The role of CodeChi and chitin-associated proteins in the cell-cell interactions of free-living protists

鉴于原生生物的巨大多样性，几丁质生物聚合物及其在非真菌微真核生物中的作用尚未得到充分研究。除极少数例子外，对原菌甲壳素和壳聚糖的化学特性和性质了解甚少，值得进一步研究。在我的实验室里，我们研究自由生活的原生生物，它们具有不同的系统发育关系，专门以其他真核生物的细胞内容物为食，尤其是藻类。这些所谓的“原生质体捕食者”局部降解猎物细胞壁，随后吞噬细胞内容物。我们未发表的数据提供了令人信服的证据，证明原生质体捕食者Orciraptor agilis产生一种几丁质-壳聚糖共聚物，该共聚物储存在外周挤压体中，并在捕食过程中沉积在藻类细胞壁上。这表明甲壳素物质和相关的酶在这些吞噬原生生物和它们的藻类猎物之间的细胞相互作用中的功能尚不清楚。本项目的主要目标是：1)通过质谱法详细分析Orciraptor（分泌前后）的C/CS聚合物，以揭示沉积和摄食过程中引入的修饰。2)评价COS与绿藻壁的结合及COS对盗龙摄食行为的影响。3) Orciraptor几种几丁质酶模块的生产和表征，特别参考亚位点特异性。4)在Orciraptor的生活史和摄食行为中选择几丁质酶的定位。有了这些目标，我们将创造有关原生生物C/CS聚合物的前所未有的知识，这些聚合物显然涉及跨物种相互作用。此外，我们将了解该原生生物潜在的相关C/ cs降解或C/ cs结合蛋白，以进一步完善其与微藻相互作用的图谱。这些数据将有助于了解非真菌微真核生物的生物相互作用如何依赖于C/CS聚合物（CodeChi）的化学性质和各自蛋白质特异性的变化。

Given the enormous diversity of protists, chitinous biopolymers and their roles are vastly understudied in non-fungal microeukaryotes. Except for very few examples, the chemical characteristics and properties of protistan chitin and chitosan are poorly known and deserve future study. In my lab, we study free-living protists of diverse phylogenetic affiliation that specifically feed on the cell contents of other eukaryotes, especially algae. These so-called “protoplast feeders” locally degrade the prey cell wall and subsequently phagocytose the cell contents. Our unpublished data provide compelling evidence that the protoplast feeder Orciraptor agilis produces a chitin-chitosan co-polymer, which is stored in peripheral extrusomes and deposited onto the algal cell wall during the feeding act. This points to a yet unknown function of chitinous substances and associated CAZymes in cell-cell interactions between these phagotrophic protists and their algal prey. The major objectives of the proposed project are: 1) The detailed analysis of C/CS polymers from Orciraptor (before and after secretion) by mass spectrometry to reveal modifications introduced during deposition and the feeding act. 2) Assessment of COS binding on green algal walls and the effect COS on Orciraptor’s feeding act. 3) Production and characterisation of selected chitinase modules from Orciraptor with special reference to subsite specificity. 4) Localisation of selected chitinases throughout Orciraptor’s life history and the feeding act, respectively. With these objectives, we will create unprecedented knowledge about protistan C/CS polymers which are clearly involved in cross-species interactions. In addition, we will learn about potentially associated C/CS-degrading or C/CS-binding proteins of this protist to further complete our picture of its interaction with microalgae. These data will help to understand how biotic interactions of non-fungal microeukaryotes rely on the chemical properties of C/CS polymers (CodeChi) and variations in specificity of respective proteins.