Cg-TGF-β, a TGF-β/activin homologue in the Pacific Oyster Crassostrea gigas, is involved in immunity against Gram-negative microbial infection

doi:10.1016/j.dci.2006.05.005

Developmental & Comparative Immunology

Volume 31, Issue 1, 2007, Pages 30-38

https://doi.org/10.1016/j.dci.2006.05.005 Get rights and content

Abstract

Transforming growth factor-β (TGF-β) members represent a widespread protein superfamily in the animal kingdom, but few members have been characterised in lophotrochozoans, a major clade of invertebrates. Here, we report the identification of Crassostrea gigas-TGF-β (Cg-TGF-β), a homologue of vertebrate TGF-β and activin, from the bivalve mollusc C. gigas. Phylogenetic analysis suggests an early ancestral origin of this subgroup of TGF-β superfamily member. Investigation of the spatio–temporal expression of Cg-TGF-β gene by real-time quantitative RT-PCR showed a ubiquitous pattern in all adult tissues. These findings imply that Cg-TGF-β has multiple functions as described for its vertebrate counterparts. Moreover, Cg-TGF-β was upregulated in haemocytes during infection by a Gram-negative bacterium, suggesting that it could act as a cytokine involved in immunity in molluscs.

Section snippets

Introduction.

In the economic context of controlling the reproduction and growth of bivalve molluscs, knowledge of physiological mechanisms is crucial. During culture of the Pacific oyster, Crassostrea gigas, the organisms are directly influenced by environmental factors (physical or chemical conditions and pathogens) and their physiology can be deregulated by potential xenobiotic components [1]. Consequently, a part of our work has focussed on the study of various physiological regulations including defence

Isolation and purification of gonadal cells

Gonadal tracts were extracted in January during the period of initiation of mitosis in gonadal cells. Cells were isolated from bladed tissues using a discontinuous percoll gradient and harvested by centrifugation.

Experimental stimulation by bacterial challenge and lipopolysaccharides (LPS)

To prevent the stress of manipulation and shipping, animals were bathed in seawater at 15 °C for 1 week prior to stimulation. To mimic an infection by Gram negative bacteria, a mixture of three Vibrio species (Vibrio metschnikovii, V. alginolyticus, V. harveyii) (10⁸ bacteria/100 μL) or

Molecular cloning of Cg-TGF-β cDNA and characterisation of gene

The RT-PCR with the consensual degenerated primers performed on cDNA from C. gigas gonadal cells led to identification of a novel TGF-β member, named Cg-TGF-β-related factor. Subsequently, an entire transcript was obtained by RACE-PCR with gene specific primers. The complete full-length cDNA comprises 1956 nucleotides with a coding sequence of 1161 nucleotides preceded by an in-frame stop codon. Two potential non-canonical sites of polyadenylation are located at 1384 bp (AATTAA) and 1901 bp

Discussion

In the present study, we report the characterisation of the cDNA encoding Cg-TGF-β, a peptide related to TGF-β (including myostatin and myoglianin), and to maverick and activins in both vertebrates and Drosophila. It could be cleaved by a pro-convertase at a tetrabasic site to a mature peptide. More particularly, this proteolytic site (RVKR) corresponds to a typical recognition motif of furin (RX(K/R)R), required for activation of vertebrate TGF-β [26]. The mature domain of Cg-TGF-β has all the

Acknowledgements

This work was supported by the Conseil Régional de Basse-Normandie, Agence de l’eau Seine-Normandie and FEDER Presage n°4474. The authors thank Dr. Ian Probert, University of Caen for carefully editing the manuscript and Dr. Nils Klüver for supplementary phylogenetic analysis.

