Disrupting nuclear condensates and immune evasion mechanisms as a novel antiparasitic strategy

锥虫是伪装大师：非洲锥虫在世界上一些最贫穷的人口中造成巨大的发病率和经济负担。像疟疾寄生虫一样，锥虫可以系统地改变向宿主免疫系统展示的蛋白质的身份-这种抗原变异极大地挑战了针对这些生物体的疫苗开发。事实上，目前没有疫苗可用。 成功的抗原变异的关键是一次表达单一抗原的能力，因为表达多种变体表面糖蛋白（VSG）的锥虫被宿主免疫系统快速清除。值得注意的是，一组形成核凝聚物的蛋白质控制这种单一抗原表达（Faria et al，2019，PMID：31289266; Faria et al，2021，PMID：33432154）。该项目旨在更深入地了解这些冷凝物如何运作，并探索开发一种针对抗原变异的新型战斗方法的潜力。 在锥虫中执行单一VSG表达的核体包括高度必需的解旋酶，称为VEX 2。我们寻求：-调查VEX 2凝聚物形成的生物物理机制;-确定其内源性RNA：DNA底物;-表征其功能结构域和基因组中特定位点的招募;-开发测试生物分子凝聚物的化学破坏的检测方法。 实验方法将包括：-蛋白质-DNA/RNA相互作用和抗原表达的功能研究，以及寄生虫培养，分子生物学，基因编辑（CRISPR/Cas9）和先进测序技术（RNA-Seq，ChIP-Seq，CLIP-Seq，DRIP-Seq）的培训。- 使用尖端成像技术（超分辨率和超结构扩展显微镜，FRAP）研究冷凝物形成/消融。开发体外（基于荧光）和细胞（基于图像）分析，以测试冷凝物的化学破坏。影响和新奇：值得注意的是，锥虫感染中非的人和牲畜（经济损失在10亿至12亿美元之间），迫切需要新的干预战略。疫苗开发的主要障碍之一是它们经历抗原变异的出色能力。几十年来，负责数千种可能基因中单一抗原表达的机制仍然难以捉摸，但最近首次在任何真核生物中被发现，这为特异性靶向抗原变异提供了前所未有的机会。 此外，锥虫代表了一种强大的单细胞模型系统来研究浓缩物的治疗性破坏，因为它们异常地依赖于蛋白质浓缩物的巨大组装来逃避其宿主免疫应答（Budzak等人，2022，PMID：35013170）。开发选择性溶解目前用于癌症或神经退行性疾病背景下的寄生虫特异性冷凝物的策略（Dolgin，2021，PMID：33564162），可以开辟靶向寄生虫免疫逃避的新途径。

Trypanosomes are master of disguise: African trypanosomes cause huge morbidity and economic burden amongst some of the world's poorest populations. Like malaria parasites, trypanosomes can systematically alter the identity of proteins displayed to the host immune system - such antigenic variation has greatly challenged vaccine development against these organisms. Indeed, there is no vaccine currently available. Key to successful antigenic variation is the ability to express a single antigen at a time, as trypanosomes expressing multiple variant-surface-glycoproteins (VSGs) are rapidly cleared by the host immune system. Notably, a set of proteins that form nuclear condensates, controls this singular-antigen-expression (Faria et al, 2019, PMID: 31289266; Faria et al, 2021, PMID: 33432154). This project aims to gain a deeper mechanistic understanding of how these condensates operate and explore the potential to develop a novel combative approach that targets antigenic variation. The nuclear body that enforces singular-VSG-expression in trypanosomes includes a highly essential helicase designated VEX2. We seek to:-Investigate the biophysical mechanism underpinning VEX2 condensates formation;-Identify its endogenous RNA:DNA substrates;-Characterise its functional domains and recruitment to specific loci in the genome;-Develop assays to test chemical disruption of biomolecular condensates. The experimental approaches will include:-Functional studies of protein-DNA/RNA interactions and antigen expression with training in parasite culture, molecular biology, gene editing (CRISPR/Cas9) and advanced sequencing techniques (RNA-Seq, ChIP-Seq, CLIP-Seq, DRIP-Seq). -Study of condensate formation/ablation using cutting-edge imaging techniques (super-resolution and ultra-structure expansion microscopy, FRAP).-Development of in vitro (fluorescence-based) and cellular (image-based) assays to test chemical disruption of condensates. Impact & Novelty: Notably, trypanosomes infect people and devastate livestock in central Africa (economic losses in the range of US$1.0-1.2 billion), new intervention strategies are desperately needed. One of the main obstacles to vaccine development has been their eximious ability to undergo antigenic variation. The machinery responsible for singular-antigen-expression out of thousands of possible genes remained elusive for decades but has recently been identified for the first time in any eukaryote, presenting an unprecedented opportunity to specifically target antigenic variation. Moreover, trypanosomes represent a powerful unicellular model system to investigate the therapeutic disruption of condensates, as they are unusually reliant on a huge assembly of protein condensates to evade their host immune response (Budzak et al, 2022, PMID: 35013170). Developing strategies to selectively dissolve parasite-specific condensates, currently used in the context of cancer or neurodegenerative diseases (Dolgin, 2021, PMID: 33564162), can open a novel avenue to target parasite immune evasion.