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Exaptation of two ancient immune proteins into a new dimeric pore-forming toxin in snails

Authors:
Matías L. Giglio at University of Utah
Matías L. Giglio
Santiago Ituarte at National University of La Plata
Santiago Ituarte
Veronica Milesi at National University of La Plata
Veronica Milesi
Marcos S Dreon at National University of La Plata
Marcos S Dreon
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Abstract

The Membrane Attack Complex-Perforin (MACPF) family is ubiquitously found in all kingdoms. They have diverse cellular roles but MACPF but pore-forming toxic function are very rare in animals. Here we present the structure of PmPV2, a MACPF toxin from the poisonous apple snail eggs, that can affect the digestive and nervous systems of potential predators. We report the three-dimensional structure of PmPV2, at 15 Å resolution determined by negative stain electron microscopy (NS-EM) and its solution structure by small angle X-ray scattering (SAXS). We found that PV2s differ from nearly all MACPFs in two respects: it is a dimer in solution and protomers combine two immune proteins into an AB toxin. MACPF chain is linked by a single disulfide bond to a tachylectin chain, and two heterodimers are arranged head-to-tail by non-covalent forces in the native protein. MACPF domain is fused with a putative new Ct-accessory domain exclusive to invertebrates. Tachylectin is a six-bladed β-propeller, similar to animal tectonins. We experimentally validated the predicted functions of both subunits and demonstrated for the first time that PV2s are true pore-forming toxins. The tachylectin B delivery subunit would bind to target membranes, and then its MACPF A toxic subunit disrupt lipid bilayers forming large pores altering the plasma membrane conductance. These results indicate that PV2s toxicity evolved by linking two immune proteins where their combined preexisting functions give rise to a new toxic entity with a novel role in defense against predation. This structure is an unparalleled example of protein exaptation.
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... This binary structure is unique among animals and resembles those of bacterial and plant AB toxins, where a¨B¨-moiety acts as a delivery unit of a toxic¨A¨-moiety (10,11). Unlike AB toxins from bacteria or plants, snail PV2 contains a unique arrangement of two AB toxins in a head-to-tail fashion (12). Interestingly, many of these AB toxins, such as the cholera toxin (CT), heat labile toxin (LT), and shiga toxins (Stxs) from bacteria and the type-2 ribosome inactivating proteins (RIPs) from plants, act as enterotoxins (11), an unexplored function in PV2. ...
... Comparative genomic analysis together with expression patterns and proteomic validation showed that although these lectin and MACPF are present in the genomes of four species of the family, as well as in the genomes of other Mollusks, only in Pomacea these two proteins experienced extensive gene expansion by tandem duplication and neofunctionalization into the PV2 complex, which is expressed as such only in an accessory gland of females and transferred to eggs (30). Although the immune role of these two proteins are largely unexplored in snails, a PV2-67-like protein found in the kidney of the snail Littorina littorea showed overexpression when infected with a trematode parasite (31), indicating a putative immune function in the common ancestor of mollusks MACPF (12). In addition to their immune role, another prominent role of animal MACPFs is in the embryonic development of several organisms, ranging from sea urchins to mammals (32). ...
... Similar to those proteins PmPV2 is maternally transferred to the eggs, where it is massively accumulated during the early developing stages, before the embryo consumes it (33). However, PV2 structure lacks some key structural features described in developmental MACPFs, such as absence of ancillary domains and shorter TMH1 (12,32). Finally, a less-extended group of animal MACPFs also act as toxins such as those from some cnidarians and the stone fish, where they play a role in prey capture (22,34). ...
Novel Role for Animal Innate Immune Molecules: Enterotoxic Activity of a Snail Egg MACPF-Toxin
Article
Full-text available
  • Mar 2020
Gastropod Molluscs rely exclusively on the innate immune system to protect from pathogens, defending their embryos through maternally transferred effectors. In this regard, Pomacea snail eggs, in addition to immune defenses, have evolved the perivitellin-2 or PV2 combining two immune proteins into a neurotoxin: a lectin and a pore-forming protein from the Membrane Attack Complex/Perforin (MACPF) family. This binary structure resembles AB-toxins, a group of toxins otherwise restricted to bacteria and plants. Many of these are enterotoxins, leading us to explore this activity in PV2. Enterotoxins found in bacteria and plants act mainly as pore-forming toxins and toxic lectins, respectively. In animals, although both pore-forming proteins and lectins are ubiquitous, no enterotoxins have been reported. Considering that Pomacea snail eggs ingestion induce morpho-physiological changes in the intestinal mucosa of rodents and is cytotoxic to intestinal cells in culture, we seek for the factor causing these effects and identified PmPV2 from Pomacea maculata eggs. We characterized the enterotoxic activity of PmPV2 through in vitro and in vivo assays. We determined that it withstands the gastrointestinal environment and resisted a wide pH range and enzymatic proteolysis. After binding to Caco-2 cells it promoted changes in surface morphology and an increase in membrane roughness. It was also cytotoxic to both epithelial and immune cells from the digestive system of mammals. It induced enterocyte death by a lytic mechanism and disrupted enterocyte monolayers in a dose-dependent manner. Further, after oral administration to mice PmPV2 attached to enterocytes and induced large dose-dependent morphological changes on their small intestine mucosa, reducing the absorptive surface. Additionally, PmPV2 was detected in the Peyer's patches where it activated lymphoid follicles and triggered apoptosis. We also provide evidence that the toxin can traverse the intestinal barrier and induce oral adaptive immunity with evidence of circulating antibody response. As a whole, these results indicate that PmPV2 is a true enterotoxin, a role that has never been reported to lectins or perforin in animals. This extends by convergent evolution the presence of plant- and bacteria-like enterotoxins to animals, thus expanding the diversity of functions of MACPF proteins in nature.
