RECEPTOR MEDIATED EFFECT OF INTERMEDILYSIN ON HUMAN POLYMORPHONUCLEAR LYMPHOCYTE

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 A.具体目标：这笔赠款的具体目标没有修改，具体说明如下。 胆固醇依赖细胞溶血素(CDCs)是一类由多种革兰氏阳性病原体产生的致病因子，是一类大孔道毒素。尽管这些多聚体致孔毒素的细胞溶解机制在各种产生CDC的细菌中很大程度上是保守的，但越来越明显的是，CDC可能在发病机制中进化出了额外的作用。细胞溶解机制需要形成一个由大约30-40个单体组成的大的寡聚体结构(在(Tweeten等人，2001)中回顾)。然而，在感染部位内，随着毒素扩散到距离感染部位更远的地方，毒素浓度降低，随后形成完整寡聚体结构的可能性降低。然而，这些“非溶解”结构在疾病中的作用还没有得到充分的研究。中间链球菌是产生CDC的唯一病原体，因为它的CDC，中间溶素(ILY)，需要一种特定的细胞受体来发挥细胞溶解功能。已被证明是人类细胞特异性的，需要其细胞受体huCD59的存在。CD59是一种重要的表面表达糖基磷脂酰肌醇(GPI)锚定蛋白，存在于多种细胞类型中，并在激活的多形核白细胞(PMN)上上调(Gordon等人，1994)。它与许多重要的细胞功能有关，包括抑制宿主细胞上膜攻击复合体(MAC)的形成(Rollins和Sims，1990)，作为候选C1q受体(C1qRO2-)的适配分子(Otabor等人，2004)，以及作为细胞信号分子。目前还不清楚ILY为什么会进化出CD59的这种细胞特异性，以及这在发病机制中可能起到什么作用。有趣的是，从深层脓肿中分离的中间链球菌产生的ILY水平是从周围部位感染中分离的菌株的6-10倍，这表明它在发病机制中的重要性(Nagamune等人，2000年)。由于PMN是脓肿中存在的主要免疫细胞，我们建议检测PMN对ILY非裂解突变体受体介导的相互作用的反应。我们假设ILY受体介导的与huCD59的相互作用可以干扰CD59细胞的功能，如MAC抑制和C1q激活的超氧化物产生(特异性目标1)。我们进一步假设，ILY与CD59的结合可以不同于其他不与特定受体结合的CDC来调节PMN的生理学(特定目的2)。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. A. Specific Aims: The specific aims of this grant have not been modified and are described below. The cholesterol dependent cytolysins (CDCs) are a family of large pore forming toxins that are virulence factors produced by a variety of Gram-positive pathogens. Although the cytolytic mechanism of these multimeric pore-forming toxins has been largely conserved among the various CDC producing bacteria, it is becoming increasingly apparent that the CDCs may have evolved additional roles in pathogenesis. The cytolytic mechanism requires the formation of a large oligomeric structure that is comprised of approximately 30-40 monomers (reviewed in (Tweten et al., 2001). However, within the site of infection, as the toxins diffuse farther away from the site of the infection the concentration of toxin decreases and the likelihood of the formation of the complete oligomeric structures subsequently decreases. Yet a role for these "non-lytic" structures in disease has not been fully examined. Streptococcus intermedius is unique among CDC producing pathogens because its CDC, intermedilysin (ILY), requires a specific cellular receptor for cytolytic function. ILY has been shown to be human cell specific and requires the presence of its cellular receptor huCD59. CD59 is an important surface expressed glycosylphosphatidylinositol (GPI)- anchored protein that is found on many cell types and is up regulated on activated polymorphonuclear leukocytes (PMNs) (Gordon et al., 1994). It has been associated with a number of important cellular functions including inhibiting the formation of the membrane attack complex (MAC) on host cells (Rollins and Sims, 1990), serving as an adaptor molecule for a candidate C1q receptor (C1qRO2-) (Otabor et al., 2004), and acting as a cell signaling molecule . It is unclear why ILY has evolved this cellular specificity for CD59 and what role this may play in pathogenesis. Interestingly, Streptococcus intermedius isolated from deep-seated abscesses has been shown to produce levels of ILY 6-10 times greater than strains isolated from peripheral site infections indicating its importance in pathogenesis (Nagamune et al., 2000). As PMNs are the major immune cell present in abscesses we propose to examine the PMN response to the receptor-mediated interaction of non-lytic mutants of ILY. We hypothesis that the ILY receptor mediated interaction with huCD59 can interfere with CD59 cellular functions such as MAC inhibition and C1q activated superoxide production (specific aim 1). We further hypothesis that ILY binding to CD59 can modulate PMN physiology differently than other CDCs that do not bind to a specific receptor (specific aim 2).