References (39)

W.S. Fisher et al.
Stimulation of defense factors for oysters deployed to contaminated sites in Pensacola Bay, Florida
Aquat Toxicol
(2003)
H.R. Nicholas et al.
Responses to infection and possible recognition strategies in the innate immune system of Caenorhabditis elegans
Mol Immunol
(2004)
A.C. Millet et al.
Immunity in Caenorhabditis elegans
Curr Opin Immunol
(2004)
R.K. Pipe
Generation of reactive oxygen metabolites by the haemocytes of the mussel Mytilus edulis
Dev Comp Immunol
(1992)
T.C. Cheng et al.
Lysosomal and other enzymes in the hemolymph of Crassostrea virginica and Mercenaria mercenaria
Comp Biochem Physiol B
(1975)
G. Mitta et al.
Original involvement of antimicrobial peptides in mussel innate immunity
FEBS Lett
(2000)
Y. Gueguen et al.
Characterization of a defensin from the oyster Crassostrea gigas: recombinant production, folding, solution structure, antimicrobial activities and gene expression
J Biol Chem
(2006)
M.J. Lieber et al.
Transforming growth factor-betas and related gene products in mosquito vectors of human malaria parasites: signaling architecture for immunological crosstalk
Mol Immunol
(2004)
A. Herpin et al.
Transforming growth factor-beta-related proteins: an ancestral and widespread superfamily of cytokines in metazoans
Dev Comp Immunol
(2004)
K. Okabayashi et al.
Tissue generation from amphibian animal caps
Curr Opin Genet Dev
(2003)

G. Kutty et al.

Identification of a new member of transforming growth factor-beta superfamily in Drosophila: the first invertebrate activin gene

Biochem Biophys Res Commun

(1998)

M. Nguyen et al.

Identification of maverick, a novel member of the TGF-beta superfamily in Drosophila

Mech Dev

(2000)

A. Herpin et al.

Gene structure and expression of cg-ALR1, a type I activin-like receptor from the bivalve mollusc Crassostrea gigas

Gene

(2002)

A. Herpin et al.

Structural and functional evidences for a type 1 TGF-beta sensu stricto receptor in the lophotrochozoan Crassostrea gigas suggest conserved molecular mechanisms controlling mesodermal patterning across bilateria

Mech Dev

(2005)

F. Rodet et al.

Molecular cloning of a molluscan gonadotropin-releasing hormone receptor orthologue specifically expressed in the gonad

Biochim Biophys Acta

(2005)

M.S. Jiang et al.

Characterization and identification of the inhibitory domain of GDF-8 propeptide

Biochem Biophys Res Commun

(2004)

H.W. Kim et al.

Characterization of a myostatin-like gene from the bay scallop, Argopecten irradians

Biochim Biophys Acta

(2004)

R. Sakai et al.

Involvement of activin in the regulation of bone metabolism

Mol Cell Endocrinol

(2001)

B. Munz et al.

The roles of activins in repair processes of the skin and the brain

Mol Cell Endocrinol

(2001)

Cited by (44)

