Protist, Vol. 171, 125717, xx 2020 http://www.elsevier.de/protis Published online date 7 February 2020 ORIGINAL PAPER Unique Dynamics of Paramylon Storage in the Marine Euglenozoan Diplonema papillatum a,b a a,c Ingrid Skodová-Sverákovᡠ, Galina Prokopchuk , Priscila Pena-Diaz˜ , a,c d b d a,e,1 Kristína Záhonová , Martin Moos , Anton Horváth , Petr Simekˇ , and Julius Lukesˇ a ˇ Institute of Parasitology, Biology Centre, Czech Academy of Sciences, Ceské Budejoviceˇ (Budweis), Czech Republic b Faculty of Natural Sciences, Comenius University, Bratislava, Slovakia c Faculty of Science, Charles University, BIOCEV, Vestec, Czech Republic d ˇ Institute of Entomology, Biology Centre, Czech Academy of Sciences, Ceské Budejoviceˇ (Budweis), Czech Republic e ˇ Faculty of Sciences, University of South Bohemia, Ceské Budejoviceˇ (Budweis), Czech Republic Submitted September 10, 2019; Accepted February 2, 2020 Monitoring Editor: Michael Ginger Diplonemids belong to the most diverse and abundant marine protists, which places them among the key players of the oceanic ecosystem. Under in vitro conditions, their best-known representa- tive Diplonema papillatum accumulates in its cytoplasm a crystalline polymer. When grown under the nutrient-poor conditions, but not nutrient-rich conditions, D. papillatum synthesizes a ␤-1,3-glucan polymer, also known as paramylon. This phenomenon is unexpected, as it is in striking contrast to the accumulation of paramylon in euglenids, since these related flagellates synthesize this polymer solely under nutrient-rich conditions. The capacity of D. papillatum to store an energy source in the form of polysaccharides when the environment is poor in nutrients is unexpected and may contribute to the wide distribution of these protists in the ocean. © 2020 Elsevier GmbH. All rights reserved. Key words: ␤-1,3-glucan; paramylon; trehalose; diplonemids; paramylon synthase. Introduction 2015). The capacity of protists to inhabit virtually every niche in the world ocean is reflected in their metabolism, which is often highly complex and ver- Protists represent the bulk of extant eukaryotic satile due to their gene-rich genomes (Keeling et al. diversity, yet their abundance and ecological sig- 2014; Keeling and del Campo 2017). nificance in the oceans is being appreciated only One of the prominent groups are diplonemids, a recently (de Vargas et al. 2015; Worden et al. sister clade of the mostly parasitic kinetoplastids and the free-living euglenids, all belonging to the 1 phylum Euglenozoa (Adl et al. 2018). Diplonemids Corresponding author; th are the most diverse and the 6 most species-rich e-mail [email protected] (J. Lukes).ˇ https://doi.org/10.1016/j.protis.2020.125717 1434-4610/© 2020 Elsevier GmbH. All rights reserved. ˇ 2 I. Skodová-Sveráková, G. Prokopchuk, P. Pena-Diaz˜ et al. group of marine protists (de Vargas et al. 2015; survival in the mammalian host (Ralton et al. 2003; Flegontova et al. 2016). The available sequence Sernee et al. 2006). Thus, the polymer synthesis data shows that they are non-photosynthetic, which and remodeling mostly depend on the parasite life is consistent with their preference for mesopelagic stage, rather than on carbon availability. and bathypelagic habitats (Flegontova et al. 2016; Out of the diverse euglenozoans with lifestyles Lara et al. 2009). So far, the only diplonemid exper- ranging from parasitism to photosynthesis (Adl et al. imentally studied to some extent is Diplonema 2018), only the euglenophytes carrying the sec- papillatum, best-known for the unprecedented ondary plastid are known to deposit glucose via ␤ complexity of its mitochondrial genome and tran- -1,3-glycosidic linkage, forming a storage polysac- scriptome (Valach et al. 2017). Moreover, studies charide known as paramylon (Kiss and Triemer implementing genetic modifications in D. papillatum 1988). However, outside of the euglenozoans, lin- have begun to turn this flagellate into a genetically ear ␤-1,3-glucans are widely distributed in bacteria, tractable organism (Kaur et al. 2018). haptophytes, fungi, lichens and some plants, where D. papillatum has also been the subject of the the polymer carries different names, such as curd- only metabolic study performed on diplonemids so lan, pachyman, laricinan or callose (Archibald et al. far, which revealed that they compartmentalize five 1963; Aspinall and Kessler 1957; Clarke and Stone of the first seven enzymes of the gluconeogenic 1960; Harada et al. 1968; Warsi and Whelan 1957). pathway into peroxisomes, which are thus reminis- Paramylon is an insoluble, linear ␤-1,3-glucan of cent of glycosomes (Morales et al. 2016). While high molecular mass, which occurs naturally in the hexokinase and phosphofructokinase do not seem crystalline form (Booy et al. 1981). Euglenids accu- to be expressed, D. papillatum was shown to per- mulate paramylon in granules, which may be widely form gluconeogenesis. Consequently, it has been distributed in the cytoplasm, may form caps over hypothesized that this protist is incapable of per- the pyrenoids or