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组成部分。正如环境测序所表明的那样，这些高度专业化的相互作用中有许多仍然知之甚少。这特别适用于食藻原生质体饲养者，即吞噬藻类细胞壁并消耗细胞内容物的噬菌体微真核生物。在过去的几年里，我们取得了进展，了解系统发育多样性，细胞结构和生态的原生质体饲养。然而，目前还不清楚原生质体饲养者如何准确地识别猎物并覆盖猎物细胞壁。关于绿盗龙类变形虫Orciraptor agilis（Rhizaria）的转录组学数据表明，碳水化合物活性酶（CAZymes）介导与猎物细胞壁的结合和细胞壁溶解。这些CAZymes的蛋白质序列代表了采取下一个实验步骤的理想基础，以建立一个关于原生质体饲养者如何识别和识别其猎物细胞壁的精细模型。利用异源表达系统，我们将生产重组候选CAZymes并测定它们在人工和天然底物上的活性。为了建立这些蛋白质在盗龙中的细胞定位，将产生抗体并用于免疫荧光显微镜检查。此外，我们将通过探索盗龙的食菌亲戚的转录组，特别是筛选纤维素酶和碳水化合物结合蛋白来研究原生质体饲养者的进化。最后，我们将把我们的研究扩展到吸血虫变形虫，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。我们将比较饲养生态和CAZyme表达在一个广泛的系统发育框架和测试与差异表达分析，如果通才vampyrellids调节CAZyme表达的猎物细胞壁生物化学。通过比较几个遥远的系统发育谱系的原生质体饲养者的分子工具包，我们将最终了解这种迷人的喂养策略是如何在生命树中独立进化多次的。

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.