Invasion of Endothelial Cells by Tissue-invasive M3 Type Group A Streptococci Requires Src Kinase and Activation of Rac1 by a Phosphatidylinositol 3-Kinase-independent Mechanism
2009; Elsevier BV; Volume: 284; Issue: 30 Linguagem: Inglês
10.1074/jbc.m109.016501
ISSN1083-351X
AutoresAndreas Nerlich, Manfred Rohde, Susanne R. Talay, Harald Genth, Ingo Just, Gursharan S. Chhatwal,
Tópico(s)Neonatal and Maternal Infections
ResumoStreptococcus pyogenes can cause invasive diseases in humans, such as sepsis or necrotizing fasciitis. Among the various M serotypes of group A streptococci (GAS), M3 GAS lacks the major epithelial invasins SfbI/PrtF1 and M1 protein but has a high potential to cause invasive disease. We examined the uptake of M3 GAS into human endothelial cells and identified host signaling factors required to initiate streptococcal uptake. Bacterial uptake is accompanied by local F-actin accumulation and formation of membrane protrusions at the entry site. We found that Src kinases and Rac1 but not phos pha tidyl ino si tol 3-kinases (PI3Ks) are essential to mediate S. pyogenes internalization. Pharmacological inhibition of Src activity reduced bacterial uptake and abolished the formation of membrane protrusions and actin accumulation in the vicinity of adherent streptococci. We found that Src kinases are activated in a time-de pend ent manner in response to M3 GAS. We also demonstrated that PI3K is dispensable for internalization of M3 streptococci and the formation of F-actin accumulations at the entry site. Furthermore, Rac1 was activated in infected cells and accumulated with F-actin in a PI3K-independent manner at bacterial entry sites. Genetic interference with Rac1 function inhibited streptococcal internalization, demonstrating an essential role of Rac1 for the uptake process of streptococci into endothelial cells. In addition, we demonstrated for the first time accumulation of the actin nucleation complex Arp2/3 at the entry port of invading M3 streptococci. Streptococcus pyogenes can cause invasive diseases in humans, such as sepsis or necrotizing fasciitis. Among the various M serotypes of group A streptococci (GAS), M3 GAS lacks the major epithelial invasins SfbI/PrtF1 and M1 protein but has a high potential to cause invasive disease. We examined the uptake of M3 GAS into human endothelial cells and identified host signaling factors required to initiate streptococcal uptake. Bacterial uptake is accompanied by local F-actin accumulation and formation of membrane protrusions at the entry site. We found that Src kinases and Rac1 but not phos pha tidyl ino si tol 3-kinases (PI3Ks) are essential to mediate S. pyogenes internalization. Pharmacological inhibition of Src activity reduced bacterial uptake and abolished the formation of membrane protrusions and actin accumulation in the vicinity of adherent streptococci. We found that Src kinases are activated in a time-de pend ent manner in response to M3 GAS. We also demonstrated that PI3K is dispensable for internalization of M3 streptococci and the formation of F-actin accumulations at the entry site. Furthermore, Rac1 was activated in infected cells and accumulated with F-actin in a PI3K-independent manner at bacterial entry sites. Genetic interference with Rac1 function inhibited streptococcal internalization, demonstrating an essential role of Rac1 for the uptake process of streptococci into endothelial cells. In addition, we demonstrated for the first time accumulation of the actin nucleation complex Arp2/3 at the entry port of invading M3 streptococci. Streptococcus pyogenes or group A streptococcus (GAS) 2The abbreviations used are: GASgroup A streptococciEGM2endothelial cell growth medium 2FBSfetal bovine serumGFPgreen fluorescence proteinHUVEChuman umbilical vein