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First published online 26 April 2005
doi: 10.1242/jcs.02328

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Cellular invasion by Staphylococcus aureus reveals a functional link between focal adhesion kinase and cortactin in integrin-mediated internalisation

¹ Zentrum für Infektionsforschung, Universität Würzburg, Röntgenring 11, 97070 Würzburg, Germany
² Institut für Molekulare Infektionsbiologie, Universität Würzburg, Röntgenring 11, 97070 Würzburg, Germany
³ Gesellschaft für Biotechnologische Forschung, Mascheroder Weg 1, 38124 Braunschweig, Germany

View larger version (59K): [in a new window]   Fig. 1. S. aureus internalisation requires host cell actin dynamics. (A) 293 cells were infected with S. aureus or S. carnosus at a MOI 20 for 2 hours in the presence of the indicated concentrations of latrunculin, cytochalasin D, or jasplakinolide. Living intracellular bacteria were counted by gentamicin/lysostaphin protection assays. Values are means±s.d. of three independent experiments done in triplicate. (B) 293 cells were transfected with GFP-actin and infected with rhodamine-labelled S. aureus. Using time-lapse microscopy, the dynamics of GFP-actin in the transfected 293 cells 30 minutes after infection were recorded. Invasive bacteria induced transient local accumulation of polymerised actin (arrows). In some cases, massive actin-filled membrane protrusions wrapped around invasive bacteria (arrowheads). Elapsed time, in seconds, from the beginning of the recording is indicated.

View larger version (39K): [in a new window]   Fig. 2. Accumulation of focal contact marker proteins in the vicinity of attached S. aureus. (A) 293 cells were transfected with GFP-tensin and infected for 1 hour with rhodamine-labelled S. aureus or S. carnosus at a MOI 20. After fixation, samples were examined by confocal microscopy. Cell-associated S. aureus (arrows) or S. carnosus (arrowheads) are indicated. Bars, 10 µm. (B) Local recruitment of GFP-tensin (green line) to cell-associated bacteria (red line) was quantified by plotting the fluorescence intensity as detected in the GFP or rhodamine channels, respectively, against the distance. (C) 293 cells were transfected with GFP-zyxin, infected and processed as in A. Bars, 10µm.

View larger version (26K): [in a new window]   Fig. 3. Interference with FAK function blocks uptake of pathogenic S. aureus. (A) 293 cells were transfected with HA-FAK K454M, HA-FAK Y397F, HA-FAK Pro(–), or the empty expression vector (pcDNA). After 2 days, cells were infected with S. aureus for 2 hours at MOI of 20 and the number of internalised bacteria was determined by gentamicin/lysostaphin protection assays. Values are means±s.d. of three independent experiments done in triplicate. (Lower panel) Western blotting of whole cell lysates (WCL) with anti-HA-tag antibody shows the expression of HA-tagged FAK constructs. (B) 293 cells were transfected with GFP-FRNK or the empty expression vector (pcDNA) and used as in A. Values are means±s.d. of two independent experiments done in triplicate. (Lower panel) WCLs were probed with anti-GFP antibody.

View larger version (62K): [in a new window]   Fig. 4. FAK-deficient cells do not support S. aureus invasion. (A) Internalisation of S. aureus or S. carnosus by FAK-re-expressing fibroblasts was measured at the indicated times following infection by gentamicin/lysostaphin protection assays. Values are means±s.d. of two independent experiments done in triplicate. (B) FAK-re-expressing [FAK(+)] or FAK-deficient [FAK(–)] mouse fibroblasts were infected with S. aureus for 2 hours and employed in gentamicin/lysostaphin protection assays. Values are means±s.d. of three independent experiments done in triplicate (C) FAK(+) or FAK(–) fibroblasts were infected with FITC- and biotin-labelled S. aureus for 2 hours. After fixation, extracellular bacteria were detected by addition of streptavidin-Cy5. Extracellular bacteria (arrowheads) stain positive with both FITC and Cy5, whereas intracellular bacteria (arrows) are labelled with FITC only. Bars, 10 µm. (D) FAK(–) and FAK(+) cells were infected for 1 hour with S. aureus and analysed by scanning electron microscopy. Pseudocoloured images depict bacteria in red and the fibroblast surface in green.

View larger version (59K): [in a new window]   Fig. 5. Recruitment and phosphorylation of FAK accompanies S. aureus invasion. (A) FAK-deficient fibroblasts were transfected with GFP-FAK. Transfected cells were infected with rhodamine-labelled S. aureus at MOI 20 for 1 hour. The arrowheads point to accumulation of GFP-FAK in the vicinity of cell-attached S. aureus. Local recruitment of GFP-FAK (green line) to cell-associated bacteria (red line) was quantified by plotting the fluorescence intensity as detected in the GFP or rhodamine channels, respectively, against the distance. Bar, 10 µm. (B) FAK re-expressing fibroblasts were plated on poly-L-lysine, and either kept uninfected or infected at MOI 50 for 1 hour with S. aureus or S. carnosus, respectively. Samples were treated with pervanadate 10 minutes prior to lysis, and whole cell lysates (WCL) were analysed by western blotting with a monoclonal anti-phosphotyrosine (P-tyr) antibody (upper panel). Membranes were stripped and reprobed with anti-HA antibody (middle panel) or anti-cortactin antibody (lower panel). (C) FAK re-expressing cells were treated as in B and infected for the indicated times. WCLs were analysed by western blotting with anti-P-tyr antibody (upper panel) or anti-desmin antibody (middle panel). The same samples were immunoprecipitated (IP) with anti-cortactin antibodies and precipitates were analysed with anti-P-tyr antibody (lower panel).

