SUMMARY Group A Streptococcus (GAS) is a leading cause of infection-related mortality in humans. All GAS serotypes express the Lancefield group A carbohydrate (GAC), comprising a polyrhamnose backbone with an immunodominant N-acetylglucosamine (GlcNAc) side chain, which is the basis of rapid diagnostic tests. No biological function has been attributed to this conserved antigen. Here we identify and characterize the GAC biosynthesis genes,gacA-L. An isogenic mutant of the glycosyltransferase gacI, which is defective for GlcNAcside chain addition, is attenuated for virulence in two infection models, in association with increased sensitivity to neutrophil killing, platelet-derived antimicrobials in serum and the cathelicidin antimicrobial peptide LL-37. Antibodies to GAC lacking the GlcNAc side chain and containing only polyrhamnose promoted opsonophagocytic killing of multiple GAS serotypes and protected against systemic GAS challenge after passive immunization. Thus, the Lancefield antigen plays a functional role in GAS pathogenesis and its understanding has implications for vaccine development.

Adhesion of T cells after stimulation of the T-cell receptor (TCR) is mediated via signaling processes that have collectively been termed inside-out signaling. The molecular basis for inside-out signalingis not yet completely understood. Here, we show that a signaling module comprising the cytosolic adapter proteins ADAP and SKAP55 is involved in TCR-mediated inside-out signaling and, moreover, that the interaction between ADAP and SKAP55 is mandatory for integrin activation. Disruption of the ADAP/SKAP55 module leads to displacement of the small GTPase Rap1 from the plasma membrane without influencing its GTPase activity. These findings suggest that the ADAP/SKAP55 complex serves to recruit activated Rap1 to the plasma membrane. In line with this hypothesis is the finding that membrane targeting of the ADAP/SKAP55 module induces T-cell adhesion in the absence of TCR-mediated stimuli. However, it appears as if the ADAP/SKAP55 module can exert its signaling function outside of the classical raft fraction of the cell membrane.Within the immune system, integrins play important roles in regulating the interaction of T cells with other cells and with proteins of the extracellular matrix. By mediating T-cell adhesion, integrins control the homing and the trafficking of T cells as well as the interaction between T cells and antigen-presenting cells (34, 41). The major integrins expressed on T cells are the ␤2-integrin LFA-1 (␣L␤2) as well as members of the ␤1-family of integrins (␣4␤1, ␣5␤1, ␣6␤1, and VLA) (25). The physiologic ligands of LFA-1 include the intercellular adhesion molecule 1 (ICAM-1), ICAM-2, and ICAM-3 (25), whereas ligands for ␤1-integrins are vascular cell adhesion molecule 1 (VCAM-1) or proteins of the extracellular matrix, such as fibronectin (13,54).On resting T cells, ␤1-and ␤2-integrins are expressed in an inactive state. However, ligation of the T-cell receptor (TCR) by antigen/major histocompatibility complexes results in a rapid increase in the activity of ␤1-and ␤2-integrins, thereby enhancing ligand binding (15,46,50). Two distinct mechanisms mediate the activation of integrins. First, the affinity of an integrin for its ligand is enhanced, and second, the lateral mobility becomes altered, which results in integrin clustering (avidity regulation) (14). The processes leading to the activation of integrins have collectively been termed inside-out signaling (14, 15, 28).Several molecules have been suggested to play critical roles during TCR-mediated activation of ␤1-and ␤2-integrins (14, 28). Among these is the small GTPase Rap1, whose role for integrin activation has been a matter of intense research during the last few years (4, 29). The mechanisms for how Rap1 becomes activated are not yet completely understood (4). Rap1 activation has been shown to be mediated by particular guanine nucleotide exchange factors (GEFs), such as C3G, and Epac (5,8,11). It has been proposed that Rap1 is associated with CalDAG-GEFI and that TCR-induced Rap1 activation is dependent upon the activation of phosphol...