endothelial cell(s)GSTglutathione S-transferasePBSphosphate-buffered salinePTKsprotein tyrosine kinasesVEGFRvascular endothelial growth factor receptorPI3Kphosphatidylinositol 3-kinaseMES4-morpholineethanesulfonic acid. 2The abbreviations used are: GASgroup A streptococciEGM2endothelial cell growth medium 2FBSfetal bovine serumGFPgreen fluorescence proteinHUVEChuman umbilical vein endothelial cell(s)GSTglutathione S-transferasePBSphosphate-buffered salinePTKsprotein tyrosine kinasesVEGFRvascular endothelial growth factor receptorPI3Kphosphatidylinositol 3-kinaseMES4-morpholineethanesulfonic acid. is an important human pathogen that causes localized infections of the respiratory tract and the skin but also severe invasive disease, sepsis, and toxic shock-like syndrome. Group A streptococci, although traditionally viewed as extracellular pathogens, are able to adhere to and invade into several eukaryotic cell types (1LaPenta D. Rubens C. Chi E. Cleary P.P. Proc. Natl. Acad. Sci. U.S.A. 1994; 91: 12115-12119Crossref PubMed Scopus (230) Google Scholar, 2Greco R. De Martino L. Donnarumma G. Conte M.P. Seganti L. Valenti P. Res. Microbiol. 1995; 146: 551-560Crossref PubMed Scopus (82) Google Scholar, 3Cue D. Dombek P.E. Lam H. Cleary P.P. Infect. Immun. 1998; 66: 4593-4601Crossref PubMed Google Scholar, 4Molinari G. Talay S.R. Valentin-Weigand P. Rohde M. Chhatwal G.S. Infect. Immun. 1997; 65: 1357-1363Crossref PubMed Google Scholar, 5Cywes C. Wessels M.R. Nature. 2001; 414: 648-652Crossref PubMed Scopus (167) Google Scholar). group A streptococci endothelial cell growth medium 2 fetal bovine serum green fluorescence protein human umbilical vein endothelial cell(s) glutathione S-transferase phosphate-buffered saline protein tyrosine kinases vascular endothelial growth factor receptor phosphatidylinositol 3-kinase 4-morpholineethanesulfonic acid. group A streptococci endothelial cell growth medium 2 fetal bovine serum green fluorescence protein human umbilical vein endothelial cell(s) glutathione S-transferase phosphate-buffered saline protein tyrosine kinases vascular endothelial growth factor receptor phosphatidylinositol 3-kinase 4-morpholineethanesulfonic acid. Localized S. pyogenes infections may lead to dissemination of bacteria through the vascular system, resulting in bacteremia and sepsis. For evasion of the vascular system, S. pyogenes may directly interact with the endothelium, which lines the inner surface of blood vessels. M3 type streptococci are, besides the M1 and M28 strains, most commonly associated with invasive GAS infections (6Stevens D.L. Clin. Infect. Dis. 1992; 14: 2-11Crossref PubMed Scopus (626) Google Scholar) and have been shown to be internalized into human umbilical vein endothelial cells (HUVEC) in vitro (7Burns Jr., E.H. Lukomski S. Rurangirwa J. Podbielski A. Musser J.M. Microb. Pathog. 1998; 24: 333-339Crossref PubMed Scopus (34) Google Scholar). S. pyogenes can express several invasins, but only the signal transduction pathways of two streptococcal factors, SfbI/prtF1 and M1 protein, respectively, have been studied in more detail. Both invasins trigger bacterial uptake by binding to soluble fibronectin, which acts as a bridging molecule and induces the clustering of host integrins, which in turn activates host signaling pathways. In the case of M1-mediated internalization, activation of PI3K, ILK, paxillin, and focal adhesion kinase has been shown, which promotes actin polymerization-based zipper-like bacterial uptake into epithelial cells (8Purushothaman S.S. Wang B. Cleary P.P. Infect. Immun. 2003; 71: 5823-5830Crossref PubMed Scopus (38) Google Scholar, 9Wang B. Yurecko R.S. Dedhar S. Cleary P.P. Cell. Microbiol. 2006; 8: 257-266Crossref PubMed Scopus (60) Google Scholar, 10Wang B. Li S. Dedhar S. Cleary P.P. Cell. Microbiol. 