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

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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.