Biopolymer degradation by phagotrophic protoplast feeders (Viridiraptoridae) and associated carbohydrate-active enzymes

The heterotrophic protist Orciraptor agilis (Viridiraptoridae, Rhizaria) is a naked amoeboflagellate, which inhabits fens and bogs and feeds on dead freshwater green algae. Orciraptor exhibits mainly three different life history stages: 1) free gliding/searching for food, 2) attacking/feeding and 3) dividing/digesting. The amoeboflagellate performs a very specialized feeding strategy called protoplast feeding. After the recognition of a suitable prey cell and adhesion to the algal cell wall, Orciraptor perforates the cell wall and subsequently extracts the algal protoplast. The green algal cell wall composition differs strongly between organisms, but consists mainly of carbohydrates, like cellulose and pectins. An enzymatic degradation of the foreign cell wall would require the action of carbohydrate-active enzymes (CAZymes), as it is known for many phytopathogenic fungi, which express a large toolbox of cell wall-degrading CAZymes. A comparative transcriptome of Orciraptor revealed a large CAZyme repertoire, including an enzyme annotated as GH5_5 endocellulase. This enzyme was also the highest expressed and upregulated during the attacking stage. Furthermore, a surprisingly high number of proteins with a potential chitin-related function was found, with multiple factors being upregulated during the attacking stage. While Orciraptor’s prey is not known to utilize chitin. This thesis follows a workflow of transcriptome-informed cell biology, to substantiate indications given by the comparative transcriptome with experimental data. The following questions were addressed in this thesis: (1) Which eukaryotic and prokaryotic organisms are consumed by Orciraptor and which feeding strategies are used? (2) Is the cell wall degradation driven by enzymatic dissolution? (3) Is Orciraptor capable of producing chitinous material and what is its function? In this thesis, Orciraptor was established as broad-range, obligatory necrophage with a great flexibility concerning its feeding strategies. The broad prey range, including cells with a variety of different cell envelopes, correlates well with the diversity of CAZyme genes found in previous studies. The highest expressed enzyme was characterized and confirmed to act as endocellulase. Utilizing custom antibodies, the endocellulase was found to localise directly to the perforation zone on the algal cell wall and additionally to intracellular granules. With this, the first experimental evidence of an enzyme-driven cell wall degradation was provided. Furthermore, Orciraptor was shown to synthesize a novel, highly acetylated chitosan, which is stored in extrusomes. The chitosan polymer is likely secreted onto the algal cell wall, functioning as adhesive material during the attacking stage. Overall, the transcriptome-informed cell biology applied to the non-model protist Orciraptor elucidated fascinating aspects of the interactions of a protoplast feeder with algal cell walls.