be packed together (Monfils et al. forming glycolysis, with the gluconeogenic flux 2011). The various types of paramylon were sepa- originating from the metabolism of amino acids rated into six morphological categories, all present being routed through the pentose phosphate path- in the diverse euglenids (Monfils et al. 2011). way (Morales et al. 2016). In trypanosomes and The formation of this polysaccharide follows related flagellates, the first seven of 10 glycolytic a general pathway where a uridine diphosphate enzymes are also compartmentalized in glyco- glucose (UDP-Glc) donates the glucose moiety somes, a hallmark of these protists (Bringaud et al. that is subsequently linked to form the grow- 2006; Opperdoes and Borst 1977). They metab- ing polysaccharide. Glucose for the UDP-Glc olize glucose and amino acids, with proline and precursor originates from various sources and threonine being essential for energy production the formation of the sugar nucleotide depends (Coustou et al. 2003; Millerioux et al. 2013). Gluco- on UTP:␣-D-glucose uridylyltransferase or UDP- neogenesis has been reported from trypanosomes Glc pyrophosphorylase (Muchut et al. 2018). In as a flux that may be directed through the pen- euglenids, paramylon formation requires the activ- tose phosphate pathway for the regeneration of ity of paramylon synthase (Bäumer et al. 2011; cytosolic NADPH (Allmann et al. 2013). This unique Marechal and Goldemberg 1964; Tanaka et al. compartmentalization of glycolytic enzymes has 2017), which is a membrane-bound enzyme com- been proposed as an adaptation to their special- plex of approximately 670 kDa belonging to the ized lifestyles (Haanstra et al. 2016; Szöor et al. eukaryotic family of glycosyltransferases 48 (GT48) 2014). (Bäumer et al. 2011). So far, two glucan synthase- Although trypanosomes lack the capability of like genes, called EgGSL1 and EgGSL2, were storing carbohydrates (Bakker et al. 2000), they found in Euglena gracilis. They code for proteins may store carbon sources in the form of lipid of 304 and 258 kDa, with 15 and 19 transmem- droplets (Allmann et al. 2014; Smith et al. 2017). brane domains, respectively (Tanaka et al. 2017). Moreover, members of the genus Leishmania dif- The synthesis of paramylon, however, relies on the fer from trypanosomes in that they synthesize a activity of EgGSL2, which forms a complex with carbohydrate polymer that resembles a mannose 37 and 54 kDa UDP-binding proteins (Tanaka et al. polymer or mannan (Blum 1993). Leishmania mex- 2017). The activity of paramylon synthase is associ- icana was subsequently shown to synthesize and ated with the membrane fraction that surrounds the ␤ store -1,2-mannan, a polymer of mannose. How- paramylon granules (Bäumer et al. 2011). Degra- ever, glycopolymers in Leishmania species are dation of ␤-1,3-glucan involves the action of endo- linked to the assembly of the glycocalyx coat and and exo-␤-1,3-glucanases found in various glyco- have an essential function in their infectivity and Paramylon in Diplonema 3 side hydrolase families (Henrissat 1991; Henrissat medium. Since tryptone is the main carbon source and Bairoch 1996; Henrissat and Davies 1997). in this medium, and to a lower extent, of nitro- Paramylon has been extensively studied in gen, hereafter we refer to them as carbon-rich and E. gracilis, where its synthesis is usually trig- carbon-poor media. Both media had 3.6% salinity gered by growth under the nutrient-rich conditions and contained 1% (v/v) horse serum. Inoculation (Calvayrac et al. 1981), represented by either pho- of the cells into the carbon-poor medium resulted tosynthetic carbon assimilation or heterotrophic in a significant growth retardation and a switch growth on a suitable carbon source, such as sug- of the cells into what may be considered a per- ars or organic acids (Briand and Calvayrac 1980). manent stationary phase (Fig. 1A), as compared The dynamics of paramylon synthesis in E. gracilis to the carbon-rich culture condition, under which is influenced by light, as the metabolite accumu- the cells grew exponentially for 11 days (264 hrs) lated during the dark phase may be consumed (Fig. 1A). A significant accumulation of cytosolic for chlorophyll synthesis in the presence of light vesicles occurred in cells grown in the carbon-poor (Calvayrac et al. 1981; Dwyer and Smillie 1970; medium (Fig. 1B). When viewed with a light micro- Schwartzbach et al. 1975). When E. gracilis is cul- scope, diplonema cells appeared to have higher tured under carbon starvation, the breakdown of motility in the carbon-poor medium as compared paramylon is triggered by light, while this process to the carbon-rich one, with their morphology unal- is slower in cells grown in the dark (Dwyer and tered (Fig. 1B). Smillie 1970). Paramylon hydrolysis may also be triggered by several factors other than metabolic Characterization of Paramylon requirements, such as osmotic stress or environ- The carbohydrate polymer isolated from approx- mental adaptations (Takenaka et al. 1997). 6 7 imately 8 × 10 and 4 × 10 E.