The molecular mechanism for trypanosome cell death induced by ApoLI and its inactivation in human infective T. b. rhodesiense.

锥虫是一种致命的单细胞寄生虫，被受感染的采采蝇叮咬后被注射到人类、家畜和野生动物的血液中。它们在人类中引起昏睡病，在牲畜中引起消耗性疾病，包括牛的长须病。这些疾病对撒哈拉以南非洲造成重大影响。它们导致成千上万人死亡，并通过降低牲畜的生产力给人类造成巨大痛苦。大多数种类的非洲锥虫不能感染人类，因为在人类血液中发现了两种分子复合物，称为锥虫溶解因子。它们含有一种共同的成分，载脂蛋白LI (ApoLI)，它可以杀死锥虫。ApoLI可以在寄生虫的膜上打孔，导致细胞死亡。感染人类并引起昏睡病的锥虫的两个亚种能够使ApoLI失活，因此可以在人类血液中存活并引起疾病。ApoLI如何杀死锥虫尚不清楚。已经确定ApoLI在膜上穿孔，但尚不清楚锥虫细胞内的哪些膜被破坏。一些研究表明，ApoLI破坏了溶酶体，这是细胞中用于降解细胞成分的一种隔室。另一些人认为，它会在外细胞膜上打洞，而进一步的研究表明，受损的是细胞的能量“发电站”线粒体。我们将使用最新的显微镜方法来观察ApoLI在细胞周围的移动，并观察细胞的哪些部分受到影响，以及它们是如何被杀死的。ApoLI用来打孔的机制也没有得到很好的理解。它是如何与膜相互作用的，又是如何形成毛孔的？这些毛孔是如何调节的？这些问题对于理解ApoLI如何杀死锥虫很重要。它们对于了解ApoLI在人体内的自然功能也很重要，因为已知ApoLI的变化与晚发性肾脏疾病有关。我们将使用结构生物学方法来了解ApoLI的样子，并进行各种研究，以了解它形成孔隙的机制。我们还将研究两种人类感染的锥虫之一，布氏罗得西亚锥虫。这种寄生虫可以抵抗ApoLI的毒性作用，因为存在一种单一分子，即血清抗性相关蛋白SRA。我们已经证明SRA可以在各种条件下直接与ApoLI相互作用，现在的目标是确定它是如何使ApoLI失活的。它是否与ApoLI结合以阻止ApoLI进入细胞中介导毒性作用的区域？或者它与ApoLI结合并阻止其形成孔？或者两者兼而有之？我们将再次使用结构生物学方法来准确了解ApoLI如何与SRA相互作用，以及ApoLI如何阻止孔隙形成。我们还将使用最新的显微镜方法来研究SRA如何在暴露于ApoLI的锥虫体内移动。这些研究将提供分子细节，使我们能够了解ApoLI如何杀死锥虫，以及人类传染性锥虫如何对这种毒素产生耐药性。这有可能帮助我们设计新的治疗方法来预防昏睡病。通过了解SRA如何结合和抑制ApoLI，我们可以设计出新的ApoLI版本，这些ApoLI可以抵抗SRA介导的失活。这些药物可直接提供给昏睡病患者以杀死锥虫。我们还可以开发出阻断SRA-ApoLI相互作用的小分子，让人体天然的apoli介导的防御来杀死寄生虫。最后，正在开发含有ApoLI的转基因牛，以防止锥虫的生长。使用对sra介导的失活具有抗性的ApoLI版本将减少这些牛被感染的机会，并为人类感染性锥虫的生长和发育提供一个储存库。

Trypanosomes are deadly, single celled parasites that are injected into the blood of humans, domestic livestock and wildlife when bitten by infected tsetse flies. They cause sleeping sickness in humans and wasting diseases in livestock, including nagana in cattle. These diseases have significant impact in sub-Saharan Africa. They lead to thousands of human deaths and cause significant human suffering by reducing the productivity of livestock. Most species of African trypanosomes cannot infect humans, due to two molecular complexes found in human blood, known as trypanolytic factors. These contain a shared component, apolipoprotein LI (ApoLI) which kills the trypanosomes. ApoLI can punch holes in the membranes of the parasite, leading to death of the cell. The two subspecies of trypanosome that infect humans and cause sleeping sickness are able to inactivate ApoLI and can therefore survive in human blood and cause disease.How ApoLI kills trypanosomes is not well understood. It is well established that ApoLI punches holes in membranes but it is not clear which membranes within the trypanosome cell are damaged. Some studies suggest that ApoLI bursts the lysosome, a compartment of the cell used to degrade cellular components. Others suggest that it punches holes in the outer cell membrane, while further studies suggest that it is the mitochondria, the energy 'power-houses' of the cell, that are damaged. We will use the latest microscopy methods to observe ApoLI as it moves around the cell, and to see what parts of the cell are affected, and how they are killed.The mechanism used by ApoLI to punch holes is also not well understood. How does it interact with membranes and how does it form pores? How are these pores regulated? These questions are important for understanding how ApoLI kills trypanosomes. They are also important in understanding the natural function of ApoLI in the human body, as it is known that changes in ApoLI are associated with late-onset kidney disease. We will use structural biology methods to understand what ApoLI looks like, and to perform a variety of studies to see the mechanism by which it forms pores.We will also study one of the two human infective species of trypanosome, Trypanosoma brucei rhodesiense. This parasite can resist the toxic effects of ApoLI because of the presence of a single molecule, the serum resistance associated protein, SRA. We have shown that SRA can interact directly with ApoLI under a variety of conditions and now aim to determine exactly how it inactivates ApoLI. Does it bind to ApoLI to prevent it from getting to the region of the cell in which it mediates its toxic effects? Or does it bind to ApoLI and prevent it from forming pores? Or perhaps it is both? We will again use structural biology methods to understand exactly how ApoLI interacts with SRA, and how it stops pore formation by ApoLI. We will also use the latest microscopy methods to investigate how SRA moves around within trypanosomes that are exposed to ApoLI.These studies will provide molecule detail to allow us to understand how ApoLI kills trypanosomes and how human infective trypanosomes become resistant to this toxin. This has the potential to help us to design new therapeutics to prevent sleeping sickness. By understanding how SRA binds to and inhibits ApoLI, we can design new versions of ApoLI that are resistant to SRA-mediated inactivation. These can be supplied directly to sleeping sickness patients to kill trypanosomes. We can also develop small molecules that block the SRA-ApoLI interaction, allowing the bodies natural ApoLI-mediated defenses to kill the parasite. Finally, transgenic cattle are being developed which contain ApoLI to prevent trypanosome growth. Using versions of ApoLI which are resistant to SRA-mediated inactivation will decrease the chance of these cattle being infected and providing a reservoir for the growth and development of human infective trypanosomes.