Mechanisms of Resistance and Susceptibility to African Trypanosome Infection

描述(由申请人提供)：布氏锥虫会引起牛的纳格纳，但由于其对正常人类血清的细胞毒活性敏感，因此对人类不具传染性。这种先天免疫活性是由于高密度脂蛋白(HDL)的一个小亚类，称为锥虫裂解因子(TLF)。TLF含有载脂蛋白A-1(apoA-1)，这是一种存在于所有HDL中的蛋白质，以及两种灵长类特有的蛋白质，即结合珠蛋白相关蛋白(HPR)和载脂蛋白L-1(apoL-1)。在体外和转基因小鼠模型中，HPR和APOL-1都被证明是最大限度地杀死锥虫所必需的。HPR是一种血红蛋白(Hb)结合蛋白，Hb是TLF杀灭锥虫的重要辅助因子。TLF介导的布鲁氏杆菌裂解的细胞途径始于TLF与位于鞭毛袋的高亲和力受体(Tb927.6.440)的结合。结合后，TLF被迅速摄取并定位于溶酶体。在酸化的溶酶体中，TLF被激活，并通过溶酶体膜的不稳定杀死布鲁氏杆菌。人类昏睡病寄生虫布氏锥虫已经进化出对TLF的防御。罗德氏锥虫产生一种有效的TLF抑制剂，血清抗性相关蛋白(SRA)，它与APOL-1结合，中和TLF的活性。布氏冈比亚锥虫也会感染人类，但缺乏SRA，正如这项建议所示，它通过下调TLF受体来抵抗TLF的杀伤。在这项提案中，我们概述了一些实验，这些实验将提供关于TLF组装机制的重要信息，以及如何改变其组成以产生具有抗人类昏睡病寄生虫活性的“变异TLF”(目标1)。为了更好地了解天然TLF及其组成蛋白导致膜失稳的生物物理基础，我们将在基于荧光的分析中使用特定组成的模型脂质体来测量TLF诱导的脂质动力学变化(目标2)。最后，我们将研究SRA依赖和非SRA依赖的TLF抗性的细胞、分子和生化基础。除了解决有关SRA如何抑制TLF活性的长期问题外，我们还将研究冈比亚结核杆菌对TLF的抗性机制(目标3)。我们的长期目标是更好地了解TLF杀死的机制，以及人类昏睡病寄生虫如何逃避这一活动。这可能使我们能够开发新的方法来治疗这种重要的人类疾病。 公共卫生相关性：非洲昏睡病是一种在撒哈拉以南非洲重新出现的人类疾病。目前估计有超过3500万人处于危险之中，感染人数可能超过30万人。目前还没有治疗非洲昏睡病的疫苗，而且大多数药物都有严重的毒性问题。对天然免疫复合体TLF作用机制的拟议研究可能会导致识别治疗开发的新方法。

DESCRIPTION (provided by applicant): Trypanosoma brucei brucei causes Nagana in cattle but is non-infectious to humans because of its susceptibility to the cytotoxic activity of normal human serum. This innate immune activity is due to a minor subclass of high-density lipoprotein (HDL) termed the Trypanosome Lytic Factor (TLF). TLF contains apolipoprotein A-1 (apoA-1), a protein found in all HDLs, and two primate specific proteins, haptoglobin related protein (Hpr) and apolipoprotein L-1 (apoL-1). Both Hpr and apoL-1 have been shown to be necessary for maximal trypanosome killing in vitro and in a transgenic mouse model. Hpr is a hemoglobin (Hb) binding protein and Hb is an essential co-factor for trypanosome killing by TLF. The cellular pathway of TLF mediated lysis of T. b. brucei initiates with binding of TLF to a high affinity receptor (Tb927.6.440) located in the flagellar pocket. Following binding TLF is rapidly taken up and localized to the lysosome. Within the acidified lysosome TLF is activated and kills T. b. brucei by destabilization of the lysosomal membrane. The human sleeping sickness parasite Trypanosoma brucei rhodesiense has evolved a defense against TLF. T. b. rhodesiense produces a potent inhibitor of TLF, Serum Resistance Associated protein (SRA), which binds apoL-1 and neutralizes the activity of TLF. Trypanosoma brucei gambiense also infects humans but lacks SRA and, as shown in this proposal, resists TLF killing by down regulation of the TLF receptor. In this proposal, we outline a number of experiments that will provide important information on the mechanism of assembly of TLF and how its composition might be altered to produce "variant TLFs" with activity against the human sleeping sickness parasites (Aim 1). To better understand the biophysical basis for membrane destabilization by native TLF and its constituent proteins we will use model liposomes of defined composition in fluorescence based assays to measure TLF induced changes in lipid dynamics (Aim 2). Finally, we will investigate the cellular, molecular and biochemical basis of SRA-dependent and SRA-independent resistance to TLF. In addition to resolving a longstanding question concerning how SRA inhibits TLF activity we will investigate the mechanism of T. b. gambiense resistance to TLF (Aim 3). Our long-term goals are to develop a better understanding of the mechanism of TLF killing and how the human sleeping sickness parasites evade this activity. This may allow us to develop novel approaches for the treatment of this important human disease. PUBLIC HEALTH RELEVANCE: African sleeping sickness is a re-emerging human disease in sub- Saharan Africa. It is currently estimated that over 35 million people are at risk and the number of infected people may exceed 300,000. There is no vaccine for African sleeping sickness and most of the drugs have serious toxicity problems. The proposed studies on the mechanism of action of the innate immune complex, TLF, may lead to the identification of novel approaches for therapeutic development.