2007; 9: 1519-1528Crossref PubMed Scopus (26) Google Scholar). In contrast to this, caveolae were shown to act as entry port for SfbI-expressing S. pyogenes (11Rohde M. Müller E. Chhatwal G.S. Talay S.R. Cell. Microbiol. 2003; 5: 323-342Crossref PubMed Scopus (95) Google Scholar), a mechanism distinct from the zipper-like uptake mechanism employed by strains expressing M1 protein (12Dombek P.E. Cue D. Sedgewick J. Lam H. Ruschkowski S. Finlay B.B. Cleary P.P. Mol. Microbiol. 1999; 31: 859-870Crossref PubMed Scopus (107) Google Scholar). SfbI/protein F1-expressing streptococci form a focal complex-like structure that consists of focal adhesion kinase, Src kinases, paxillin, and Rho GTPases, resulting in uptake of the bacteria (13Ozeri V. Rosenshine I. Ben-Ze'Ev A. Bokoch G.M. Jou T.S. Hanski E. Mol. Microbiol. 2001; 41: 561-573Crossref PubMed Scopus (79) Google Scholar). However, a requirement for PI3K activation, which in turn induced paxillin phosphorylation, was recently shown for M1-mediated as well as SfbI-mediated invasion (10Wang B. Li S. Dedhar S. Cleary P.P. Cell. Microbiol. 2007; 9: 1519-1528Crossref PubMed Scopus (26) Google Scholar). In contrast, M3 streptococci do not express these two well characterized invasins (14Vlaminckx B.J. Schuren F.H. Montijn R.C. Caspers M.P. Fluit A.C. Wannet W.J. Schouls L.M. Verhoef J. Jansen W.T. Infect. Immun. 2007; 75: 2603-2611Crossref PubMed Scopus (18) Google Scholar), the mechanism by which M3 streptococci are able to trigger entry into human endothelial cells is still poorly understood, and no information is currently available concerning host cell signaling factors involved in this process. In this study, we characterized the intracellular signals governing internalization of SfbI/prtF1/M1-negative M3 GAS into primary endothelial cells. We found an essential role for host cell protein-tyrosine kinases (PTKs) and identified Src family PTKs to play an essential role during the uptake process. In contrast to the already characterized receptor-mediated bacterial invasion strategies, which rely on PI3K activation, internalization of M3 GAS is PI3K-independent. In addition to Src family PTKs, the GTPase Rac1 was identified as an important factor for M3 S. pyogenes internalization. Rac1 was found to be activated in response to bacterial internalization, and genetic interference with Rac1 function significantly reduced uptake. Rac1 as well as the actin nucleation complex Arp2/3 was found to accumulate at streptococcal entry ports, strengthening the important role of this GTPase for uptake of M3 type streptococci into human endothelial cells. AG 957, c-Met kinase inhibitor II, genistein, LY294002, PP2, PP3, and VEGFR tyrosine kinase inhibitor IV were obtained from Calbiochem. Antibodies against Group A streptococci were developed in rabbits and protein A-purified. Polyclonal anti-phospho-Akt (Ser473) was obtained from Cell Signaling (Frankfurt, Germany). Polyclonal phospho-Src antibody Tyr(P)-418 (recognizing phosphorylated Tyr-419 in human c-Src) was from BIOSOURCE International (Nivelle, Belgium), and polyclonal antibodies against Src PTKs (SRC2) and Akt were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Monoclonal antibody against phosphotyrosine (clone 4G10) was purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). Monoclonal antibody against Rac1 (clone 102) was obtained from BD Biosciences (Heidelberg, Germany). Alexa® Fluor-conjugated secondary reagents and phalloidin were from Invitrogen/Molecular Probes (Leiden, The Netherlands). Cy5-conjugated goat anti-rabbit IgG was purchased from Millipore (Schwalbach/Ts, Germany). Escherichia coli HB101 harboring pGEX-6P-1, pGEX-2T-PAK-CRIB, pEGFP-wtRac1, and pEGFP-.dnRac1 were cultured in Luria-Bertani broth with antibiotic selection. The M3 serotype S. pyogenes strains A60 and A128 were grown in tryptic soy broth (BD Biosciences) at 37 °C. The green fluorescence protein (GFP) expression vector pAT18-GFP (kindly provided by I. Nakagawa) containing the recA promoter region (15Nakagawa I. Nakata M. Kawabata S. Hamada S. Cell. Microbiol. 