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... This binary structure is unique among animals and resembles those of bacterial and plant AB toxins, where a¨B¨-moiety acts as a delivery unit of a toxic¨A¨-moiety (10,11). Unlike AB toxins from bacteria or plants, snail PV2 contains a unique arrangement of two AB toxins in a head-to-tail fashion (12). Interestingly, many of these AB toxins, such as the cholera toxin (CT), heat labile toxin (LT), and shiga toxins (Stxs) from bacteria and the type-2 ribosome inactivating proteins (RIPs) from plants, act as enterotoxins (11), an unexplored function in PV2. ...
... Comparative genomic analysis together with expression patterns and proteomic validation showed that although these lectin and MACPF are present in the genomes of four species of the family, as well as in the genomes of other Mollusks, only in Pomacea these two proteins experienced extensive gene expansion by tandem duplication and neofunctionalization into the PV2 complex, which is expressed as such only in an accessory gland of females and transferred to eggs (30). Although the immune role of these two proteins are largely unexplored in snails, a PV2-67-like protein found in the kidney of the snail Littorina littorea showed overexpression when infected with a trematode parasite (31), indicating a putative immune function in the common ancestor of mollusks MACPF (12). In addition to their immune role, another prominent role of animal MACPFs is in the embryonic development of several organisms, ranging from sea urchins to mammals (32). ...
... Similar to those proteins PmPV2 is maternally transferred to the eggs, where it is massively accumulated during the early developing stages, before the embryo consumes it (33). However, PV2 structure lacks some key structural features described in developmental MACPFs, such as absence of ancillary domains and shorter TMH1 (12,32). Finally, a less-extended group of animal MACPFs also act as toxins such as those from some cnidarians and the stone fish, where they play a role in prey capture (22,34). ...
Novel Role for Animal Innate Immune Molecules: Enterotoxic Activity of a Snail Egg MACPF-Toxin
Article
Full-text available
  • Mar 2020
Gastropod Molluscs rely exclusively on the innate immune system to protect from pathogens, defending their embryos through maternally transferred effectors. In this regard, Pomacea snail eggs, in addition to immune defenses, have evolved the perivitellin-2 or PV2 combining two immune proteins into a neurotoxin: a lectin and a pore-forming protein from the Membrane Attack Complex/Perforin (MACPF) family. This binary structure resembles AB-toxins, a group of toxins otherwise restricted to bacteria and plants. Many of these are enterotoxins, leading us to explore this activity in PV2. Enterotoxins found in bacteria and plants act mainly as pore-forming toxins and toxic lectins, respectively. In animals, although both pore-forming proteins and lectins are ubiquitous, no enterotoxins have been reported. Considering that Pomacea snail eggs ingestion induce morpho-physiological changes in the intestinal mucosa of rodents and is cytotoxic to intestinal cells in culture, we seek for the factor causing these effects and identified PmPV2 from Pomacea maculata eggs. We characterized the enterotoxic activity of PmPV2 through in vitro and in vivo assays. We determined that it withstands the gastrointestinal environment and resisted a wide pH range and enzymatic proteolysis. After binding to Caco-2 cells it promoted changes in surface morphology and an increase in membrane roughness. It was also cytotoxic to both epithelial and immune cells from the digestive system of mammals. It induced enterocyte death by a lytic mechanism and disrupted enterocyte monolayers in a dose-dependent manner. Further, after oral administration to mice PmPV2 attached to enterocytes and induced large dose-dependent morphological changes on their small intestine mucosa, reducing the absorptive surface. Additionally, PmPV2 was detected in the Peyer's patches where it activated lymphoid follicles and triggered apoptosis. We also provide evidence that the toxin can traverse the intestinal barrier and induce oral adaptive immunity with evidence of circulating antibody response. As a whole, these results indicate that PmPV2 is a true enterotoxin, a role that has never been reported to lectins or perforin in animals. This extends by convergent evolution the presence of plant- and bacteria-like enterotoxins to animals, thus expanding the diversity of functions of MACPF proteins in nature.
View
Evidence of a Lytic Pathway in an Invertebrate Complement System: Identification of a Terminal Complement Complex Gene in a Colonial Tunicate and Its Evolutionary Implications
Article
Full-text available
The complement system is a pivotal component of innate immunity, extensively studied in vertebrates but also present in invertebrates. This study explores the existence of a terminal complement pathway in the tunicate Botryllus schlosseri, aiming to understand the evolutionary integration of innate and adaptive immunity. Through transcriptome analysis, we identified a novel transcript, BsITCCP, encoding a protein with both MACPF and LDLa domains—a structure resembling that of vertebrate C9 but with a simpler organization. Phylogenetic reconstruction positions BsITCCP between invertebrate perforins and vertebrate terminal complement proteins, suggesting an evolutionary link. Localization studies confirmed that bsitccp is transcribed in cytotoxic morula cells (MCs), which are also responsible for producing other complement components like BsC3, BsMBL, BsMASP, and BsBf. Functional assays demonstrated that bsitccp transcription is upregulated in response to nonself challenges and is dependent on BsC3 activity; inhibition of BsC3 led to a significant reduction in BsITCCP expression. Electron microscopy revealed that MCs form contact with perforated yeast cells, indicating a possible mechanism of cell lysis similar to the immunological synapse observed in vertebrates. These findings suggest that a C3-governed lytic complement pathway exists in B. schlosseri, challenging the assumption that a C5 ortholog is necessary for such a pathway. This work enhances our understanding of the evolution of the complement system and suggests that invertebrates possess a terminal complement complex capable of mediating cell lysis, regulated by C3. Future studies will focus on confirming the pore-forming ability of BsITCCP and its role in the immunological synapse.
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