the cercozoan flagellate Orciraptor agilis, specifically feed on the contents of dead algal cells, which usually do not burst upon attack. Some vampyrellid amoebae, in particular, are able to simultaneously take up the ejected material in a large food vacuole, which resembles a sucking motion and might be responsible for their figurative name. After the local weakening of the wall by the protoplast feeder, the internal pressure (turgor) of the algal cell causes the cell to burst, i.e. vacuoles, plastids) disintegrate during the attack by a protoplast feeder, which is very obvious in freshwater green algae. In most known cases, the contents of the prey cell (e.g. Some species extract the cell contents of their prey by pseudopodial movements, while others infiltrate, feed and complete their life history inside the prey cell. The highly specialised feeding processes of protoplast feeders typically involves (1) prey cell recognition, (2) attachment, (3) local dissolution of the cell wall and (4) phagocytosis of the cell contents. Idionectes vortex, Schizoplasmodiidae), and Opisthokonta (e.g. vampyrellid amoebae, viridiraptorid flagellates), Amoebozoa (e.g. They are phylogenetically diverse and can be found in several eukaryotic supergroups for example, in the Rhizaria (e.g. Some phagotrophic protists, referred to as “protoplast feeders”, specifically feed on the cell contents of other eukaryotes by penetrating the foreign cell wall. Microbes have evolved various strategies to capitalise on other organisms. With that, we provide evidence that the well-defined perforations produced by protoplast feeders are caused by extracellular carbohydrate-active enzymes and made a first step towards establishing the molecular basis of a fascinating, yet poorly understood microbial feeding strategy. Our experimental data from enzymatic assays, immunocytochemistry and inhibition experiments strongly suggest a key role of the GH5_5 endocellulase in cell wall dissolution by Orciraptor agilis. The cells attacked the algae, which, however, resulted in numerous incomplete perforations. Furthermore, the anti-GH5_5 antibody applied to live cells significantly reduced the feeding success of Orciraptor. Immunocytochemistry with a polyclonal antibody raised against the GH5_5 domain revealed that the native endocellulase localises to the contact zone of Orciraptor and the algal cell wall (= perforation zone) and to intracellular granules, which were enriched during attack. Crystalline cellulose was not digested by the enzyme, which suggests a typical endocellulase activity. The GH5_5 catalytic domain from Orciraptor showed pronounced activity on soluble cellulose derivatives and mixed-linkage glucans, with reaction optima comparable to known GH5_5 representatives. To assess the importance of this carbohydrate-active enzyme in the feeding act of Orciraptor, we recombinantly produced its catalytic domain and studied the enzymatic activity, cellular localisation and function. Differential expression analyses of the algivorous flagellate Orciraptor agilis (Viridiraptoridae, Cercozoa, Rhizaria) suggested the involvement of a highly expressed putative glycoside hydrolase of family GH5_5. Although their fascinating feeding behaviour has been observed for the last 150 years, it is still unknown how protoplast feeders produce the well-defined and species-specific perforations in biochemically diverse cell walls. These phagotrophic “protoplast feeders” represent an interesting mechanistic intermediate between predators and parasites and pose a number of cell biological questions. Several protists have evolved the ability to perforate the cell walls of algae and fungi to specifically feed on their cell contents.
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