IntroductionThe initial adhesion of platelets to the extracellular matrix of injured blood vessels is mediated at high shear rates by von Willebrand factor (VWF) interaction with glycoprotein (GP) Ib-IX-V. 1 Additionally, engagement of GP Ib-IX-V by VWF is thought to contribute to stable platelet adhesion by generating intracellular signals necessary for activation of ␣IIb␤3. Indeed, ␣IIb␤3 activation and platelet thrombus formation have been observed under a number of experimental conditions following platelet interaction with VWF. [2][3][4][5][6] Furthermore, specific biochemical responses have been documented under the same conditions, including induction of Ca 2ϩ fluxes and activation of tyrosine, serine-threonine, and lipid kinases. 7,8 Consequently, GP Ib-IX-V may function as a signaling receptor and an adhesion receptor.GP Ib-IX-V is a complex of 4 transmembrane polypeptides. 9-11 Although the cytoplasmic tail of each subunit lacks a catalytic domain, each may interact directly or indirectly with proteins that can transmit intracellular signals. For example, the cytoplasmic tail of GP Ib␣ can interact directly with filamin, GP Ib␣ and Ib␤ with 14-3-3-, and GP Ib␤ and GP V with calmodulin. 12-15 GP Ib-IX-V can be coimmunoprecipitated from platelets with signaling molecules, including Src family kinases, 16 phosphatidylinositol 3-kinase (PI 3-kinase) 17 and Src homology 2 domain-containing inositol polyphosphate 5-phosphatase-2 (SHIP-2). 18 Furthermore, VWF-dependent platelet activation may require localization of GP Ib-IX-V to lipid rafts, membrane structures implicated in cellular signaling. 19 Although there is good evidence for a functional link between GP Ib-IX-V and ␣IIb␤3, 2 critical questions remain: Is GP Ib-IX-V itself capable of transducing signals in platelets, and, if so, to what extent do these signals participate in the activation of ␣IIb␤3? Several factors have conspired to make it difficult to answer these questions. First, a subpopulation of GP Ib-IX-V in platelets may be associated with immunoreceptor tyrosine activation motif (ITAM)-bearing proteins that can signal in their own right, including the Fc␥RIIA receptor and the FcR ␥-chain. 20,21 Second, platelets express numerous receptors for soluble and matrix-associated agonists, and some of the agonists (eg, adenosine diphosphate [ADP], thromboxane A 2 ) are released by adherent platelets. 22 Third, VWF not only interacts with GP Ib-IX-V through its A1 domain but also with ␣IIb␤3 through its C1 domain. 1 Thus, outside-in signals stimulated by VWF binding to ␣IIb␤3 can confound analysis of GP Ib-IX-V signaling. 23 Finally, studies of GP Ib-IX-V signaling under static conditions have frequently utilized nonphysiological mediators, such as botrocetin or ristocetin, to promote VWF binding to GP Ib-IX-V, complicating data interpretation further.Thus, despite the publication of many important studies on the molecular contributors to signaling responses downstream of GP Ib-IX-V, most to date have failed to consistently employ conditions to avoi...

Increased ligand binding to cellular integrins (“activation”) plays important roles in processes such as development, cell migration, extracellular matrix assembly, tumor metastasis and hemostasis and thrombosis[1-5]. Integrin activation encompasses both increased integrin monomer affinity and increased receptor clustering[6] and depends on integrin-talin interactions[5]. Loss of kindlins results in reduced activation of integrins[7-13]. Kindlins might promote talin binding to integrins through a cooperative mechanism[5, 14-16]; however, kindlins do not increase talin association with integrins[17]. Here we report that, unlike talin head domain (THD), kindlin-3 caused little effect on the affinity of purified monomeric αIIbβ3, and it didn’t enhance activation by THD. Furthermore, studies with ligands of varying valency showed that kindlins primarily increased cellular αIIbβ3 avidity rather than monomer affinity. In platelets or nucleated cells, loss of kindlins markedly reduced αIIbβ3 binding to multivalent but not monovalent ligands. Finally, silencing of kindlins reduced the clustering of ligand-occupied αIIbβ3 as revealed by total internal reflection fluorescence (TIRF) and electron microscopy. Thus, in contrast to talins, kindlins have little primary effect on integrin αIIbβ3 affinity for monovalent ligands and increase multivalent ligand binding by promoting the clustering of talin-activated integrins.