RECEPTOR MEDIATED EFFECT OF INTERMEDILYSIN ON HUMAN POLYMORPHONUCLEAR LYMPHOCYTE

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目及 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 a.具体目标：尽管由于遇到技术困难，蛋白质组学特征的特定靶细胞系已从PMN变更为肝细胞系HepG2细胞，但该资助的具体目的尚未修改。 这一变化的具体情况如下所述。 胆固醇依赖性溶细胞素（CDC）是一类大孔形成毒素，是由多种革兰氏阳性病原体产生的毒力因子。尽管这些多聚体孔形成毒素的细胞溶解机制在各种CDC产生细菌中基本上是保守的，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由大约30 - 40个单体组成的大的寡聚结构（综述于（Tweten等人，2001年）。 然而，在感染部位内，随着毒素扩散远离感染部位，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“非溶解性”结构在疾病中的作用尚未得到充分研究。 中间链球菌在产生CDC的病原体中是独特的，因为它的CDC，中间溶素（ILY），需要特异性细胞受体用于细胞溶解功能。 ILY已被证明是人细胞特异性的，并且需要其细胞受体huCD59的存在。 CD59是一种重要的表面表达的糖基磷脂酰肌醇（GPI）锚定蛋白，其存在于许多细胞类型中并且在活化的多形核白细胞（PMN）上上调（Gordon et al.，1994年）。 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物（MAC）的形成（Rollins和西姆斯，1990），作为候选C1q受体（C1qRO2-）的衔接分子（Otabor et al.，2004），并作为细胞信号分子。 目前还不清楚为什么ILY已经进化出这种对CD59的细胞特异性，以及这在发病机制中可能起什么作用。有趣的是，分离自深部脓肿的中间链球菌已显示产生比分离自外周部位感染的菌株高6 - 10倍的ILY水平，表明其在发病机制中的重要性（Nagamune等人，2000年）。 由于中性粒细胞是主要的免疫细胞，目前在pneumonesses，我们建议检查中性粒细胞的反应，受体介导的相互作用的非裂解突变体的ILY。 我们假设ILY受体介导的与huCD59的相互作用可以干扰CD59细胞功能，例如MAC抑制和C1q激活的超氧化物产生（具体目的1）。我们进一步假设ILY与CD59的结合可以调节PMN的生理学，与其他不与特定受体结合的CDC不同（特异性目的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 although due to technical difficulties encountered the specific target cell line for the proteomic profile has been changed from PMNs to a liver cell line, HepG2 cells. The specifics of this change 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 hysiology differently than other CDCs that do not bind to a specific receptor (specific aim 2).