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，有各种各样的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，许多高度专业化的相互作用仍然鲜为人知。这尤其适用于吞噬原生质体食性的微真核生物，它们穿透藻类的细胞壁并消耗细胞内容物。在过去的几年里，我们在了解原生质体取食者的系统发育多样性、细胞结构和生态学方面取得了进展。然而，目前还不清楚原生质体摄食者到底是如何识别猎物并穿透猎物细胞壁的。关于病毒纲变态鞭毛虫OrciRaptor Agilis(Rhizaria)的转录数据表明，碳水化合物活性酶(CAZymes)介导与猎物细胞壁的结合和细胞壁的溶解。这些CAZyme的蛋白质序列为采取下一步实验步骤提供了理想的基础，以建立一个关于原生质体摄食者如何识别和穿孔猎物细胞壁的精炼模型。利用异源表达系统，我们将生产重组候选CAZymes，并检测其在人工和天然底物上的活性。为了确定这些蛋白质在OrciRaptor中的细胞定位，将提高抗体并用于免疫荧光显微镜。此外，我们将通过探索食菌恐龙近亲的转录本，特别是纤维素酶和碳水化合物结合蛋白的筛选，来研究原生质体摄食者的进化。最后，我们将把我们的研究扩展到金龟子变形虫，以研究这些多才多艺的捕食性变形虫如何适应不同的猎物类型。我们将在广泛的系统发育框架中比较取食生态和CAZyme的表达，并使用差异表达分析来检验通用型金龟子是否调节CAZyme的表达以响应猎物细胞壁的生化反应。通过比较几个遥远的系统发育谱系的原生质体取食者的分子工具包，我们最终将理解这种迷人的取食策略是如何在生命树上独立进化的几次。

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.