2001; 3: 395-405Crossref PubMed Scopus (70) Google Scholar) was transformed into GAS strain A128 by electroporation as described (16McLaughlin R.E. Ferretti J.J. Methods Mol. Biol. 1995; 47: 185-193PubMed Google Scholar). HUVEC were obtained from PromoCell and cultured in endothelial cell growth medium 2 (EGM2; PromoCell, Heidelberg, Germany) containing 2% fetal bovine serum (FBS) at 37 °C, 5% CO2, and 90% humidity. For infection assays, cells were seeded onto gelatin-coated coverslips at a concentration of ∼1 × 105 cells/ml (500 μl/well) and gelatin-coated 6-cm diameter tissue culture dishes at a concentration of 2 × 106 cells/ml and grown to confluence, respectively. Wild-type Rac1 and dominant negative Rac1 were expressed as GFP fusions. HUVEC were transfected with the Amaxa nucleofection system (Amaxa, Cologne, Germany), applying the standard protocol as specified by the manufacturer. Plasmids were purified with the EndoFree® plasmid purification kit from Qiagen (Hilden, Germany). Two hours before adding the bacteria, the EGM2 medium with growth factors and antibiotics was replaced by antibiotic-free EGM2 medium containing 2% FBS. Bacteria were grown in tryptic soy broth, harvested by centrifugation, washed once with PBS, and resuspended in EGM2 with 2% FBS. Bacteria were added to the cells to give a multiplicity of infection of 1:20, plates were incubated for defined times, and wells were washed with PBS to remove non-adherent bacteria and fixed in appropriate buffers before microscopic analysis. For biochemical analysis, cells were serum-starved in EGM2 containing 0.1% FBS, and infections were synchronized using a modified protocol, as described previously (17Tran Van Nhieu G. Ben-Ze'ev A. Sansonetti P.J. EMBO J. 1997; 16: 2717-2729Crossref PubMed Scopus (170) Google Scholar). Briefly, bacteria were allowed to bind to confluent HUVEC grown in 6-cm diameter dishes for 10 min at 21 °C at a multiplicity of infection of 1:50 in EGM2. Following incubation, medium was removed, fresh medium prewarmed to 37 °C was added, and samples were shifted to 37 °C. This procedure gave a final multiplicity of infection of 1:15. The cells were incubated for the indicated times and processed for biochemical analysis. In inhibition experiments, pharmacological inhibitors were added to HUVEC for 15 min (PP2 and PP3), 30 min (AG 957, c-Met kinase inhibitor II, VEGFR tyrosine kinase inhibitor IV), 45 min (LY294002), or 60 min (genistein) prior to infection. For immunolabelings, cells were fixed with 3% paraformaldehyde in PBS for 15 min, quenched with 10 mm glycine in PBS, and permeabilized with 0.1% Triton X-100 in PBS. For phalloidin stainings, cells were fixed with a mixture of 3% paraformaldehyde and 0.2% Triton X-100 in modified cytoskeleton buffer (10 mm MES (pH 7.0), 150 mm NaCl, 5 mm EGTA, 5 mm MgCl2, 5 mm sucrose) for 20 min and quenched with 10 mm glycine in PBS. Double immunofluorescence staining of streptococci was done as described previously (11Rohde M. Müller E. Chhatwal G.S. Talay S.R. Cell. Microbiol. 