was determined by immunofluorescence microscopy. Results are presented as the mean ± s.d. of at least three independent experiments. *P<0.01. (C) Dose-dependent inhibition by heparin of vitronectin-mediated pneumococcal invasion into Detroit 562 cells was measured by the antibiotic protection assay, and the number of internalized bacteria was determined after 4 hours. Results are presented as mean ± s.d. of at least three independent experiments.

View larger version (20K): [in this window] [in a new window]   Fig. 4. Integrin-dependent vitronectin-mediated pneumococcal adherence to and invasion into host cells. (A) Vitronectin-mediated adherence of pneumococci to Detroit 562 cells in the absence (Ctrl) or presence of RGD-peptide (H-Gly-Arg-Gly-Asp-Asn-Pro-OH) was monitored after 4 hours of infection by immunofluorescence microscopy. Results are presented as the mean ± s.d. of at least three independent experiments. *P<0.01. (B) Vitronectin-mediated invasion of pneumococci into host cells was followed in the absence (Ctrl) or presence of soluble vβ3 integrin or 5β1 integrin as indicated after 4 hours using the antibiotic protection assay. Results are presented as the mean ± s.d. of at least three independent experiments. *P<0.05. (C) Influence of various blocking antibodies directed against host adhesion receptors on vitronectin-mediated internalization of pneumococci into host cells was investigated by antibiotic protection assay. Results are presented as the mean ± s.d. of at least three independent experiments. *P<0.05. none, infections performed in the absence of vitronectin; VN, infections performed in the presence of vitronectin.

View larger version (72K): [in this window] [in a new window]   Fig. 5. Vitronectin-mediated pneumococcal invasion requires the dynamics of the actin cytoskeleton. (A) Vitronectin-mediated infection of Detroit 562 cells (4 hours) with pneumococci was followed in the absence (none) or presence of inhibitors of the actin cytoskeleton, including cytochalasin D (Cyto D, 10 µM), latrunculin B (Lat B, 1 µM) and jasplakinolide (Jspk, 1 µM), by antibiotic protection assays. Results are presented as the mean ± s.d. of at least three independent experiments. *P<0.05. (B) Immunofluorescence microscopy of host-cell-attached and intracellular pneumococci. Host cells were pre-treated with vitronectin and infected with pneumococci. (Ba) Intracellular pneumococci were stained with Alexa Fluor 568 (red) and are indicated by arrows, whereas adherent bacteria appear yellow (green/red stain). Illustration of microspike-like structures (Bb,Bc) was achieved by phalloidin staining of the actin cytoskeleton (green). (Bd) Magnification of Bc, illustrating pneumococci attached to microspike-like structures on the cell surface. (C) Expression of the microspike-associated proteins frabin (upper row) and profilin (lower row) is induced upon vitronectin-mediated invasion of pneumococci. F-actin is stained with phalloidin (green), whereas profilin and frabin are stained with protein-specific antibodies and Alexa Fluor 568 (red). Boxed areas indicate frabin in microspikes; arrows indicate the association of profilin with microspike structures. (D) Scanning electron microscopy of Detroit 562 cells that were pre-treated with vitronectin (3.0 µg) and subsequently infected with pneumococci for 4 hours (a,c,e) or 6 hours (b,d,f). The inset in panel De shows pneumococci invading the host cell (arrow). (E) Scanning electron microscopy (top) and immunofluorescence microscopy (bottom) after staining the actin cytoskeleton of untreated Detroit 562 cells (a,d), Detroit 562 cells incubated with multimeric vitronectin (b,e) or host cells infected with pneumococci in the absence of vitronectin (c,f).

View larger version (63K): [in this window] [in a new window]   Fig. 6. Vitronectin–vβ3-integrin-mediated invasion of host cells by pneumococci depends on ILK expression. (A) Vitronectin-mediated invasion of pneumococci into ILK-expressing fibroblasts (fl/fl) or ILK–/– fibroblasts was determined by the antibiotic protection assay. Results are presented as the mean ± s.d. of at least three independent experiments. *P<0.004; n.s., non-significant. (B) Immunoblot analysis of ILK expression in ILK-expressing fibroblasts (fl/fl) or ILK-deficient cells (ILK–/–). (C) Vitronectin-mediated pneumococcal internalization into fibroblasts overexpressing ILK (ILK/ILK) or fibroblasts expressing the N-terminal ankyrin repeats (ILK-ANK). ILK-ANK cells lack the C-terminal kinase domain. Wild-type (Ctrl), ILKfl/fl (fl/fl) and ILK–/– fibroblasts were used as controls, and pneumococcal invasion was examined in the presence and absence of the Akt1/2 inhibitor VIII (Akti1/2, 10 µM). Pneumococcal invasion indicates the relative number of intracellular bacteria compared with non-treated fibroblasts (Ctrl). Data are shown as mean ± s.d. of at least three independent experiments. (D) Vitronectin-dependent pneumococcal invasion of non-transfected A549 cells (mock) or cells transfected with the ILK-specific siRNA (ILK-H) or with a control siRNA (Ctrl) was performed 48 hours post-transfection. The number of intracellular and recovered pneumococci was monitored by the antibiotic protection assay. Results are presented as the mean ± s.d. of at least three independent experiments. *P<0.004. (E) Reduction of ILK expression was controlled by immunoblot analysis of transfected cell lysates with specific anti-ILK antibodies to verify knock down of ILK by siRNA. Erk1/2 served as loading control. (F, top row) ILK ablation leads to an impaired F-actin cytoskeleton, as demonstrated by immunofluorescence microscopy of ILK-expressing A549 cells (mock) and ILK-knockdown A549 cells (siRNA ILK-H). F-actin is stained with phalloidin and the impact of ILK on the cytoskeleton is also shown for ILK-knockout fibroblasts (ILK–/–; bottom-right panel). The middle row shows A549 cells treated with vitronectin and infected with pneumococci. Bacteria were stained with Alexa Fluor 488 and Alexa Fluor 568, and appear yellow (green/red stain).

View larger version (24K): [in this window] [in a new window]   Fig. 7. PI3K- and Akt-dependent pneumococcal invasion via the vitronectin–vβ3-integrin pathway. (A) Vitronectin-dependent internalization of pneumococci into Detroit 562 cells in the absence (none) or presence of the PI3K inhibitors Wortmannin (WM, 50 nM) or LY294002 (LY, 50 nM), or the Akt1/2 inhibitor VIII (Akti1/2, 10 µM) was measured after 4 hours of infection by using the antibiotic protection assay. Results are presented as mean ± s.d. of three independent experiments. *P<0.05. (B) 48 hours prior to infection, A549 cells were transfected with ILK-specific siRNA (ILK-H) or control siRNA (Ctrl). The phosphorylation of Akt (pAkt) in A549 cells was analyzed after 30 minutes and 90 minutes of infection with pneumococci by western blot analysis in the absence or presence of host-cell-bound vitronectin (VN) and with or without bacteria (Sp), as indicated. Simultaneously, the protein Rac served as loading control.

View larger version (30K): [in this window] [in a new window]   Fig. 8. Schematic model of the vitronectin–vβ3-integrin-mediated invasion mechanism of S. pneumoniae and of the involved signalling pathway.

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