Parameters that govern initiation of VSG switching in T.brucei

描述(申请人提供)：寄生原生动物布鲁氏锥虫是非洲锥虫病的病原体，非洲锥虫病在人类引起昏睡病，在农场动物引起Nagana。世界卫生组织预测，目前人类非洲锥虫病的疾病负担约为200万“残疾调整寿命年”(每年新增30万例)。再加上Nagana对经济造成的深刻影响，布鲁氏锥虫是非洲大部分地区发展的主要障碍之一。虽然相关寄生虫在进入哺乳动物宿主后利用受保护的生态位，但布鲁氏毛滴虫在哺乳动物的血液中茁壮成长，在那里它必须在体液免疫反应的攻击下生存下来。寄生虫通过其致密的表面涂层做到这一点，表面涂层由约5×106个可变表面糖蛋白(VSG)同源二聚体组成。当这种VSG外壳暴露在抗体下并被抗体识别时，布氏锥虫会定期替换它(在一个被称为“VSG转换”的过程中)，从而使一群寄生虫通过逃避补体介导的裂解而存活下来。关于VSG在分子水平上的开关，人们知之甚少。过去25年的研究已经确定：(A)大多数转换是通过复制的基因转换发生的，(B)关于感染后出现的VSG的类型，存在半可预测的等级。我们最近建立了一个强大的实验系统，我们已经用它成功地复制了体内感染的许多特征。在这里，我们建议使用该系统来实现两个特定的目标：1)确定启动VSG切换的机制。长期以来，有一种假设认为，VSG的转换是由内切酶启动的。或者，转换可能是自发但频繁的DNA断裂的结果，这些DNA断裂可能来自染色体末端特有的内源过程(所有表达的VSG位于染色体末端)。我们提出了实验来评估这两种假设。2)了解支配施主VSG选择的规则，从而导致观察到的切换层次。在VSG转换的早期阶段，感染过程中产生的新的(供体)VSG不是随机的。例如，表达VSG221的细胞倾向于切换到VSG224，无论是在体内(Robinson等人，1999)还是在体外(我们的初步数据)。我们将评估两种机制来解释这一一致的发现：(A)最初的偏好可能是由于表达位点的保守元件(如70bp重复区段)的序列相似性，以及(B)这种偏好可能是两个表达位点在核的背景下接近的结果。除非接受治疗，否则非洲锥虫病总是致命的。确实存在的为数不多的治疗方法昂贵，有严重的副作用，随着耐药布鲁氏菌株的开始出现，治疗效果越来越低。我们提出的工作集中在了解并最终扰乱这种寄生虫免疫逃避的主要已知途径，这也是病原体持续存在的原因。因此，从长远来看，我们提出的这项研究可能会导致一种新的有效方法来预防这种被忽视的疾病。 公共卫生相关性：布鲁氏锥虫寄生虫是非洲人类昏睡病和牛Nagana病的病原体，是撒哈拉以南非洲大部分地区发展的主要障碍之一。布鲁氏毛滴虫通过Tse-Tse载体的咬合传播到哺乳动物的血液中，一旦到达那里，它就会迅速引发抗体介导的免疫反应，通过与暴露在表面的毛皮结合来消除寄生虫。然而，这种寄生虫已经进化出一种生存策略，这取决于它是否有能力改变它的表面皮毛，以逃避宿主的免疫反应。我们提出的工作旨在了解布氏毛滴虫表面毛皮转换的机制，这是病原体持续存在和慢性感染的唯一原因。

DESCRIPTION (provided by applicant): The parasitic protozoan Trypanosoma brucei is the causative agent of African trypanosomiasis which causes sleeping sickness in humans and Nagana in farm animals. The World Health Organization projects that the current disease burden of human African trypanosomiasis is about two million "Disability Adjusted Life Years" (with 300,000 new cases each year). Coupled with the profound effect on the economy caused by Nagana, T. brucei is one of the leading impediments to development in much of Africa. While related parasites utilize protected niches after they enter their mammalian host, T. brucei thrives in the mammalian bloodstream, where it must survive the onslaught of the humoral immune response. The parasite does this by virtue of its dense surface coat, consisting of ~5 x 106 variable surface glycoprotein (VSG) homodimers. While this VSG coat is exposed to and recognized by antibodies, T. brucei periodically replaces it (in a process called "VSG switching") enabling a subpopulation of parasites to survive by evading complement-mediated lysis. Little is known about VSG switching at the molecular level. Work over the last 25 years has determined (a) that the majority of switching occurs by duplicative gene conversion and (b) that there is semi-predictable hierarchy with regard to the types of VSGs that appear early vs. late after infection. We have recently established a robust experimental system, which we have used to successfully reproduce many of the features of in vivo infection. Here, we propose to use this system toward two specific aims: 1) Determine the mechanism that initiates VSG switching. There is a long-standing hypothesis that VSG switching is initiated by an endonuclease. Alternatively, switching could be a result of spontaneous but frequent DNA breaks that can arise from endogenous processes peculiar to chromosome ends (which is where all expressed VSGs are located). We propose experiments to evaluate both hypotheses. 2) Understand the rules that govern choice of donor VSG, leading to the observed hierarchy of switching. In the early stages of VSG switching, the new (donor) VSGs arising in the course of infection are not random. For example, cells expressing VSG221 tend to switch to VSG224, both in vivo (Robinson et al., 1999) and in vitro (our preliminary data). We will evaluate two mechanisms that could explain this consistent finding: (a) initial preference could be due to sequence similarity in conserved elements (such as the 70-bp repeat tracts) of expression sites, and (b) this preference could be the result of proximity of the two expression sites in the context of the nucleus. African trypanosomiasis is always fatal unless treated. The few therapeutic treatment options that do exist are expensive, have serious side effects and are increasingly inefficient as drug resistant T. brucei strains begin to emerge. The work we propose centers upon understanding and eventually disrupting the major known pathway of immune evasion by this parasite which is also the cause of pathogen persistence. As such, in the long term the research we propose could lead to a novel and effective way to protect against this neglected disease. PUBLIC HEALTH RELEVANCE: The parasite Trypanosoma brucei is the causative agent of African sleeping sickness in humans and of Nagana in cattle, and one of the leading impediments to development in much of sub-Saharan Africa. T. brucei is transmitted to the mammalian bloodstream through the bite of the tse-tse vector, and once there, it quickly elicits an antibody mediated immune response which is capable of eliminating the parasite by binding to its exposed surface coat. However, the parasite has evolved a survival strategy that hinges upon its ability to change its surface coat, to evade the host immune response. The work we propose aims to understand the mechanism of T. brucei surface coat switching, which is the sole cause of pathogen persistence and chronic infection.