New aspects of integrin regulation and the role of membrane microdomains and phosphatidylinositol phosphates in the integrin-mediated uptake of pathogens

Abstract

Integrins are crucial for many cellular processes including cell spreading, migration, and proliferation. Their engagement by extracellular ligands or intracellular stimuli leads to the activation of integrins which is under normal conditions tightly regulated and results in a variety of signaling processes. However, integrins are often exploited by pathogens to mediate host cell contact. Staphylococcus aureus engages integrin beta 1 via fibronectin as a molecular bridge. This triggers the clustering and activation of integrin beta 1 causing the formation of a focal adhesion-like complex surrounding the bacterium. This leads to the reorganization of the actin cytoskeleton and finally to the uptake of S.aureus into the host cell facilitating colonization and, thereby, a broad range of infections.In this work, we focused on the regulation of integrin beta 1 by its interaction partner alpha-actinin and on the molecular uptake mechanism of S. aureus via integrins employing membrane microdomains and phosphatidylinositol phosphates.We discovered that alpha-actinin is an inhibitor of integrin activation.RNAi-mediated knockdown of alpha-actinin deregulated important cellular functions such as cell spreading and migration. Additionally, the composition of focal adhesions was disturbed as talin was more abundant. We could show that integrin activation is increased in the absence of alpha-actinin correlating with more integrin-associated talin. We conclude, that alpha-actinin inhibits integrins by displacing talin from integrins by competing for the same binding site on integrin.We also found that, besides integrin beta1, the internalization of S. aureus employs membrane microdomains. However, caveolin-rich membrane microdomains impeded the uptake. We could show that caveolin immobilizes membrane microdomains which correlates directly with the uptake efficiency. Mobilizing membrane microdomains led to an enhanced internalization.Additionally, we could clarify the role of phosphatidylinositol phosphates during staphylococcal infections. Phosphatidylinositol-4,5-bisphosphate (PtdIns-4,5-P2) and phosphatidylinositol-3,4,5-trisphosphate (PtdIns-3,4,5-P3) were both generated at the site of attachment. By RNAi-mediated knockdowns, we could identify that phosphatidylinositol-5-kinase Igamma (PIP5KIgamma) is essential at the site of bacterial uptake. Furthermore, overexpression of a-PtdIns-3,4,5-P3 blocking pleckstrin homolog domain and phosphatidylinositol-3-kinase (PI3K) inhibitors demonstrated that an elevated level of the local PtdIns-3,4,5-P3 is also necessary for staphyloccal uptake.Altogether, these findings allow new insights in integrin regulation, describe for the first time that membrane microdomain mobility correlates with bacterial uptake and decipher the role of phosphatidylinositol phosphates in the uptake of S. aureus.