Biomphalysin, a New β Pore-forming Toxin Involved in Biomphalaria glabrata Immune Defense against Schistosoma mansoni

a New b Pore-forming Toxin Involved in Biomphalaria glabrata Immune Defense against Schistosoma mansoni. PLoS Pathog 9(3): e1003216. doi:10.1371/journal.ppat.1003216 Biomphalysin, a New b Pore-forming Toxin Involved in Biomphalaria glabrata Immune Defense against Schistosoma mansoni Richard Galinier 0 Julien Portela 0 Yves Mone 0 Jean Franc ois Allienne 0 He le` ne Henri 0 Ste phane Delbecq 0 Guillaume Mitta 0 Benjamin Gourbal 0 David Duval 0 Chris Bayne, Oregon State University, United States of America 0 1 CNRS, UMR 5244, Ecologie et Evolution des Interactions (2EI) , Perpignan , France , 2 Universite de Perpignan Via Domitia, Perpignan, France, 3 Universite de Lyon, Lyon; Universite Lyon 1; CNRS, UMR 5558, Laboratoire de Biome trie et Biologie Evolutive , Villeurbanne, France, 4 EA 4558, Vaccination Antiparasitaire , Laboratoire de Biologie Cellulaire et Mole culaire UFR Pharmacie , Montpellier , France Aerolysins are virulence factors belonging to the b pore-forming toxin (b-PFT) superfamily that are abundantly distributed in bacteria. More rarely, b-PFTs have been described in eukaryotic organisms. Recently, we identified a putative cytolytic protein in the snail, Biomphalaria glabrata, whose primary structural features suggest that it could belong to this b-PFT superfamily. In the present paper, we report the molecular cloning and functional characterization of this protein, which we call Biomphalysin, and demonstrate that it is indeed a new eukaryotic b-PFT. We show that, despite weak sequence similarities with aerolysins, Biomphalysin shares a common architecture with proteins belonging to this superfamily. A phylogenetic approach revealed that the gene encoding Biomphalysin could have resulted from horizontal transfer. Its expression is restricted to immune-competent cells and is not induced by parasite challenge. Recombinant Biomphalysin showed hemolytic activity that was greatly enhanced by the plasma compartment of B. glabrata. We further demonstrated that Biomphalysin with plasma is highly toxic toward Schistosoma mansoni sporocysts. Using in vitro binding assays in conjunction with Western blot and immunocytochemistry analyses, we also showed that Biomphalysin binds to parasite membranes. Finally, we showed that, in contrast to what has been reported for most other members of the family, lytic activity of Biomphalysin is not dependent on proteolytic processing. These results provide the first functional description of a mollusk immune effector protein involved in killing S. mansoni. - Funding: This work was supported by funds from the Centre National de la Recherche (CNRS) and the Universite de Perpignan Via Domitia (UPVD), and by a grant by the ANR (25390 Schistophepigen). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. . These authors contributed equally to this work. Schistosomiasis, or bilharzia, is a tropical disease caused by worms of the genus Schistosoma. The main disease-causing species are Schistosoma haematobium, Schistosoma japonicum, and Schistosoma mansoni. An estimated 200 million people in 74 countries suffer from schistosomiasis [1,2]. The World Health Organization expert committee (WHO Technical Report Series 912: prevention and control of schistosomiasis and soil transmitted helminthiasis (WHO, Geneva, 2002)) concluded that yearly deaths could be as high as 200,000 making schistosomiasis the most severe tropical disease after malaria in terms of mortality [1]. No vaccines are yet available to fight S. mansoni, and current chemotherapy relies on a single drug, praziquantel, for which resistant cases have been reported [1,2]. The life cycle of the parasite requires contamination of surface water by excrement, specific freshwater snails as intermediate hosts, and human-to-water contact. Because of their medical and epidemiological importance as intermediate hosts for Schistosoma parasites, freshwater snails of the Biomphalaria genus have garnered considerable research attention. Given the limited options for treating S. mansoni infections, a better understanding of the immunobiological interactions between the invertebrate host Biomphalaria glabrata and its parasite S. mansoni could be invaluable in developing new strategies for preventing and/or controlling Schistosomiasis diseases. A number of studies published over the last two decades have contributed greatly to our understanding of B. glabrata innate immune mechanisms involved in the defense against pathogens. The discovery of recognition molecules such as lectins contributed to a better understanding of the mechanisms involved in pathogen recognition. Among this family of recognition molecules, the discovery of the somatically diversified FREPs (fibrinogen-related proteins) was an important advance in elucidating the immunerecognition step [3,4]. Recently, FREPs were shown to play a crucial role in the fate of the interaction between B. glabrata and its trematode parasites [5]. A recent study described the putative involvement of the cytokine-like molecule, BgMIF (B. glabrata Schistosomiasis is the second most widespread tropical parasitic disease after malaria. It is caused by flatworms of the genus Schistosoma. Its life cycle is complex and requires certain freshwater snail species as intermediate host. Given the limited options for treating S. mansoni infections, much research has focused on a better understanding of the immunobiological interactions between the invertebrate host Biomphalaria glabrata and its parasite S. mansoni. A number of studies published over the last two decades have contributed greatly to our understanding of B. glabrata innate immune mechanisms involved in the defense against parasite. However, most studies have focused on the identification of recognition molecules or immune receptors involved in the host/ parasite interplay. In the present study, we report the first functional description of a mollusk immune effector protein involved in killing S. mansoni, a protein related to the b pore forming toxin that we named Biomphalysin. macrophage migration inhibitory factor) in the anti-parasite response of B. glabrata [6]. A number of studies have analyzed the response of B. glabrata to different immune challenges, allowing the identification of numerous putative immune genes that could play a key role in B. glabrata immune processes [7,8,9,10,11,12]. Other studies based on comparisons of resistant and susceptible strains of B. glabrata to different trematode species from Schistosoma and Echinostoma genera [13,14,15,16,17] have also made a large contribution to the identification of factors putatively involved in the success or failure of parasite infection. Still other studies have explored mechanisms underlying compatibility polymorphism characteristics in certain B. glabrata/S. mansoni (...truncated)