Identification validation and therapeutic potential of cis-trans interactions that direct coordinated gene expression in Plasmodium falciparum

这项研究提出了一个简单的问题，对我们理解疟疾病理学具有广泛的影响-疟疾寄生虫恶性疟原虫如何控制在其复杂的生命周期中打开和关闭其基因的分子过程。疟疾每年造成200多万人死亡，但这一毁灭性的统计数字只代表了“疟疾冰山”的一角-每年有5亿多病例给疟疾流行地区造成重大的健康和财政负担。对寄生虫至关重要的过程，如人类的感染和发育，抗药性和免疫逃避，都依赖于寄生虫遗传组成中存在的5300个基因的协调控制。我们已经知道，疟疾基因表达的分子控制是协调的，与寄生虫发育的不同阶段密切相关;然而，驱动这一协调过程的分子机制（或更可能是它们的组合）仍然困扰着我们。我们所知道的是，它们与人类中存在的那些非常不同。正是这些差异的本质，以及它们如何控制寄生虫特异性的适应，让我感兴趣，因为它们提供了一个有吸引力的机会--差异可以被利用！我的假设是，编码蛋白质的基因一起被打开，一起工作，也一起被控制。我想确定这是如何发生的。这项研究将从一项基于计算机的调查开始，该调查利用了疟疾基因组计划的产品--我们知道所有可能包含基因调控元件的DNA序列，只是不知道它们在哪里。我实验室的模拟实验已经确定了基因组中最有可能包含调控基序的区域，这些基序参与了基因表达的协调控制（打开基因的过程）。这个丰富的DNA序列库将被搜索，以找到我们预测将指导协调表达的共同序列基序。在这一点上，该项目进入实验室。在这里，我们将在转基因寄生虫中测试这些基于计算机的预测，我们将替换，删除或突变这些拟议的调控序列，以了解它们对基因表达的影响-特别是，它们在改变基因表达的水平和/或时间方面的影响。我们还将对与这些拟议的调控序列结合的核蛋白进行测序，从而试图完成我们对不同分子机制在控制这种生物体中遗传信息流动方面的相对贡献的理解。这项研究很重要。我不仅想更好地了解控制基因表达的分子机制如何共同驱动人类和蚊子体内的寄生虫发育，而且还想帮助确定这种基因表达程序对宿主-寄生虫相互作用的致病性的贡献。但是如何将这些信息转化为新的药物或疗法呢？合理药物设计的过程从根本上得到了这种描述性研究的支持，正如格言“了解你的敌人”所最好的例证。为此，这项研究的目的不仅是确定基因调控过程的组成部分，而且还在分子水平上剖析它们如何共同工作。通过这种方式，我想更好地了解寄生虫感染，定植并最终导致其宿主发病的基因调控动态相互作用，确定这些过程如何被颠覆，以获得治疗这种毁灭性人类疾病的急需的替代途径。

This research proposal asks a simple question that has wide ranging implications to our understanding of malaria pathology - how does the malarial parasite Plasmodium falciparum control the molecular process that turn its genes on and off as it progresses through its complex life cycle. Malaria kills over two million people each year, yet this devastating statistic only represents the tip of the 'malaria iceberg' - with over 500 million cases each year resulting in significant health and financial burdens to malaria endemic regions. Processes that are essential to the parasite such as infection and development in humans, drug resistance and immune evasion all rely on the concerted control of the 5300 genes present in the parasite's genetic make-up. We already know that the molecular control of malarial gene expression is coordinated and intimately linked to different stages of parasite development; yet the molecular mechanisms (or more likely their combination) that drive this coordinated process still elude us. What we do know is that they are quite distinct to those present in humans. It is the nature of these differences, and how they control parasite-specific adaptations that interest me as they offer an attractive opportunity - differences can be exploited! My hypothesis is that genes that encode proteins that are turned on together and work together are also controlled together. I want to establish how this happens. The research will start with a computer-based investigation that exploits the product of the malaria genome project - we know all the DNA sequences that could possibly contain gene regulatory elements, we just don't know where they are. Modelling experiments in my laboratory have identified areas of the genome most likely to contain regulatory motifs involved in the coordinated control of gene expression (the process of turning a gene 'on'). This enriched pool of DNA sequences will be searched to find common sequence motifs that we predict will direct coordinated expression. At this point, the project moves into the laboratory. Here we will test these computer-based predictions in genetically-modified parasites where we will replace, delete or mutate these proposed regulatory sequences to see what effect they have on gene expression - specifically, what is their impact in altering the level and/or timing of gene expression. We will also characterise the nuclear proteins that bind to these proposed regulatory sequences, thus attempting to complete our understanding of the relative contributions that different molecular mechanisms make in controlling the flow of genetic information in this organism. This study is important. I want to not only to better understand how the molecular mechanisms that control gene expression come together to drive parasite development in humans and mosquitoes, but also help establish the contribution that this programme of gene expression make to the pathogenicity of the host-parasite interaction. But how can this information be translated into a new drug or therapy? The process of rational drug design is fundamentally underpinned by this type of descriptive research, as is perhaps best exemplified by the adage 'know thine enemy'. Towards this end, this proposed investigation aims not only identify the component parts of the gene regulatory process, but also to dissect at the molecular level how they work together. In this way, I want to better understand the dynamic interplay in gene regulation as the parasite infects, colonizes and often ultimately causes pathogenesis of its host, identifying how these processes may be subverted for much needed alternative routes to treat this devastating human disease.