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

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)² 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 (1–5).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 (6) and have been shown to be internalized into human umbilical vein endothelial cells (HUVEC) in vitro (7).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 (8–10). In contrast to this, caveolae were shown to act as entry port for SfbI-expressing S. pyogenes (11), a mechanism distinct from the zipper-like uptake mechanism employed by strains expressing M1 protein (12). 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 (13). However, a requirement for PI3K activation, which in turn induced paxillin phosphorylation, was recently shown for M1-mediated as well as SfbI-mediated invasion (10). In contrast, M3 streptococci do not express these two well characterized invasins (14), 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.