Genome-wide identification and expression profiles of sex-related gene families in the Pacific abalone Haliotis discus hannai
2024, Comparative Biochemistry and Physiology - Part D: Genomics and Proteomics
In recent years, members of the Dmrt family, TGF-β superfamily and Sox family have been recognized as crucial genes for sex determination/differentiation across diverse animal species. Nevertheless, knowledge regarding the abundance and potential functions of these genes in abalone remains limited. In this study, a total of 5, 10, and 7 members of the Dmrt family, the TGF-β superfamily and the Sox family, respectively, were identified in the Pacific abalone Haliotis discus hannai. Sequence characteristics, phylogenetic relationships and spatiotemporal expression profiles of these genes were investigated. Notably, HdDmrt-04 (Dmrt1/1L-like) emerged as a potential mollusc-specific gene with a preponderance for expression in the testis. Interestingly, none of the TGF-β superfamily members exhibited specific or elevated expression in the gonads, highlighting the need for further investigation into their role in abalone sex differentiation. The Sox proteins in H. discus hannai were categorized into 7 subfamilies: B1, B2, C, D, E, F, and H. Among them, HdSox-07 (SoxH-like) was observed to play a crucial role in testis development, while HdSox-03 (SoxB1-like) and HdSox-04 (SoxC-like) probably cooperate in abalone ovary development. Taken together, the results of the present study suggested that HdDmrt-04 and HdSox-07 can be used as male-specific markers for gonad differentiation in H. discus hannai and imply conservation of their functions across invertebrates and vertebrates. Our findings provide new insights into the evolution and genetic structure of the Dmrt family, the TGF-β superfamily and the Sox family in abalone and pave the way for a deeper understanding of sex differentiation in gastropods.
The different expression patterns of cytokines in Pacific oyster Crassostrea gigas response against bacterial stimulation
2023, Aquaculture
Cytokines can be induced by pathogen stimulation for the information exchange between cells, and their expression patterns indicate the response status of cells. In the present study, the expression patterns of ten cytokines (CgIL17–1, −2, −3, −4, −5, −6, CgTNF-1/2, CgTGF-β and CgIFNLP) in haemocytes were analyzed in the Pacific oyster Crassostrea gigas after Vibrio splendidus stimulation in the indoor and outdoor experiments to compare the induction patterns of different cytokines and evaluate the response status of haemocytes. After the indoor V. splendidus immersion stimulation, the mRNA abundances of six cytokines (CgIL17–1, −3, −4, −5, CgTGF-β and CgIFNLP) in haemocytes were significantly downregulated at 48 h compared with that in the control group, among which CgIL17–4 was the most down-regulated with 0.009-fold of that in the control group (p < 0.05). The mRNA abundances of CgIL17–2, CgIL17–6, CgTNF-1 and CgTNF-2 showed no significant change at 48 h compared with that in the control group. The apoptosis rate of haemocytes reached the maximum level of 1.138% at 48 h after the stimulation. In the oysters cultured outdoor from April to September 2021, the mRNA abundances of all ten cytokines in haemocytes were found to decrease in June (0.270-fold on average) and increase in July (5.402-fold on average) compared with those in May. After the in situ V. splendidus immersion stimulation in the outdoor cultured oysters in June and July, seven cytokines (CgIL17–1, CgIL17–2, CgIL17–4, CgIL17–5, CgTNF-1, CgTGF-β and CgIFNLP) exhibited upregulated mRNA abundance in June, while in July, the mRNA abundances of five cytokines (CgIL17–1, CgIL17–3, CgTNF-2, CgTGF-β, and CgIFNLP) remained constant, and four cytokines (CgIL17–2, CgIL17–4, CgIL17–5 and CgTNF-1) were suppressed after the stimulation. CgIL17–4 had the highest fold-change in mRNA abundance among all cytokines in June after the stimulation, which was 85.599-fold compared with that in the control group (p < 0.05). The score of haemocyte response status (SH) was evaluated using the mRNA abundance database of all ten cytokines. It decreased to 124.860 at 48 h after the indoor V. splendidus stimulation, and it decreased to 66.793 in June and increased to 451.092 in July in the cultured oysters prior to the stimulation. Collectively, the expression patterns of ten cytokines in haemocytes varied against bacterial stimulation, among which CgIL17–4 was found to response the most significantly, and SH indicated by the mRNA abundances of cytokines suggested an abnormal response status of haemocytes in the oysters at 48 h after indoor V. splendidus stimulation and the oysters cultured outdoor during June and July. These findings will further our understanding of the cytokine variation and haemocyte response of the Pacific oyster against bacterial stimulation.
