Universität zu Köln

Moye, Jannika, Schenk, Tobias and Hess, Sebastian (2022). Experimental evidence for enzymatic cell wall dissolution in a microbial protoplast feeder (Orciraptor agilis, Viridiraptoridae). BMC Biol., 20 (1). LONDON: BMC. ISSN 1741-7007

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Abstract

Background: Several protists have evolved the ability to perforate the cell walls of algae and fungi to specifically feed on their cell contents. These phagotrophic protoplast feeders represent an interesting mechanistic intermediate between predators and parasites and pose a number of cell biological questions. 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. 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. 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. Results: 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. Crystalline cellulose was not digested by the enzyme, which suggests a typical endocellulase activity. 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. Furthermore, the anti-GH5_5 antibody applied to live cells significantly reduced the feeding success of Orciraptor. The cells attacked the algae, which, however, resulted in numerous incomplete perforations. Conclusions: 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. 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.

Item Type:	Journal Article
Creators:	Creators Email ORCID ORCID Put Code Moye, Jannika UNSPECIFIED UNSPECIFIED UNSPECIFIED Schenk, Tobias UNSPECIFIED UNSPECIFIED UNSPECIFIED Hess, Sebastian UNSPECIFIED UNSPECIFIED UNSPECIFIED
URN:	urn:nbn:de:hbz:38-660846
DOI:	10.1186/s12915-022-01478-x
Journal or Publication Title:	BMC Biol.
Volume:	20
Number:	1
Date:	2022
Publisher:	BMC
Place of Publication:	LONDON
ISSN:	1741-7007
Language:	English
Faculty:	Unspecified
Divisions:	Unspecified
Subjects:	no entry
Uncontrolled Keywords:	Keywords Language HYDROLASE FAMILY 5; ESCHERICHIA-COLI; SP VAMPYRELLIDAE; FINE-STRUCTURE; PERFORATION; CLONING; ENDOGLUCANASE; CELLULASE; NOV; PURIFICATION Multiple languages Biology Multiple languages
URI:	http://kups.ub.uni-koeln.de/id/eprint/66084