Over the last decade, research interest in defining how extracellular vesicles (EVs) shape cross-species communication has grown rapidly. Parasitic helminths, worm species found in the phyla... Show moreOver the last decade, research interest in defining how extracellular vesicles (EVs) shape cross-species communication has grown rapidly. Parasitic helminths, worm species found in the phyla Nematoda and Platyhelminthes, are well-recognised manipulators of host immune function and physiology. Emerging evidence supports a role for helminth-derived EVs in these processes and highlights EVs as an important participant in cross-phylum communication. While the mammalian EV field is guided by a community-agreed framework for studying EVs derived from model organisms or cell systems [e.g., Minimal Information for Studies of Extracellular Vesicles (MISEV)], the helminth community requires a supplementary set of principles due to the additional challenges that accompany working with such divergent organisms. These challenges include, but are not limited to, generating sufficient quantities of EVs for descriptive or functional studies, defining pan-helminth EV markers, genetically modifying these organisms, and identifying rigorous methodologies for in vitro and in vivo studies. Here, we outline best practices for those investigating the biology of helminth-derived EVs to complement the MISEV guidelines. We summarise community-agreed standards for studying EVs derived from this broad set of non-model organisms, raise awareness of issues associated with helminth EVs and provide future perspectives for how progress in the field will be achieved. Show less
alpha-galactosidase (alpha-GAL) and alpha-N-acetylgalactosaminidase (alpha-NAGAL) are two glycosyl hydrolases responsible for maintaining cellular homeostasis by regulating glycan substrates on... Show morealpha-galactosidase (alpha-GAL) and alpha-N-acetylgalactosaminidase (alpha-NAGAL) are two glycosyl hydrolases responsible for maintaining cellular homeostasis by regulating glycan substrates on proteins and lipids. Mutations in the human genes encoding either enzyme lead to neurological and neuromuscular impairments seen in both Fabry- and Schindler/Kanzaki- diseases. Here, we investigate whether the parasitic blood fluke Schistosoma mansoni, responsible for the neglected tropical disease schistosomiasis, also contains functionally important alpha-GAL and alpha-NAGAL proteins. As infection, parasite maturation and host interactions are all governed by carefully-regulated glycosylation processes, inhibiting S. mansoni's alpha-GAL and alpha-NAGAL activities could lead to the development of novel chemotherapeutics. Sequence and phylogenetic analyses of putative alpha-GAL/alpha-NAGAL protein types showed Smp_089290 to be the only S. mansoni protein to contain the functional amino acid residues necessary for alpha-GAL/alpha-NAGAL substrate cleavage. Both alpha-GAL and alpha-NAGAL enzymatic activities were higher in females compared to males (p<0.05; alpha-NAGAL > alpha-GAL), which was consistent with smp_089290's female biased expression. Spatial localisation of smp_089290 revealed accumulation in parenchymal cells, neuronal cells, and the vitellaria and mature vitellocytes of the adult schistosome. siRNA-mediated knockdown (>90%) of smp_089290 in adult worms significantly inhibited alpha-NAGAL activity when compared to control worms (siLuc treated males, p<0.01; siLuc treated females, p<0.05). No significant reductions in alpha-GAL activities were observed in the same extracts. Despite this, decreases in alpha-NAGAL activities correlated with a significant inhibition in adult worm motility as well as in egg production. Programmed CRISPR/Cas9 editing of smp_089290 in adult worms confirmed the egg reduction phenotype. Based on these results, Smp_089290 was determined to act predominantly as an alpha-NAGAL (hereafter termed SmNAGAL) in schistosome parasites where it participates in coordinating movement and oviposition processes. Further characterisation of SmNAGAL and other functionally important glycosyl hydrolases may lead to the development of a novel anthelmintic class of compounds.Author summary Schistosomiasis is a parasitic disease caused by infection with blood flukes, which leads to acute and chronic pathology in millions of infected individuals located in deprived tropical and subtropical regions. Elucidating the function of schistosome genes has provided a clearer view on their roles in various molecular pathways, which are critical to successful parasitism. This information is invaluable when progressing novel drug and vaccine candidates. Here, we add to the existing knowledge of the Schistosoma mansoni parasitic glycan processing and modification machinery by functionally characterising a glycosyl hydrolase (S. mansoni alpha-N-acetylgalactosaminidase, SmNAGAL). We demonstrate that this protein is enzymatically active and important in coordinating parasite movement in adult male and female schistosomes. Additionally, we provide evidence that this protein regulates pathways associated with egg production in female schistosomes, which is responsible for inducing pathological reactions. Developing drugs that inhibit SmNAGAL enzymatic activity could provide a novel approach for controlling schistosomiasis. Show less
Egesa, M.; Lubyayi, L.; Tukahebwa, E.M.; Bagaya, B.S.; Chalmers, I.W.; Wilson, S.; ... ; Cose, S. 2018