2003; 5: 323-342Crossref PubMed Scopus (95) Google Scholar). For detecting tyrosine-phosphorylated proteins, permeabilized cells were blocked in PBS with 5% horse serum, 1% bovine serum albumin (blocking buffer) for 30 min at room temperature and subsequently incubated with monoclonal mouse anti-phosphotyrosine (1:200, clone 4G10) in blocking buffer for 45 min at room temperature. Samples were washed twice in PBS and incubated with Alexa® Fluor 568-conjugated goat anti-mouse IgG (Invitrogen) for 30 min at room temperature. Antibody dilutions were as follows. For Src kinase labeling, polyclonal rabbit IgG antibodies were 1:50 diluted (0.2 mg/ml stock solution); for Arp2/3 labeling, anti-p16 hybridoma supernatant (kindly provided by T. Stradal, Helmholtz-HZI, Braunschweig) was used undiluted. The bound primary antibodies were visualized with Alexa® Fluor 568-conjugated goat anti-mouse IgG (Invitrogen). F-actin was labeled with Alexa® Fluor 488-conjugated phalloidin and Alexa® Fluor 568-conjugated phalloidin (Invitrogen), respectively, for 20 min at room temperature. Coverslips were washed three times in PBS and then mounted using ProLong® Gold with 4′,6-diamidino-2-phenylindole (Invitrogen). Mounted samples were examined using a Bio-Rad MRC1024UV (Bio-Rad), as described previously (11Rohde M. Müller E. Chhatwal G.S. Talay S.R. Cell. Microbiol. 2003; 5: 323-342Crossref PubMed Scopus (95) Google Scholar), or a Zeiss LSM 510 Meta (Zeiss, Jena, Germany) confocal laser-scanning microscope equipped with a 100×, 1.3 numerical aperture Plan-NEOFLUAR objective (Zeiss). Epifluorescence images were acquired on an inverted microscope (Axiovert 200 M; Zeiss), which was equipped with a 100×, 1.3 numerical aperture Plan-NEOFLUAR objective (Zeiss) and an interline transfer, progressive scan CCD camera (CoolsnapHQ; Photometrics, Tucson, AZ) driven by Metamorph software (Molecular Devices Corp., Downingtown, PA) All images were deconvolved using Huygens® Essential (Hilversum, The Netherlands). Image stacks are represented as extended depth of field (18Forster B. Van De Ville D. Berent J. Sage D. Unser M. Microsc. Res. Tech. 2004; 65: 33-42Crossref PubMed Scopus (289) Google Scholar) or maximum intensity projection and were processed for contrast and brightness with ImageJ. Live cell imaging was performed at 37 °C in 5% CO2 with cells grown in μ-Slides (μ-Slide 8 well; Ibidi, Munich, Germany). Cells were infected with EGFP-expressing M3 GAS (A128) with a multiplicity of infection of 20, and samples were mounted in an incubation chamber (Zeiss) for temperature and CO2 control. Imaging was performed on an inverted microscope (Axiovert 200 M; Zeiss), which was equipped with a 100×, numerical aperture 1.3 Plan-NEOFLUAR objective (Zeiss) and an interline transfer, progressive scan CCD camera (CoolsnapHQ; Photometrics) driven by Metamorph software (Molecular Devices) attached to a MultiSpec-ImagerTM (Visitron Systems, Puchheim, Germany) for simultaneous imaging in the enhanced GFP and phase contrast channel. A 100-watt halogen lamp was used for illumination. Video frames were deconvolved using Huygens® Essential (Hilversum, The Netherlands) and processed for contrast and brightness using ImageJ. Following infection, cells were fixed with a solution containing 2% glutaraldehyde and 3% formaldehyde in cacodylate buffer for 1 h on ice and washed with cacodylate buffer. After washing several times in Tris/EDTA buffer, samples were dehydrated with a graded series of acetone (10, 30, 50, 70, 90, 100%) on ice, each step 15 min, followed by critical point drying with liquid CO2. Samples were sputter-coated with an ∼10-nm-thick gold film before examining in a Zeiss field emission scanning electron microscope DSM982 Gemini (Zeiss) at an acceleration voltage of 5 kV using the Everhart Thornley SE detector and the inlens-SE detector in a 50:50 ratio. Images were digitally recorded on MO-disks and processed for contrast and brightness using ImageJ. At the indicated times, infected HUVEC were washed once with PBS containing 1 mm MgCl2 and 1 mm CaCl2 and lysed in ice-cold modified radioimmune precipitation assay buffer (25 mm Hepes (pH 7.4), 1% Nonidet P-40, 0.25% sodium deoxycholate, 10% glycerol, 