Singh Shishir, Murillo-León Mateo, Bastidas Quintero Aura María, Melbert Florence, Pfeffer Klaus, Degrandi Daniel, Steinfeldt Tobias
Institute of Virology, Medical Center University of Freiburg, Freiburg, Germany.
Faculty of Biology, University of Freiburg, Freiburg, Germany.
Front Immunol. 2025 Jul 9;16:1593785. doi: 10.3389/fimmu.2025.1593785. eCollection 2025.
() virulence in mice depends on different multiprotein complexes that assemble at the parasitophorous vacuole membrane (PVM) of the parasite. Individual rhoptry proteins within these complexes inhibit different Immunity-Related GTPases (IRG proteins). The rhoptry pseudokinase ROP5 is a central element to achieve IRG-specific rhoptry kinase activity and/or efficient complex formation. The locus of each of the canonical strains encodes three major isoforms, ROP5A, ROP5B and ROP5C, and was shown to have the largest impact on virulence. By reverse genetics, we have generated strains expressing either ROP5A, ROP5B or ROP5C in a RHΔ genetic background and demonstrate that ROP5B is mainly responsible for heightened virulence of type I in laboratory strains of mice. virulence correlates with diminished vacuolar IRG protein loading and parasite control only in presence of ROP5B but not ROP5A or ROP5C. Our results suggest that ROP5A and ROP5C isoforms might have co-evolved with IRG proteins or other host cell resistance factors in evolutionarily important intermediate hosts beyond . The same parasite effectors that inhibit IRG protein accumulation and function reduce the vacuolar amount of Guanylate Binding Proteins (GBP proteins). However, a parasite effector targeting a GBP protein at the PVM has not been described yet. Using two different approaches, Yeast Two-Hybrid analysis and Protein-fragment complementation assay, we here identified three heterologous IRG:GBP pairs, GBP6:Irgb10, GBP5:Irgb10, GBP5:Irgb6, and demonstrate that the accumulation of these GTPases at the PVM is interdependent. Our results offer a novel perspective on the IRG and GBP protein-mediated control of infections and may further advance the investigation of GBP-specific effectors.
（）在小鼠中的毒力取决于在寄生虫的寄生泡膜（PVM）上组装的不同多蛋白复合物。这些复合物中的单个棒状体蛋白会抑制不同的免疫相关GTP酶（IRG蛋白）。棒状体假激酶ROP5是实现IRG特异性棒状体激酶活性和/或有效复合物形成的核心要素。每个经典菌株的位点编码三种主要异构体，ROP5A、ROP5B和ROP5C，并且已证明其对毒力的影响最大。通过反向遗传学，我们在RHΔ遗传背景下产生了表达ROP5A、ROP5B或ROP5C的菌株，并证明ROP5B主要负责I型在实验室小鼠菌株中的高毒力。只有在存在ROP5B而不是ROP5A或ROP5C的情况下，毒力才与液泡IRG蛋白负载减少和寄生虫控制相关。我们的结果表明，ROP5A和ROP5C异构体可能在进化上重要的中间宿主中与IRG蛋白或其他宿主细胞抗性因子共同进化。抑制IRG蛋白积累和功能的相同寄生虫效应器会减少鸟苷酸结合蛋白（GBP蛋白）的液泡量。然而，尚未描述在PVM处靶向GBP蛋白的寄生虫效应器。使用两种不同的方法，酵母双杂交分析和蛋白质片段互补分析，我们在此鉴定出三对异源IRG：GBP对，GBP6：Irgb10、GBP5：Irgb10、GBP5：Irgb6，并证明这些GTP酶在PVM处的积累是相互依赖的。我们的结果为IRG和GBP蛋白介导的弓形虫感染控制提供了新的视角，并可能进一步推进对GBP特异性弓形虫效应器的研究。
