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

Abstract

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 phosphatidylinositol 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-dependent 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.

FIGURE 1.

Microscopic analysis of the interaction between M3 type S. pyogenes and HUVEC. A, electron microscopic analysis of HUVEC infected with M3 type S. pyogenes A60 and A128 for 30 min showing the formation of membrane protrusions, which engulf the streptococcal chains by a zipper-like mechanism. Bars, 1 μm. B, fluorescence microscopic analysis of the actin cytoskeleton of A128-GFP-infected HUVEC 30 min after infection. Invading streptococci induced F-actin rearrangements (arrows) as shown by phalloidin staining. The arrowhead points to an adherent chain without F-actin accumulation. Bar, 10 μm.

FIGURE 2.

Internalization of M3 S. pyogenes requires protein-tyrosine kinases of the host cell. A, HUVEC were infected with GFP-expressing S. pyogenes A128 (green) for 30 min and stained for phosphotyrosine (PY, blue) and F-actin (red). Fluorescence analysis showed accumulation of phosphotyrosine and F-actin in the vicinity of invading streptococci (arrow). The arrowhead indicates adherent bacteria without accumulation. Bar, 5 μm. B, HUVEC were pretreated with the indicated concentrations of genistein and infected for 1 h with S. pyogenes A128. Intracellular bacteria were determined using the double immunofluorescence assay. The graph shows mean values ± S.E. of three independent experiments (**, p < 0.01; ***, p < 0.001). C, HUVEC were infected with GFP-expressing S. pyogenes A128 (green) for 30 min. After fixation and permeabilization, samples were stained for Src kinase (blue) and F-actin (red). Fluorescence analysis showed accumulation of Src kinase and F-actin in the vicinity of invading streptococci (arrow). The arrowhead indicates adherent bacteria without accumulation. Bar, 5 μm. D, HUVEC were pretreated with the indicated concentrations of PP2 and its inactive homolog PP3, respectively, and infected for 1 h with S. pyogenes. Intracellular bacteria were determined using the double immunofluorescence assay. The graph shows mean values ± S.E. of three independent experiments (**, p < 0.01; ***, p < 0.001).

FIGURE 3.

Src family kinases are essential for membrane protrusion formation and F-actin accumulation. HUVEC were pretreated with 10 μm PP2 or vehicle alone and subsequently infected with S. pyogenes A128. After fixation, the samples were processed for electron microscopy and immunofluorescence microscopy, respectively. A, formation of membrane protrusions is visible in control cells, whereas cells pretreated with the Src kinase inhibitor PP2 were impaired in the formation of membrane protrusions. The arrows indicate the formation of microspike-like structures in PP2-treated HUVEC in the vicinity of adherent bacteria. Bar, 2 μm. B, accumulation of F-actin in the vicinity of adherent streptococci in control cells is shown. Local recruitment of F-actin (green line) to cell-associated bacteria (red line) was quantified by plotting the fluorescence intensities as detected in the green and red channel, respectively, against the distance. Bar, 5 μm. C, lack of F-actin accumulation in the vicinity of adherent streptococci in cells pretreated with PP2 is shown. By plotting the fluorescence intensities in the green and red channel, respectively, against the distance no local recruitment of F-actin (green line) to cell-associated bacteria (red line) was detected. Bar, 5 μm. Plotted data in B and C are mean intensity values assessed in different sites (n = 7).

FIGURE 4.

Activation of Src family kinases in M3 S. pyogenes-infected HUVEC. A, serum-starved HUVEC were infected with S. pyogenes A128 for the indicated times or left uninfected. After cell lysis, samples were analyzed in Western blots with phosphospecific anti-Src Tyr(P)-419 antibodies (top) and polyclonal anti-Src antibodies (bottom). Infection with streptococci resulted in an increase of phosphorylated Src during the course of infection. B, densitometry showed a maximal 3.5-fold increase of phosphorylated Src after 60 min. The graph depicts the ratio between phosphorylated Src and the total amount of Src present in the sample (mean values ± S.E. of three independent experiments).

FIGURE 5.