150 mm NaCl, 1 mm EDTA, 10 mm sodium pyrophosphate, 50 mm NaF, 2 mm NaVO3, 2 mm phenylmethylsulfonyl fluoride, Complete MiniTM (Roche Applied Science). Equivalent amounts of the cleared lysates were added to an equal volume of reducing 2× SDS sample buffer, and the proteins were separated by SDS-PAGE. Proteins were transferred to nitrocellulose and blocked (5% bovine serum albumin and 5% skim milk, respectively, in Tris-buffered saline containing 0.05% Tween (TBS/T)) for 1 h at room temperature. The membranes were incubated with a 1:1000 dilution of the polyclonal antibodies against Src kinase and phospho-Src kinase (Tyr(P)-419) overnight at 4 °C and visualized by enhanced chemiluminescent detection methods. For Rac activation assays, glutathione S-transferase (GST) fusion of PAK-CRIB was immobilized on glutathione-Sepharose 4B (Amersham Biosciences) in bacterial lysis buffer (10 mm Tris-HCl (pH 8.0), 20% sucrose, 10% glycerol, 2 mm MgCl2, 2 mm dithiothreitol, 2 mm phenylmethylsulfonyl fluoride. Lysates from infected HUVEC were prepared as follows. Infected cells were washed once with PBS containing 1 mm MgCl2 and 1 mm CaCl2 and lysed in 150 μl of ice-cold lysis buffer (50 mm Tris-HCl (pH 7.4), 1% Nonidet P-40, 10% glycerol, 100 mm NaCl, 2 mm MgCl2, 2 mm phenylmethylsulfonyl fluoride, Complete MiniTM (Roche Applied Science) at the indicated times. Samples were centrifuged for 10 min at 15,000 × g, and the supernatant was used for pull-down assay. 30 μl of GST-PAK-CRIB beads were added to each sample and rotated at 4 °C for 30 min. Beads were collected by centrifugation and washed twice with lysis buffer. To each sample, 20 μl of reducing SDS sample buffer were added, and a 15% SDS-PAGE with subsequent transfer of proteins onto nitrocellulose was performed. After blocking (5% skim milk in TBS/T) for 1 h at room temperature, the membranes were incubated with a 1:1000 dilution of monoclonal anti-Rac1 antibody overnight at 4 °C, and visualized by enhanced chemiluminescent detection methods. The intensity of the bands was quantified using ImageJ. Statistical comparisons were made using Student's t test and Tukey multiple comparison post hoc test, respectively, with R 2.5.0 software. Values are expressed as means ± S.E. for at least three independent experiments. S. pyogenes has evolved multiple mechanisms for invasion of a wide variety of mammalian cells, which show remarkable differences at the ultrastructural level. For M1 protein-expressing GAS, an increase of microvilli-like structures in the vicinity of adherent streptococci that appear to entrap the bacteria was shown (12Dombek P.E. Cue D. Sedgewick J. Lam H. Ruschkowski S. Finlay B.B. Cleary P.P. Mol. Microbiol. 1999; 31: 859-870Crossref PubMed Scopus (107) Google Scholar). We have recently shown that SfbI-expressing streptococci induce the accumulation of small ω-shaped structures, so-called caveolae, in the host cell membrane, near adherent streptococci, which are subsequently taken up by large membrane invaginations (11Rohde M. Müller E. Chhatwal G.S. Talay S.R. Cell. Microbiol. 2003; 5: 323-342Crossref PubMed Scopus (95) Google Scholar). However, the interaction of the M3 serotype strains A60 and A128 with the surface of HUVEC revealed that following attachment to the apical surface of the cells, M3 GAS induced the formation of membrane protrusions that tightly engulf the invading streptococci. Morphologically, this mechanism resembles a receptor-ligand interaction and does not show any accumulation of caveolae or microvilli (Fig. 1A). On the level of the actin cytoskeleton, M3 streptococci induced F-actin-rich structures that surrounded invading GFP-expressing streptococci (Fig. 1B), as shown by phalloidin staining. These observations suggest the occurrence of kinase and GTPase