Effects of invasin and YopH of Yersinia pseudotuberculosis on host cell signaling

Sammanfattning: Integrins are a large family of membrane-spanning heterodimeric (??) receptors that bind to ligands on other cells or to extracellular matrix (ECM) proteins. These receptors mediate bidirectional signaling over the cell membrane to induce signaling cascades mediating functions as cell adhesion, spreading and migration. This signaling takes place at cell-matrix adhesions, which are sites where clustered and ligand-bound integrins connect to and mediate stabilization of the actin cytoskeleton, and induce signaling cascades. Integrins have a short cytoplasmic tail that is crucial for the bidirectional signaling, and the ?1-integrin subunit exists in five splice variants only differing in the membrane-distal part of the cytoplasmic tail. This region of the almost ubiquitously expressed ?1-integrin, ?1A, contains two protein tyrosine motifs (NPXYs) interspaced with a threonine-rich region, while this region of the ?1B splice variant is completely different and lacks known motifs. In contrast to the ?1A-integrin, the ?1B variant cannot mediate cell-matrix adhesion formation following binding to ECM ligands.The enteropathogenic bacterium Yersinia pseudotuberculosis binds to ?1-integrins on the host cell with invasin, and this stimulates uptake of the bacterium. However, upon binding to the host cell, pathogenic Yersinia strains inject virulence effectors that block uptake. One effector responsible for the blocking is a tyrosine phosphatase, YopH. We identified the targets for this effector in the macrophage-like cell line J774A.1, which represent a professional phagocyte and thus is the likely target cell for the antiphagocytic effect of Yersinia. Two YopH target proteins were p130Cas and ADAP, of which the latter interestingly is an adapter protein specifically expressed in hematopoietic cells. ADAP has previously been implicated to participate in Fc-receptor-mediated phagocytosis and in communication between T-cell receptors and integrins.We also studied the importance of the cytoplasmic tail of ?1-integrin for uptake of Yersinia. The GD25 cell line, which is a fibroblast-like cell line that lacks endogenous ?1-integrins, was used together with GD25 cells transfected with ?1B, ?1? or cytoplasmic tail mutants of ?1A. These studies revealed that ?1B-integrins could bind to invasin but not mediate uptake of Yersinia, while ?1A both bound to invasin and mediated uptake. The first NPXY motif (unphosphorylated) and the double-threonines of the unique part of ?1A were important for the ability of integrin to mediate uptake of Yersinia. These studies lead to the interesting finding that, when these cells were allowed to spread on invasin, those that expressed ?1A spread as normal fibroblasts while for ?1B-integrin-expressing cells, only finger-like protrusions of filopodia were formed. This provided us with a tool to study formation of filopodia without interference of the tightly linked process of lamellipodia formation. Initially, proteins that localized to the tip complex of these filopodia were identified. These were talin, VASP and interestingly the p130Cas-Crk-DOCK180 scaffold, while FAK, paxillin and vinculin were absent. In addition, VASP, p130Cas and Crk were shown to be important for the filopodia formation in GD25?1B. Further, the role of the actin motor myosin X, which previously has been implicated in formation of filopodia, was studied in the GD25?1B cells and it was shown that myosin X not was important for filopodia formation, but that it recruited FAK and vinculin to the tip complexes of filopodia.