G C A T T A C G G C A T genes Article Biomphalaria glabrata Granulin Increases Resistance to Schistosoma mansoni Infection in Several Biomphalaria Species and Induces the Production of Reactive Oxygen Species by Haemocytes Jacob R. Hambrook 1, Abdullah A. Gharamah 1 , Emmanuel A. Pila 1, Solomon Hussein 2 and Patrick C. Hanington 1,* 1 School of Public Health, University of Alberta, Edmonton, AB T6G 2R3, Canada; [email protected] (J.R.H.); [email protected] (A.A.G.); [email protected] (E.A.P.) 2 Department of Biological Sciences, University of Alberta, Edmonton, AB T6G 2R3, Canada; [email protected] * Correspondence: [email protected] Received: 22 November 2019; Accepted: 27 December 2019; Published: 28 December 2019 Abstract: Gastropod molluscs, which have co-evolved with parasitic digenean trematodes for millions of years, utilize circulating heamocytes as the primary method of containing and killing these invading parasites. In order to do so, they must generate suitable amounts of haemocytes that are properly armed to kill parasitic worms. One method by which they generate the haemocytes required to initiate the appropriate cell mediated immune response is via the production and post-translational processing of granulins. Granulins are an evolutionarily conserved family of growth factors present in the majority of eukaryotic life forms. In their pro-granulin form, they can elicit cellular replication and differentiation. The pro-granulins can be further processed by elastase to generate smaller granulin fragments that have been shown to functionally differ from the pro-granulin precursor. In this study, we demonstrate that in vivo addition of Biomphalaria glabrata pro-granulin (BgGRN) can reduce Schistosoma mansoni infection success in numerous Biomphalaria sp. when challenged with different S. mansoni strains. We also demonstrate that cleavage of BgGRN into individual granulin subunits by elastase results in the stimulation of haemocytes to produce reactive oxygen species. Keywords: granulin; Biomphalaria glabrata; Schistosoma mansoni; host-parasite compatibility; invertebrate immunology 1. Introduction Schistosomiasis is a serious parasitic disease caused by trematodes of the genus Schistosoma. It affects over 206 million people worldwide, especially in tropical and sub-tropical regions [1,2]. After malaria and intestinal helminthiasis, schistosomiasis is the third most devastating tropical disease in the world, representing a major source of morbidity and mortality for developing countries in Africa, South America, the Caribbean, the Middle East, and Asia [1]. Freshwater snails of the genus Biomphalaria act as obligate intermediate hosts for Schistosoma mansoni, which is the predominant causative agent of intestinal schistosomiasis in Africa and South America. This association with human schistosomiasis has made Biomphalaria sp. some of the most extensively studied gastropods in terms of immunobiology and host–parasite interactions. Although numerous species are capable of transmitting S. mansoni, B. glabrata has emerged as the predominant model for studying the intra-molluscan aspects of the S. mansoni lifecycle, with hopes that this research will prove fruitful towards disease control efforts [3]. While B. glabrata is a strong model, it does not Genes 2020, 11, 38; doi:10.3390/genes11010038 www.mdpi.com/journal/genes Genes 2020, 11, 38 2 of 12 transmit as much S. mansoni as its African counterparts. Thus, studying other species of Biomphalaria is important to understand whether information gleaned from B. glabrata studies translates across all Biomphalaria snails. Seeing as the majority of S. mansoni transmission worldwide occurs through Genes 2020, 11, x FOR PEER REVIEW 2 of 13 Biomphalaria sudanica and Biomphalaria pfefferi, these two snail species are essential to include in such comparisonsresearch [4]. Inwill addition, prove fruitful there towards are di diseasefferent control strains efforts of B. [3]. glabrata While B.that glabrata display is a strong differing model, compatibility it phenotypesdoes to notS. transmit mansoni asinfection. much S. mansoni Some as its strains African display counterparts. high Thus, levels studying of resistance other species such of as BS-90, Biomphalaria is important to understand whether information gleaned from B. glabrata studies 13-16-R1, 10-R2 [5,6] while some show high levels of susceptibility such as the M-line and NMRI translates across all Biomphalaria snails. Seeing as the majority of S. mansoni transmission worldwide strains [7,8occurs]. Understanding through Biomphalaria the molecularsudanica and diBiomphalariafferences pfefferi between, these these two snail strains species and arespecies essential allowsto for a more thoroughinclude understanding in such