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 timedependent 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 ...

The M1 Protein of <b><i>Streptococcus pyogenes</i></b> Triggers an Innate Uptake Mechanism into Polarized Human Endothelial Cells

“…Several reports have shown that serotype M1 S. pyogenes efficiently invades and survives within various cell types including phagocytic and non-phagocytic cells [4,5,6,8,10,12,19,26]. In this study, we provide evidence that serotype M1 streptococci efficiently invade human blood EC and that the M1 protein is a key molecule in this process.…”

“…Although the invasion factors of M3 and M1 S. pyogenes are distinct, both trigger similar uptake processes with phago-lysosomal destination (fig. 2) [19,20]. However, there are also other important factors that may have a strong impact on the EC invasion by GAS.…”

“…Another potentially important cell type is the blood endothelial cells (EC) lining the lumen of the blood vessels. We previously identified a new invasion mechanism of serotype M3 S. pyogenes into EC [19], and identified the fibronectin-binding protein FbaB as a major invasin of M3 S. pyogenes [20]. However, a direct interaction of the most invasive serotype, i.e.…”

Invasion of Eukaryotic Cells byBorrelia burgdorferiRequires β1Integrins and Src Kinase Activity

“…Given that several intracellular pathogens utilize Src kinase for internalization and dissemination (16,32,51,61,78,90), we tested whether specific Src family kinase (SFK) inhibitors inhibited B. burgdorferi internalization in vitro. Specifically, Src kinase activity is required for the internalization of Neisseria spp.…”

CCCTC-Binding Factor Controls Its Own Nuclear Transport via Regulating the Expression of Importin 13

“…The invasion and migration of tumor is a multi-procedure and multistage process including shedding from of tumors from primary focus of infection, degradation of extracellular matrix (ECM), invasion into the blood vessel or absorbent vessel, transferring through the circulation system, escaping from the blood vessel or absorbent vessel, and formation of fresh focus of infection through proliferation and angiogenesis. This process requires complex molecular mechanisms including the participation of multiple cytokines, growth factors, adhesion factors and proteases (Lash et al, 2006;Mihaljevic et al, 2010;Nerlich et al, 2009;Wong and Gumbinger, 2003). Through wide-scale screening for CTCF binding site by CHIP, CTCF might also regulate the expression of proteins similar to cadherin like 26 and MMP23B (Arpanahi et al, 2009;Mukhopadhyay et al, 2004).…”