activation that in turn activate the actin polymerization machinery to provide the driving forces for the dynamic membrane rearrangements during the invasion process. Binding of bacteria to eukaryotic cells involves the interaction of bacterial factors with cellular receptors that subsequently leads to receptor activation and recruitment of signaling enzymes. Protein tyrosine phosphorylation is one of the early cellular responses following receptor stimulation. Fluorescence microscopy of HUVEC infected with S. pyogenes A128 and stained with an anti-phosphotyrosine antibody revealed an accumulation of tyrosine-phosphorylated proteins at sites of actin rearrangement in the vicinity of cell-associated bacteria (Fig. 2A and Fig. S1A). Western blot analysis of cell extracts showed an increase of tyrosine-phosphorylated proteins in infected cells, compared with non-infected cells (data not shown). To further address the role of cellular PTKs in S. pyogenes internalization, we incubated HUVEC with genistein, a general inhibitor of PTK activity. Pretreatment of HUVEC with increasing concentrations of genistein blocked S. pyogenes internalization in a dose-dependent manner, resulting in 70% inhibition at 50 μm genistein (Fig. 2B). Several host PTKs have been shown to be activated in during interaction of pathogens with host cells (19Agerer F. Michel A. Ohlsen K. Hauck C.R. J. Biol. Chem. 2003; 278: 42524-42531Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 20Shen Y. Naujokas M. Park M. Ireton K. Cell. 2000; 103: 501-510Abstract Full Text Full Text PDF PubMed Scopus (397) Google Scholar, 21Poppe M. Feller S.M. Römer G. Wessler S. Oncogene. 2007; 26: 3462-3472Crossref PubMed Scopus (138) Google Scholar, 22Meyer M. Clauss M. Lepple-Wienhues A. Waltenberger J. Augustin H.G. Ziche M. Lanz C. Büttner M. Rziha H.J. Dehio C. EMBO J. 1999; 18: 363-374Crossref PubMed Scopus (408) Google Scholar). To investigate whether particular non-receptor and receptor PTKs are involved in M3 GAS internalization, we blocked Src family PTKs (PP2; 1 μm) and the PTK c-Abl (AG 957, 10 μm) as well as the receptor PTKs c-Met (250 nm) and VEGFR (250 nm) with specific inhibitors (Fig. S2). These concentrations were at least 8 times above the reported IC50 value. Clearly, inhibition of Src PTKs by PP2 significantly impaired uptake of S. pyogenes A128 (p < 0.01, n = 3 experiments), whereas AG 957, c-Met inhibitor, and VEGFR kinase inhibitor had no effect (Fig. S2). These findings show the importance of host PTKs and Src family PTKs in particular in the regulation of S. pyogenes internalization into endothelial cells. The Src kinase family has been shown to be activated during internalization of pathogenic bacteria into non-phagocytic cells (19Agerer F. Michel A. Ohlsen K. Hauck C.R. J. Biol. Chem. 2003; 278: 42524-42531Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 23Eitel J. Heise T. Thiesen U. Dersch P. Cell. Microbiol. 2005; 7: 63-77Crossref PubMed Scopus (44) Google Scholar), and the Src PTK inhibitor PP2 significantly blocked the internalization of M3 GAS. Fluorescence microscopy of HUVEC infected with M3 S. pyogenes revealed an accumulation of Src kinase together with F-actin at bacterial entry sites (Fig. 2C and Fig. S1B). To further address the role of Src PTKs in S. pyogenes invasion, we preincubated HUVEC with increasing concentrations of the Src PTK inhibitor PP2 and with its inactive homolog PP3, respectively. Inhibition of Src PTKs by PP2 blocked uptake of S. pyogenes in a dose-dependent manner, resulting in more than 85% inhibition at 10 μm inhibitor, whereas its inactive homolog PP3 had no effect (Fig. 2D). To study the effect of Src inhibition on membrane protrusions and actin accumulation, we preincubated HUVEC with 10 μm PP2, subsequently infected