Increased abundances of potential pathogenic bacteria and expressions of inflammatory cytokines in the intestine of oyster Crassostrea gigas after high temperature stress
2023, Developmental and Comparative Immunology
High temperature stress is a significant threat to the health of oysters, but the effects on their intestinal performances are not well understood. In this study, the effects of high temperature stress on the intestinal histology, immune response and associated microbiota were investigated in Crassostrea gigas after rearing at 20, 25 and 28 °C for 21 days. With the increase of temperature, shortened and shed microvilli as well as increased goblet cells were observed in the intestines of oysters. The transcripts of cytokines CgIL17-5, CgTNF-2 and CgTGF-β and apoptosis-related gene CgCaspase-3 in intestine increased with the increasing temperature. Further, the diversity and composition of the oyster intestinal microbiota changed after high temperature stress. The 16S rRNA gene copy number per ng of DNA in the T25 (5.16 × 10⁵) and T28 (1.63 × 10⁵) groups were higher than that in the control group (8.62 × 10⁴). The Chao 1 index in the T25 (238.00) and T28 (240.17) groups was lower than that in the control group (279.00). The Shannon index decreased progressively with the increasing temperature, with the value in the T28 group (4.44) significantly lower than that in the control group (5.40) (p < 0.05). The abundances of potential pathogenic bacteria such as Acinetobacter, Pseudomonas, Vibrio and Endozoicomonas increased while that of probiotic bacteria Bacillus decreased after high temperature exposure. Functional prediction indicated that the pathways associated with bacterial proliferation were enriched at 25 °C, while those involved in protein synthesis were blocked at 28 °C. Collectively, these results suggested that high temperature stress led to an increased abundances of potential pathogenic bacteria and expressions of inflammatory cytokines in the intestine, which may consequently affect the functional integrity of the intestinal barrier in oysters.
Identification, expression and functional analysis of activin type I receptor in common Chinese Cuttlefish, Sepiella japonica
2022, Animal Reproduction Science
Citation Excerpt :
They isolated a genomic clone from Drosophila that encoded a putative β activin subunit which displayed more than 60% similarity with vertebrate activin β sequence (Kutty et al., 1998). In mollusks, the biological activity of transforming growth factor-β (Cg-TGF-β) has been previously demonstrated in Pacific Oyster Crassostrea gigas (Lelong et al., 2007). In addition, one activin-like type I receptor (cgALR1) has also been isolated and characterized in C. gigas, which implied a potential role in the development (Herpin et al., 2002; Dewulf et al., 1995; Yelick et al., 1998).
Activin, a member of the transforming growth factor-β(TGF-β) superfamily, exerts its actions by binding to specific transmembrane serine/threonine kinase receptors, known as types II and I receptors. In this study, a full-length cDNA encoding for activin type Ⅰ receptor (SjActRⅠ) was cloned, characterized, and functionally studied in S.japonica. The full-length cDNA of SjActRⅠwas 2264 bp and encoded 505 amino acids. Subcellular localization analysis showed that SjActRⅠwas distributed in the plasma membrane in HEK293T cells. qRT-PCR showed that expressions of SjActRⅠ were ubiquitously expressed in all examined tissues, with the highest expression in the ovary. During the different ovarian development, the expression levels of SjActRⅠ in these three tissues (ovary, brain, and liver) increased from stage I to stage Ⅲ and then decreased from stage III to stage IV. Injection of exogenous GnRH significantly increased the mRNA levels of SjActRⅠ in the ovary and liver, while the expression level of sjActRI in the brain showed no significant difference. Knock-down of SjActRⅠ, by injection of double-strand RNA (dsRNA) significantly reduced the expression levels of SjActRⅠ. Furthermore, silencing SjActRⅠsignificantly reduced the expression of Vitellogenin in the ovary, suggesting that SjActRⅠ might promote ovarian development by stimulating the expression of Vitellogenin. These findings implied that SjActRⅠ might play a crucial role in ovarian development in S.japonica. In conclusion, our results provide novel insights into the evolution and roles of the ActRⅠgene in cuttlefish.
The role of Smad1/5 in mantle immunity of the pearl oyster Pinctada fucata martensii
2021, Fish and Shellfish Immunology
