Phylogenetic analysis of the caspase family in bivalves: implications for programmed cell death, immune response and development

Abstract

Background: Apoptosis is an important process for an organism's innate immune system to respond to pathogens, while also allowing for cell differentiation and other essential life functions. Caspases are one of the key protease enzymes involved in the apoptotic process, however there is currently a very limited understanding of bivalve caspase diversity and function.

Results: In this work, we investigated the presence of caspase homologues using a combination of bioinformatics and phylogenetic analyses. We blasted the Crassostrea gigas genome for caspase homologues and identified 35 potential homologues in the addition to the already cloned 23 bivalve caspases. As such, we present information about the phylogenetic relationship of all identified bivalve caspases in relation to their homology to well-established vertebrate and invertebrate caspases. Our results reveal unexpected novelty and complexity in the bivalve caspase family. Notably, we were unable to identify direct homologues to the initiator caspase-9, a key-caspase in the vertebrate apoptotic pathway, inflammatory caspases (caspase-1, - 4 or - 5) or executioner caspases-3, - 6, - 7. We also explored the fact that bivalves appear to possess several unique homologues to the initiator caspase groups - 2 and - 8. Large expansions of caspase-3 like homologues (caspase-3A-C), caspase-3/7 group and caspase-3/7-like homologues were also identified, suggesting unusual roles of caspases with direct implications for our understanding of immune response in relation to common bivalve diseases. Furthermore, we assessed the gene expression of two initiator (Cg2A, Cg8B) and four executioner caspases (Cg3A, Cg3B, Cg3C, Cg3/7) in C. gigas late-larval development and during metamorphosis, indicating that caspase expression varies across the different developmental stages.

Conclusion: Our analysis provides the first overview of caspases across different bivalve species with essential new insights into caspase diversity, knowledge that can be used for further investigations into immune response to pathogens or regulation of developmental processes.

Keywords: Apoptosis; Bivalves; Caspase; Inflammation response; Innate immune system; Programmed cell death; Pyroptosis.

Fig. 1

Schematic representation of a potential apoptotic pathways in bivalve species based on homologous genes characterised in bivalves or suggested in bivalve genomes (*not identified in a bivalve species yet) to the vertebrate’s intrinsic mitochondrial or extrinsic apoptotic pathways as well as apoptotic pathways in Drosophila melanogaster and Caenorhabditis elegans. b Pyroptotic pathways in vertebrates. (Adopted from [–16])

Fig. 2

a Phylogenetic relationship of initiator caspases in bivalves (blue) compared to other vertebrate and invertebrate homologues (black). Values above/below nodes separated by slash show bootstrap support values for Maximum Likelihood (ML) analysis as percentage of bootstrap values for the main tree with additional Bayesian Inference (BI) posterior probabilities. /x indicates the nodes obtained from the BI which were different from the ML analysis. Human caspase-3 and caspase-7 homologues used as outgroup. Phylogenetic relationship of caspase-recruitment domain (CARD) b or single/double death-effector domains (DED) c. d Schematic representation of initiator caspase structure of bivalves with the CARD, DED or death domain (DD) motifs in their prodomains and the two caspase specific domains: large p20 and small p10 domain. The p20 active sites motif (..H … ..QACXG) with the conserved histidine and cysteine residue in bold is shown for each bivalve caspase homolog. e Schematic representation of gene location for each identified C. gigas caspase on the pseudo-chromosomes (LG). Mb: megabase. Aj: Apostichopus japonicus, Bf: Branchiostoma floridae, Bl: Branchiostoma lanceolatum, Ca: Crassostrea angulata, Ce: Caenorhabditis elegans, Cg: Crassostrea gigas, Ch: Crassostrea hongkongensis, Dl: Dicentrarchus labrax, Dm: Drosophila melanogaster, Dr.: Danio rerio, Hd: Haliotis diversicolor, Hdd: Haliotis discus discus, Hl: Holothuria leucospilota, Hs: Homo sapiens, Mc: Mytilus californianus, Mco: Mytilus coruscus, Mg: Mytilus galloprovincialis, Mm: Mus musculus, Mt: Molgula tectiformis, Tt: Tubifex tubifex, Xl: Xenopus laevis. _amf: amphibian, −amp: amphioxus, _ann: annelid, _asc: ascidian, _ech: echinoderm, _fish: fish, _mam: mammal, _mol: mollusc

Fig. 3

a Phylogenetic relationship of executioner caspases in bivalves (blue) compared to other vertebrate and invertebrate homologues (black). Values above/below branches separated by slash show bootstrap support values for Maximum Likelihood (ML) analysis as percentage of bootstrap values for the main tree with additional Bayesian Inference (BI) posterior probabilities. /x indicates the nodes obtained from the BI which were different from the ML analysis. Human caspase-8 used as outgroup. b Schematic representation of caspase structure with two caspase specific domains: large p20 and small p10 domain. The p20 active sites motif (..H … ..QACXG) with the conserved histidine and cysteine residue in bold is shown for each bivalve caspase homolog. DSRM: double stranded RNA-binding motif. c Schematic representation of gene location for each identified C. gigas caspase on the pseudo-chromosomes (LG). Mb: megabase pairs. Aj: Apostichopus japonicus, Av: Anemonia viridis, Bl: Branchiostoma lanceolatum, Bf: Branchiostoma floridae, Ca: Crassostrea angulata, Cg: Crassostrea gigas, Ch: Crassostrea hongkongensis, Co: Cynops orientalis, Dm: Drosophila melanogaster, Dr.: Danio rerio, Ep: Exaiptasia pallida, Es: Eriocheir sinensis, Hd: Haliotis diversicolor, Hl: Holothuria leucospilota, Hs: Homo sapiens, Hv: Hydra vulgaris, Lm: Locusta migratoria, Meg: Meleagris gallopavo, Mg: Mytilus galloprovincialis, Mj: Marsupenaeus japonicas, Mm: Mus musculus, On: Oreochromis niloticu, Pm: Penaeus monodon, Pme: Penaeus merguiensis Sf: Spodoptera frugiperda, Sm: Schistosoma mansoni, Ss: Salmo salar, Tg: Tegillarca granosa, Xl: Xenopus laevis. _amf: amphibian, −amp: amphioxus, _art: arthropod, _bird: bird, _cni: cnidarian, _ech: echinoderm, _fish: fish, _mam: mammal, _mol: mollusc, _pla: plathelminth, _rep: reptile

Fig. 4

Relative gene expression of two initiator caspases, Cg2A and Cg8B, and four executioner caspases, Cg3/7, Cg3A, Cg3B, Cg3C, during Pacific oyster late larval development (14–17 days post fertilisation (dpf)), after 3 and 6 h post exposure start (3 hpe & 6 hpe) to a 3 h epinephrine (EPI) exposure at 10^− 4 M for metamorphosis induction, and in spat (24 hpe). Each gene expression profile is accompanied by a schematic representation of the key domains present in the protein sequence of the analysed caspase. Different lower-case letters represent significant differences with p < 0.05. CARD: caspase-recruitment domain, DED: death-effector domain, DD: death domain, Large p20: large caspase subunit p20, Small p10: small caspase subunit p10