with S. pyogenes A128, and analyzed the cells using FESEM and immunofluorescence microscopy. In non-treated cells, invading M3-type streptococci were engulfed by the typical membrane protrusions (Fig. 3A, ctrl). In contrast, inhibition of Src activity completely abolished formation of the protrusive membrane structures; only the formation of microspike-like structures could be observed in the vicinity of adherent streptococci (arrows in Fig. 3A, PP2). Quantification of the fluorescence signals obtained separately in the red or green channels clearly showed a sharp increase in the local concentration of F-actin over a distance of 1–2 μm in the vicinity of attached S. pyogenes A128, whereas in cells pretreated with the Src kinase inhibitor PP2, no F-actin accumulation was found (Fig. 3, B and C). These results clearly point to an involvement of Src family kinases during the uptake of M3 GAS. To test whether Src kinase activity is altered in HUVEC following S. pyogenes infection, we analyzed the activation status of Src using phosphospecific antibodies to detect phosphorylation of Src at regulatory tyrosine residues. Thus, cells were serum-starved for 6 h, infected for the indicated times with S. pyogenes A128, or left uninfected. Following lysis, the samples were analyzed by Western blotting with a phosphospecific antibody recognizing the phosphorylated tyrosine residue 419 in the activation loop of human c-Src. As shown in Fig. 4A, S. pyogenes induces phosphorylation of Tyr419 in a time-dependent manner compared with non-infected cells (ctrl), resulting in a maximum 3.5-fold activation after 60 min of infection (Fig. 4B). Taken together, these results provide evidence that cellular Src kinase function is required for bacterial internalization. The lipid kinase PI3K has been shown to be required for the formation of membrane protrusion and actin cytoskeleton rearrangements downstream of many growth factors (24Nobes C.D. Hawkins P. Stephens L. Hall A. J. Cell Sci. 1995; 108: 225-233Crossref PubMed Google Scholar). Importantly, PI3K is also required for M1- and SfbI-mediated entry of GAS (8Purushothaman S.S. Wang B. Cleary P.P. Infect. Immun. 2003; 71: 5823-5830Crossref PubMed Scopus (38) Google Scholar, 10Wang B. Li S. Dedhar S. Cleary P.P. Cell. Microbiol. 2007; 9: 1519-1528Crossref PubMed Scopus (26) Google Scholar). To investigate the role of PI3K in the activation of Rac by M3 streptococci, we first treated HUVEC with the PI3K inhibitor LY294002 and subsequently infected them with M3 GAS. As shown in Fig. 5A, inhibition of PI3K did not significantly (p = 0.063, n = 6 experiments) reduce the number of intracellular bacteria; nor did it alter the number of adherent bacteria (data not shown) at a concentration of 50 μm LY294002, a concentration that significantly blocked M1-mediated (8Purushothaman S.S. Wang B. Cleary P.P. Infect. Immun. 2003; 71: 5823-5830Crossref PubMed Scopus (38) Google Scholar) and SfbI-mediated 3A. Nerlich, unpublished results. uptake of S. pyogenes. To show that the inhibitor is able to block PI3K activity efficiently in the system used, we stimulated serum-starved HUVEC with 10% fetal calf serum, and the phosphorylation status of Akt1, a downstream substrate of class I PI3Ks, was monitored. As shown in Fig. 5B, stimulation with fetal calf serum induced a strong phosphorylation of Akt compared with untreated control cells, whereas in LY294002-treated cells, the phosphorylated form of Akt was undetectable. If uptake of M3 GAS is PI3K-independent, accumulation of actin at streptococcal entry site should be detectable in the presence of the inhibitor. To determine whether the inhibition of PI3K by LY294002 resulted in an inhibition of actin polymerization at strep
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