Activity, regulation and evolution of carbohydrate-active enzymes used in cell wall penetration by unicellular predators and parasitoids of microalgae

在水生和陆地生态系统中，有多种寄生微生物攻击微藻和真菌，形成微生物食物网的一个组成部分。正如环境测序所表明的那样，许多这些高度专业化的相互作用仍然鲜为人知。这尤其适用于食性原生质体食性生物，即能穿透藻类细胞壁并消耗细胞内容物的吞噬性微真核生物。近年来，我们在原生质体取食动物的系统发育多样性、细胞结构和生态学方面取得了一定的进展。然而，目前还不清楚原生质体捕食者是如何识别猎物并穿透猎物细胞壁的。关于变形虫鞭毛虫Orciraptor agilis （Rhizaria）的转录组学数据表明，碳水化合物活性酶（CAZymes）介导了与猎物细胞壁的结合和细胞壁的溶解。这些CAZymes的蛋白质序列为下一步的实验步骤提供了理想的基础，以建立原生质体捕食者如何识别和穿透猎物细胞壁的精细模型。利用异源表达系统，我们将产生重组候选酶，并测定其在人工和天然底物上的活性。为了在Orciraptor中建立这些蛋白质的细胞定位，抗体将被提高并用于免疫荧光显微镜。此外，我们将通过探索Orciraptor的细菌近亲的转录组来研究原生质体捕食者的进化，特别是筛选纤维素酶和碳水化合物结合蛋白。最后，我们将把我们的研究扩展到吸血鬼阿米巴，研究这些多才多艺的掠食性阿米巴是如何适应不同的猎物类型的。我们将在广泛的系统发育框架内比较摄食生态学和CAZyme的表达，并通过差异表达分析来测试多面手吸血鬼是否会根据猎物细胞壁的生化反应调节CAZyme的表达。通过比较几个遥远的系统发育谱系的原生质体捕食者的分子工具箱，我们将最终了解这种迷人的捕食策略是如何在生命之树中独立进化几次的。

There is a diversity of parasitoid microbes that attack microalgae and fungi in aquatic and terrestrial ecosystems, forming an integral part of the microbial food web. As suggested by environmental sequencing, many of these highly-specialised interactions are still poorly known. This applies in particular to algivorous protoplast feeders, phagotrophic microeukaryotes that perforate the cell walls of algae and consume the cell contents. During the past years we made progress in understanding the phylogenetic diversity, cellular structure and ecology of protoplast feeders. However, it is still unclear how exactly protoplast feeders recognise their prey and perforate the prey cell wall. Transcriptomic data about the viridiraptorid amoeboflagellate Orciraptor agilis (Rhizaria) suggest that carbohydrate-active enzymes (CAZymes) mediate binding to the prey cell wall and cell wall dissolution. The protein sequences of these CAZymes represent an ideal basis for taking the next experimental steps, to establish a refined model of how protoplast feeders recognise and perforate the cell walls of their prey. Employing heterologous expression systems, we will produce recombinant candidate CAZymes and assay their activity on artificial and natural substrates. To establish the cellular localisation of these proteins in Orciraptor, antibodies will be raised and used for immunofluorescence microscopy. Furthermore, we will investigate the evolution of protoplast feeders by exploring the transcriptomes of the bacterivorous relatives of Orciraptor, specifically screening for cellulases and carbohydrate-binding proteins. Finally, we will expand our research to the vampyrellid amoebae to study how these versatile predatory amoebae adapted to diverse prey types. We will compare the feeding ecology and CAZyme expression in a wide phylogenetic framework and test with differential expression analyses if generalist vampyrellids regulate CAZyme expression in response to the prey cell wall biochemistry. By comparing the molecular toolkit of protoplast feeders of several distant phylogenetic lineages, we will finally understand how this fascinating feeding strategy evolved several times independently in the tree of life.