View larger version (50K): [in a new window]   Fig. 6. Accumulation of phosphotyrosine-containing proteins and cortactin at the sites of S. aureus invasion. (A) FAK(+) or FAK(–) fibroblasts were infected with FITC-labelled S. aureus for 1 hour. After fixation and permeabilization, samples were stained with monoclonal anti-phosphotyrosine (P-tyr) antibodies. Whereas S. aureus induced massive accumulation of tyrosine-phosphorylated proteins in FAK(+) cells (arrows), no bacteria-associated tyrosine phosphorylation was evident in FAK(–) cells (arrowheads). (B) 293 cells were transfected with RFP-cortactin and infected for 1 hour with FITC-labelled staphylococci. In contrast to S. carnosus (arrowheads), S. aureus induced accumulation of cortactin (arrows). Bars, 10 µm. (C) Local recruitment of cortactin-RFP (green line) to cell-associated bacteria (red line) was quantified by plotting the fluorescence intensity along the indicated red lines as detected in the FITC- or RFP-channels, respectively, against the distance.

View larger version (41K): [in a new window]   Fig. 7. Interference with cortactin expression or function inhibits invasion of S. aureus. (A) 293 cells were transfected with siRNA directed against either cortactin (Cort) or firefly luciferase (Luc) as a control. 24 hours later, cells were employed in gentamicin/lysostaphin protection assays with S. aureus. Values are means±s.d. of two independent experiments done in triplicate. Western blotting of whole cell lysates (WCL) with anti-cortactin antibody demonstrates gene silencing of cortactin (upper panel). The same membrane was probed with anti-actin antibodies as a loading control (lower panel). (B) 293 cells were transfected with the empty control vector (pcDNA) or cortactin mutants interfering with Arp2/3 association (W22A) or dynamin binding (W525K). Transfected cells were employed in gentamicin/lysostaphin protection assays with S. aureus. Values are means±s.d. of three independent experiments done in triplicate. (Lower panel) Western blotting of whole cell lysates (WCL) of the transfected cells with anti-FLAG antibody demonstrates expression of cortactin mutants. (C) 293 cells were transfected with the empty control vector (pcDNA), cortactin mutated at tyrosine residue 421 (Y421F), or mutated at three C-terminal tyrosine residues (Y421,466,482F). Cells were infected with S. aureus for 2 hours and the number of internalised bacteria was determined in gentamicin/lysostaphin protection assays. Values are means±s.d. of three independent experiments done in triplicate. Whole cell lysates (WCL) probed with anti-FLAG antibody demonstrate the expression of transfected cortactin constructs (lower panel).

View larger version (49K): [in a new window]   Fig. 8. FAK and Src cooperate in integrin-initiated tyrosine phosphorylation of cortactin. (A) Serum-starved FAK(+) and FAK(–) cells were either kept in suspension (Sus) or seeded on fibronectin (Fn) or poly-L-lysine (PL)-coated dishes for 30 minutes. Samples were treated with pervanadate for 5 minutes before lysis. After cortactin immunoprecipitation (IP), samples were analysed by western blotting with monoclonal anti-phosphotyrosine antibody (P.Tyr; upper panel). Following stripping, the blot was re-probed with polyclonal anti-cortactin antibodies (lower panel). The graph shows the ratio between phosphorylated cortactin and the total amount of cortactin present in the immunoprecipitates. For comparison, the ratio of FAK(–) cells plated onto poly-L-lysine was set to 1. (B) Serum-starved FAK(+) or FAK(–) cells were seeded onto poly-L-lysine-coated dishes and infected with S. aureus for 1 hours or left uninfected. Whole cell lysates (WCL) were analysed by western blotting and probed with monoclonal anti-phosphotyrosine antibody (P.Ttyr; upper panel). After stripping, membranes were re-probed with polyclonal anti-cortactin antibodies (lower panel). (C) Serum-starved FAK(+) cells were seeded onto poly-L-lysine-coated dishes and either left uninfected or infected with S. aureus for 1 hour in the presence or absence of 5 µM PP2. After cortactin immunoprecipitation (IP), samples were analysed by western blotting with monoclonal anti-phosphotyrosine antibody (P.Tyr; upper panel). Following stripping, the blot was re-probed with polyclonal anti-cortactin antibodies (lower panel).