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 Our unpublished data provide compelling evidence that the protoplast feeder Orciraptor agilis produces a chitin-chitosan co-polymer, which is stored in peripheral extrusomes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 chitin/chitosan polymers from Orciraptor (before and after secretion) by mass spectrometry to reveal modifications introduced during deposition and the feeding act.

2) Assessment of chitooligosaccharide binding on green algal walls and the effect of chitooligosaccharides on Orciraptor’s feeding act.

3) Production and characterization of selected chitinase modules from Orciraptor with special reference to subsite specificity.

4) Localization of selected chitinases throughout Orciraptor’s life history and the feeding act, respectively.

With these objectives, we will create unprecedented knowledge about protistan chitin/chitosan polymers which are clearly involved in cross-species interactions. In addition, we will learn about potentially associated chitin/chitosan-degrading or -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 chitin/chitosan polymers (Code𝛘) and variations in specificity of respective proteins.