comparisons ofthe [4]. molecularIn addition, underpinningsthere are different ofstrains resistance. of B. glabrata that display A widediffering array compatibility of proteins havephenotypes been identifiedto S. mansoni and infection. studied Some in B. strains glabrata displaywith high the intentlevels of to discover resistance such as BS-90, 13-16-R1, 10-R2 [5,6] while some show high levels of susceptibility such as those factors that elicit and coordinate the immune response against S. mansoni [9,10]. One such protein the M-line and NMRI strains [7,8]. Understanding the molecular differences between these strains is B. glabrataandgranulin species allows (BgGRN). for a more Granulins thorough understand are evolutionarilying of the molecular conserved underpinnings growth factors of resistance. that are found in diverse phylaA wide ranging array fromof proteins eubacteria have been to humansidentified [and11]. studied They arein B. potent glabrata growthwith the factorsintent to that drive proliferationdiscover of immune those factors cells that in elicit organisms, and coordinate spanning the immune the animal response kingdom. against S. mansoni It has [9,10]. been One shown to be such protein is B. glabrata granulin (BgGRN). Granulins are evolutionarily conserved growth factors essential for several molecular processes in humans and other animals, such as: in tissue repair, cellular that are found in diverse phyla ranging from eubacteria to humans [11]. They are potent growth proliferation,factors development, that drive proliferation wound of healing, immune andcells inflammationin organisms, spanning [12–14 the]. animal kingdom. It has Progranulinbeen shown proteins to be essential (PGRNs) for several are secreted molecular pro-peptides processes in humans that and can other be subsequently animals, such as: cleaved in into smaller functionaltissue repair, units cellular by proliferation, elastase [15 development,]. Often, granulin wound healing, pro-peptides and inflammation appear [12–14]. to resemble a “pearl Progranulin proteins (PGRNs) are secreted pro-peptides that can be subsequently cleaved into necklace”smaller wherein functional folded units granulin by elastase domains, [15]. Often, held granulin together pro-peptides by disulfide appear bonds,to resemble are a strung “pearl together while separatednecklace” by wherein predicted folded cutgranulin sites domains, for elastase held to [gether16–20 by]. disulfide The granulin bonds, are domains strung together flanked by the elastase siteswhile are separated defined by by predicted a characteristic cut sites for 12-cysteineelastase [16–20]. repeat. The granulinB. glabrata domainsgranulin flanked isby comprised the of 4 such domainselastase withsites are predicted defined by elastase a characteristic cut sites 12-cysteine before repeat. and afterB. glabrata each granulin of these is comprised 12 cysteine of 4 domains, such domains with predicted elastase cut sites before and after each of these 12 cysteine domains, a a fact notedfact by noted Pila by et Pila al. et in al. their in their earlier earlier work work on on BgGRN BgGRN (Figure (Figure 1) [11,12,16].1)[11,12 ,16]. Figure 1. Diagram depicting the basic structure of Biomphalaria glabrata pro-granulin (BgGRN), the possibleFigure intermediate 1. Diagram depicting forms, asthe wellbasic structure as the fact of Biomphalaria that it can glabrata be cleaved pro-granulin into (BgGRN), individual the subunits. possible intermediate forms, as well as the fact that it can be cleaved into individual subunits. All All generatedgenerated granulin granulin subunits subunits feature featurea a blackblack bracket, bracket, whereas whereas a red a bracket red bracket indicates indicates an intermediate an intermediate that was notthat successfully was not successfully cloned. cloned. PreviousPrevious work has work demonstrated has demonstrated that that full-length full-length BgGRN BgGRN induces induces proliferation proliferation of B. glabrata of B. glabrata hemocytes,hemocytes, and that and these that haemocytesthese haemocytes are are largely largely ad adherentherent and and express express Bg Toll-Like Bg Toll-Like Receptor Receptor(TLR). (TLR). Both of these characteristics render haemocytes better capable of killing invading sporocysts [12,21]. Both of theseAdditionally, characteristics susceptible render B. glabrata haemocytes snails can be better made capableresistant to of infection killing with invading S. mansoni sporocysts with an [12,21]. Additionally,injection susceptible of full-lengthB.glabrata rBgGRN,snails while siRNA can be mediated made resistantknock-down to of infection BgGRN results with inS. a mansoni loss of with an injection ofresistance full-length [12]. The rBgGRN, differences