PI3K-independent internalization of M3 S. pyogenes into endothelial cells. A, HUVEC were pretreated with 50 μm LY294002 and infected for 1 h with S. pyogenes A128. Intracellular bacteria were determined using the double immunofluorescence assay. The graph shows mean values ± S.E. of six independent experiments. B, serum-starved HUVEC were treated with 50 μm LY294002 or with vehicle alone and subsequently stimulated with 10% fetal calf serum or left untreated. Phosphorylation of Akt1 (top) was completely abolished in inhibitor-treated cells, as shown by Western blotting. Total Akt served as a loading control (bottom). C, untreated HUVEC and cells treated with 50 μm LY292002 or treated with 10 μm PP2 were infected with S. pyogenes A128 for 30 min. Fluorescence microscopic analysis showed a lack of F-actin accumulation in the vicinity of streptococci only in PP2-treated cells. Bar, 5 μm.

FIGURE 6.

Rac1 is required for M3 S. pyogenes internalization into endothelial cells. HUVEC were transiently transfected with GFP-tagged N17Rac1 and wtRac1, respectively, infected with S. pyogenes A128 for 1 h, and analyzed by fluorescence microscopy. A, in HUVEC expressing GFP-dnRac1, mostly extracellular streptococci (magenta) were found, whereas GFP-wtRac1-expressing HUVEC showed numerous intracellular streptococci (yellow/red). The arrows highlight streptococcal chains with strong GFP-wtRac1 accumulation that are in the process of entering the cell. Bar, 10 μm. B, quantification showed a 85% reduction of intracellular streptococci in GFP-N17Rac1-expressing cells. The graph shows the mean number of intracellular streptococci ± S.E. of three independent experiments (*, p < 0.005).

FIGURE 7.

Rac1 recruitment during uptake of M3 S. pyogenes into endothelial cells. HUVEC were transiently transfected with GFP-tagged wtRac1 and infected with S. pyogenes for 30 min. A, immunofluorescence analysis showing an accumulation of F-actin (red), GFP-Rac1 (green), and GAS (blue) during certain stages of internalization (arrows). The arrowheads indicate bacteria that showed only Rac1 accumulation. Bar, 15 μm. B, enlarged xy sections of the indicated area in the merged image (A) with corresponding xz sections. C, local recruitment of F-actin (red line) and GFP-Rac1 (green line) to cell-associated bacteria (blue line) quantified by plotting the fluorescence intensity profile of all three channels against the distance.

FIGURE 8.

Activation of Rac1 and Rac1 dynamics in M3 GAS-infected HUVEC. Serum-starved HUVEC were infected with S. pyogenes A128 for the indicated times (in minutes) or left uninfected, and activation of Rac1 was determined by a Rac activation assay. A, an increase of active Rac1 (GTP-Rac1; top) was detected 15 min postinfection and lasted during the examined course of infection. Cellular extracts were immunoblotted with anti-Rac1 antibodies to indicate a similar concentration of Rac1 (total-Rac1) in each sample (bottom). The numbers below the representative blot show -fold increase of GTP-Rac1 normalized to total-Rac1 (means of three independent experiments). B, activation of Rac1 in HUVEC after stimulation with 100 ng ml⁻¹ epidermal growth factor (EGF) for 3 min is shown. C, using time lapse microscopy, the dynamics of GFP-wtRac1 in transfected HUVEC 15 min after starting the infection were recorded using the GFP channel and phase contrast. One streptococcal chain with partial GFP-wtRac1 accumulation that is in the process of entering the cell is shown (arrow, first frame). Attached bacteria (arrowhead, first frame) induced local accumulation of GPF-wtRac1 within minutes after contact with the cell. Elapsed time, in minutes, from the beginning of the recording is indicated. Bar, 5 μm.

FIGURE 9.

Arp2/3 recruitment during uptake of M3 S. pyogenes. HUVEC infected with S. pyogenes A128 for 30 min were stained for the p16 subunit of Arp2/3 complex and for GAS. Fluorescence microscopy revealed a distinct accumulation of Arp2/3 complex (red) in the vicinity of invading streptococci (green). The arrows indicate Arp2/3 localization in membrane ruffles at the edge of the mammalian cell. Bar, 5 μm.