Chemical proteomic mapping of redox signalling in the intracellular pathogen, Toxoplasma gondii

背景在所有原生动物寄生虫感染中，弓形虫是分布最广的，感染了88个国家30%-50%的人口。临床弓形虫病对免疫缺陷个体的健康构成严重威胁，弓形虫是一种重要的兽医病原体，给农业生产带来经济负担。然而，目前的药物存在毒性、治疗方案延长和出现耐药性等问题。因此，继续需要发现新的药物靶点。作为一种专性的细胞内病原体，弓形虫需要宿主细胞的环境才能生存和繁殖。发病机制是由无性速殖子的迭代裂解生长驱动的，这一过程与寄生虫和宿主中发生的一系列分子事件有关。钙、钾和环核苷酸等信号分子是速殖子入侵、复制和排出的关键调节因子。最近，过氧化氢(H_2O_2)和一氧化氮(NO)被认为是真核生物中重要的信号分子。虽然众所周知是压力的转导物质，但在低水平下，这些活性氧物种(ROS)可以通过氧化还原敏感的半胱氨酸残基的可逆、离散和选择性氧化来调节蛋白质的功能。对于过氧化氢，半胱氨酸氧化可以改变不同蛋白质类别的活性和/或定位，包括激酶、磷酸酶、离子通道和代谢酶。事实上，弓形虫在整个生命周期中都会遇到ROS，并在宿主巨噬细胞的氧化挑战过程中抑制过氧化氢的产生。尽管如此，氧化还原信号对寄生虫生物学的全面影响尚不清楚。作为识别弓形虫和疟原虫等细胞内病原体蛋白质组中新的可药物结节的努力的一部分，该项目的首要目标是从分子上定位弓形虫的氧化还原信号。为了实现这一目标，将使用跨越生物化学、分子、细胞和化学生物学的多学科技术的组合。利用化学蛋白质组识别与蛋白质相关的反应性半胱氨酸氧化还原信号与反应性半胱氨酸硫醇对氧化翻译后修饰的敏感性有关。为了首先描述弓形虫蛋白质组中半胱氨酸硫醇的反应性，将在已发表的平台上建立基于定量质谱学的化学蛋白质组工作流程。将对确认的HIT进行一系列生物信息学分析，以深入了解它们的生物学重要性，并优先进行下游分子询问2。活性半胱氨酸的系统遗传学验证为了系统地评估已鉴定的活性半胱氨酸对蛋白质功能和寄生虫适合性的贡献，将进行一种基于CRISPR/Cas9的新的表型筛选。那些被认为是必需的半胱氨酸将优先用于使用传统的遗传和生化方法进行验证。氧化还原感受器的鉴定和特征目标1中描述的蛋白质组工作流程将被修改，以能够检测对过氧化氢氧化敏感的活性半胱氨酸，从而具有转导氧化还原信号的能力。氧化还原传感器将得到生化验证，反向遗传方法将用于评估它们对宿主细胞入侵、复制和逃逸等基本细胞过程的贡献。广泛影响目前共价药物的复苏反映了它们在临床上的成功记录。例如抑制剂Afatinib和Ibrutinib，它们与癌症相关的激酶形成共价键。随着伊布鲁替尼的全球销售额预计在2020年达到90亿美元，工业对靶向半胱氨酸的共价抑制剂的兴趣持续存在。我们的研究有可能在临床上重要的寄生虫中发现新的可用药热点，从而对工业和健康产生广泛影响。

BackgroundOf all protozoan parasitic infections, T. gondii is the most widespread, infecting 30-50% of the human population across 88 countries. Clinical toxoplasmosis is a serious health risk for immunodeficient individuals, and T. gondii is a significant veterinary pathogen, imposing economic burden on agricultural industries. However, current drugs suffer from issues with toxicity, prolonged treatment regimens and the emergence of resistance. Hence, there is a continuing need to discover novel drug targets.As an obligate intracellular pathogen, T. gondii requires the environment of a host cell to survive and propagate. Pathogenesis is driven by iterative lytic growth of asexual tachyzoites, a process associated with a range of molecular events that occur in both the parasite and host. Signalling molecules such as Ca2, K and cyclic nucleotides are key regulators of tachyzoite invasion, replication and egress. More recently, hydrogen peroxide (H2O2) and nitric oxide (NO) have been recognised as important signalling molecules in eukaryotes. While notorious transducers of stress, at low levels these reactive oxygen species (ROS) can modulate protein function via reversible, discrete and selective oxidation of redox-sensitive cysteine residues. For H2O2, cysteine oxidation can alter the activity and/or localisation of diverse protein classes including kinases, phosphatases, ion channels and metabolic enzymes. Indeed, T. gondii encounters ROS throughout its life cycle and is known to suppress the production of H2O2 during oxidative challenge by host macrophages. Despite this, the full impact of redox signals on the parasite's biology is unclear.As part of efforts to identify novel druggable nodes in the proteomes of intracellular pathogens such as T. gondii and Plasmodium, the overarching aim of this project is to molecularly map redox signalling in T. gondii. To achieve this, a combination of multidisciplinary techniques spanning biochemistry, molecular, cell and chemical biology will be used.Objectives1. Identify protein-associated reactive cysteines using chemical proteomicsRedox signalling is associated with the sensitivity of reactive cysteine thiols to oxidative post-translational modification. To first profile cysteine thiol reactivity in the T. gondii proteome, a quantitative mass-spectrometry-based chemical proteomic workflow will be established based on a published platform. A series of bioinformatics analyses will be performed on identified hits to gain insight into their biological importance and prioritise downstream molecular interrogation.2. Systematic genetic validation of reactive cysteinesTo systematically assess the contribution of the identified reactive cysteines to protein function and parasite fitness, a novel CRISPR/Cas9-based phenotypic screen will be undertaken. Those cysteines considered essential will be prioritised for validation using traditional genetic and biochemical approaches.3. Identification and characterisation of redox sensorsThe proteomic workflow described in Objective 1 will be modified to enable detection of reactive cysteines that are sensitive to H2O2 oxidation and thus have the capacity to transduce redox signals. Redox sensors will be validated biochemically, and reverse genetic approaches will be used to assess their contribution to essential cellular processes including host-cell invasion, replication and egress.Broad impactThe current resurgence of covalent drugs reflects their track-record of success in the clinic. Examples include the inhibitors Afatinib and Ibrutinib, which form covalent bonds with cancer-associated kinases. With global sales of Ibrutinib expected to reach $9 billion in 2020, there is sustained industrial interest in covalent inhibitors targeting cysteines. Our research has the potential to uncover new druggable hotspots in clinically-important parasites, and thus has broad impact on industry and health.