The Smad protein family is an important medium for transducing BMP–Smads signals, and which have been proved that their important role in regulating shell biomineralization in Pinctada fucata martensii in our previous study. The members of TGF-β superfamily were involved in innate immunity in vertebrates and invertebrates, and Smad regulatory networks construct a balanced immune system. However, little is known about the role of Smad1/5 in immunity in P. f. martensii. The present study shows that the tissue distribution and the expression profiles of Smad1/5 at developmental stages suggested its wide distribution and crucial role in development at embryonic stages other than larval stage; the increased expression of bone morphogenetic proteins 2 (BMP2), Smad4, Smad1/5 and MSX mRNAs at mantle tissue after LPS and Poly (I:C) challenged implied the potential immune role of Smad1/5 and BMP2-Smad signals to defense against bacterial and virus infections; the reduced expression of immune gene nuclear factor kappa-B (NF-κB), matrix metalloproteinase (MMP), interleukin 17 (IL-17), CuZn-superoxide dismutase (CuZn-SOD), tissue inhibitors of metalloproteinase (TIMP) and lipopolysaccharide-induced TNF-α factor (LITAF) mRNA following knockdown of Smad1/5 indicated that Smad1/5 can regulate their expression via BMP2-Smads pathway in the immunity process; the up-regulated expression of Smad1/5 and BMP2-Smad signals genes, and immune genes during wound healing indicated that Smad1/5 and BMP2-Smad signals genes may be involved in wound healing collaborated with immune genes via a different and complex Smads signaling pathway. These results indicated Smad1/5 could regulate innate immunity via BMP2-Smads signal pathway, and which provided new insights into the relationship between BMP2-Smads signal pathway and mantle immunity.
Expansion and loss events characterized the occurrence of MIF-like genes in bivalves
2019, Fish and Shellfish Immunology
Citation Excerpt :
The involvement of cytokine-like genes in bivalve immunity has been hypothesized since 1990 [7,8] and has been corroborated over the past two decades by the identification of several sequences homologous to vertebrate cytokines in bivalve genomes, as well as of homologs of downstream signaling elements, often reported as expanded gene families [9]. The repertoire of known cytokines shared by bivalves and vertebrates currently includes transforming growth factor-beta [10], allograft inflammatory factor-1 [11], interleukin-17 [12], tumor necrosis factor-alfa (TNFα) [13] and macrophage migration inhibitory factor (MIF) [14]. Furthermore, immunological research enabled the identification of a number of other cytokines independently developed in the protostome lineage [8], including spätzle in arthropods [15], astakine in crustaceans and in the bivalve Crassostrea gigas [16,17], CCF in the annelid Eisenia foetida [18] and Vago in arthropods [19].
Macrophage migration inhibitory factor (MIF) dynamically connects innate and adaptive immune systems in vertebrate animals, allowing highly orchestrated systemic responses to various insults. The occurrence of MIF-like genes in non-vertebrate organisms suggests its origin from an ancestral metazoan gene, whose function is still a matter of debate. In the present work, by analyzing available genomic and transcriptomic data from bivalve mollusks, we identified 137 MIF-like sequences, which were classified into three types, based on phylogeny and conservation of key residues: MIF, D-DT, and the lineage-specific type MDL. Comparative genomics revealed syntenic conservation of homologous genes at the family level, the loss of D-DT in the Ostreidae family as well as the expansion of MIF-like genes in the Mytilidae family, possibly underpinning the neofunctionalization of duplicated gene copies. In M. galloprovincialis, MIF and one D-DT were mostly expressed in haemocytes and mantle rim of untreated animals, while D-DT paralogs often showed very limited expression, suggesting an accessory role or their persistence as relict genes.

View all citing articles on Scopus

View full text

Cg-TGF-β, a TGF-β/activin homologue in the Pacific Oyster Crassostrea gigas, is involved in immunity against Gram-negative microbial infection

Abstract

Section snippets

Introduction.

Isolation and purification of gonadal cells

Experimental stimulation by bacterial challenge and lipopolysaccharides (LPS)

Molecular cloning of Cg-TGF-β cDNA and characterisation of gene

Discussion

Acknowledgements

Aquat Toxicol

Mol Immunol

Curr Opin Immunol

Dev Comp Immunol

Comp Biochem Physiol B

FEBS Lett

J Biol Chem

Mol Immunol

Dev Comp Immunol

Curr Opin Genet Dev

Biochem Biophys Res Commun

Mech Dev

Gene

Mech Dev

Biochim Biophys Acta

Biochem Biophys Res Commun

Biochim Biophys Acta

Mol Cell Endocrinol

Mol Cell Endocrinol