Zhihao Jia

An integrin from oyster Crassostrea gigas mediates the phagocytosis toward Vibrio splendidus through LPS binding activity

Integrins are a family of cell adhesion molecules which play important roles in the regulation of cell adhesion, migration, proliferation, apoptosis and phagocytosis. In the present study, the immune function of an integrin from the oyster Crassostrea gigas (designated CgIntegrin) was characterized to understand the regulatory mechanism of hemocyte phagocytosis toward different microbes. The full-length cDNA of CgIntegrin was 2571 bp with an open reading frame (ORF) of 2397 bp, encoding a polypeptide of 799 amino acids. The mRNA transcripts of CgIntegrin were predominantly detected in hemocytes, gonad and adductor muscle, while lowly in hepatopancreas, mantle and gill. The mRNA expression level was up-regulated at 6 h post lipopolysaccharide (LPS) stimulation (p < 0.01), while no significant change was observed after peptidoglycan (PGN) stimulation. The oyster hemocytes with relative high CgIntegrin expression level exhibited different phagocytic abilities towards different microorganism and particles, such as Gram-positive bacteria Vibrio splendidus, Gram-negative bacteria Staphylococcus aureus and latex beads. Moreover, the phagocytic rate towards V. splendidus was significantly decreased after the blockade of CgIntegrin using the polyclonal antibody. The recombinant CgIntegrin (rCgIntegrin) displayed agglutinating activity towards V. splendidus but not S. aureus and Y. lipolytica. It also exhibited a higher binding affinity towards LPS (compared to rTrx group) in a dose-dependent manner with the apparent dissociation constant (Kd) of 5.53 × 10(-6) M. The results indicated that CgIntegrin served as a pattern recognition receptor with LPS binding activity, which could directly bind to V. splendidus and enhance the phagocytosis of oyster hemocytes.

The granulocytes are the main immunocompetent hemocytes in Crassostrea gigas

ABSTRACT Hemocytes comprise diverse cell types with morphological and functional heterogeneity and play indispensable roles in immunological homeostasis of invertebrates. The morphological classification of different hemocytes in mollusk has been studied since the 1970s, yet the involvement of the different sub‐populations in immune functions is far from clear. In the present study, three types of hemocytes were morphologically identified and separated as agranulocytes, semi‐granulocytes and granulocytes by flow cytometry and Percoll® density gradient centrifugation. The granulocytes were characterized functionally as the main phagocytic and encapsulating population, while semi‐granulocytes and agranulocytes exhibited low or no such capacities, respectively. Meanwhile, the lysosome activity and the productions of ROS and NO were all mainly concentrated in granulocytes under both normal and immune‐activated situations. Further, the mRNA transcripts of some immune related genes, including CgTLR, CgClathrin, CgATPeV, CgLysozyme, CgDefensin and CgIL‐17, were mainly expressed in granulocytes, lower in semi‐granulocytes and agranulocytes. These results collectively suggested that the granulocytes were the main immunocompetent hemocytes in oyster C. gigas, and a differentiation relationship among these three sub‐population hemocytes was inferred based on the gradual changes in morphological, functional and molecular features. HighlightsAgranulocytes, semi‐granulocytes and granulocytes were identified and separated.Phagocytosis and encapsulation were primarily executed by granulocytes, with little by others.Lysosome activity and production of ROS and NO were mostly concentrated in granulocytes.Immune genes CgTLR, CgLysozyme, CgDefensin and CgIL‐17, were mainly expressed in granulocytes.

Comparative study of two single CRD C-type lectins, CgCLec-4 and CgCLec-5, from pacific oyster Crassostrea gigas

C-type lectins (CTLs), a superfamily of Ca2+-dependent carbohydrate-recognition proteins, are involved in nonself-recognition and pathogen elimination, and play crucial roles in the innate immunity. In the present study, two single CRD C-type lectins, CgCLec-4 and CgCLec-5, were identified from oyster Crassostrea gigas. The open reading frame (ORF) of CgCLec-4 and CgCLec-5 encoded polypeptides of 152 and 150 amino acids, respectively. Both CgCLec-4 and CgCLec-5 contained one CRD with six conserved cysteines to form three disulfide bridges. The motif in Ca2+-binding site 2 of CgCLec-4 was QPE, while it was QYE, a non-a typical motif in CgCLec-5. CgCLec-4 was a secreted lectin with a signal peptide which was highly expressed in hepatopancreas, mantle and hemocytes. CgCLec-5 was an intracellular lectin which was mostly expressed in hemocytes. The lipopolysaccharide stimulation could induce the expressions of CgCLec-4 and CgCLec-5. The recombinant proteins of CgCLec-4 and CgCLec-5 (rCgCLec-4 and rCgCLec-5) could bind to various PAMPs including LPS, PGN, GLU and mannan, while the binding affinity of rCgCLec-5 was stronger than that of rCgCLec-4. Meanwhile, rCgCLec-4 and rCgCLec-5 could bind to different kinds of microorganisms, including Staphylococcus aureus, Escherichia coli and Vibro anguillarum and Yarrowia lipolytica, and the microbial agglutinating ability of rCgCLec-4 was stronger than that of CgCLec-5. Moreover, rCgCLec-4 exhibited anti-microbial activity against bacteria and fungi, but anti-microbial activity of CgCLec-5 was not obvious. All these results suggested that CgCLec-4 and CgCLec-5 could function as an important PRR involved in immune defense against invading pathogen in oyster, and the diversity and complexity of motifs in Ca2+ binding site 2 in CRDs determined their comprehensive recognition spectrum and multiple immune functions.

Functional characterisation of phagocytes in the Pacific oyster Crassostrea gigas

Invertebrates lack canonical adaptive immunity and mainly rely on innate immune system to fight against pathogens. The phagocytes, which could engulf and kill microbial pathogens, are likely to be of great importance and have to undertake significant roles in invertebrate immune defense. In the present study, flow cytometry combined with histological and lectin staining was employed to characterise functional features of phagocytes in the Pacific oyster Crassostrea gigas. Based on the cell size and cellular contents, haemocytes were categorised into three cell types, i.e., granulocytes, semigranulocytes and agranulocytes. Agranulocytes with smaller cell volume and lower cytoplasmic-to-nuclear ratio did not show phagocytic activity, while semigranulocytes and agranulocytes exhibited larger cell volume, higher cytoplasmic-to-nuclear ratio and phagocytic activity. In addition, granulocytes with higher side scatter (SSC) exhibited higher phagocytic activity than that of semigranulocytes. When β-integrin and lectin-like receptors were blocked by RGD tripeptide and carbohydrates, respectively, the phagocytic activity of both granulocytes and semigranulocytes was significantly inhibited, indicating that β-integrin and certain lectin-like receptors were involved in phagocytosis towards microbes. Moreover, lipopolysaccharide but not peptidylglycan could enhance phagocytic activity of granulocytes and semigranulocytes towards Vibrio splendidus and Staphylococcus aureus. Lectin staining analysis revealed that Lycopersicon esculentum lectin (LEL), binding the epitope polylactosamine, was highly distributed on the extracellular cell surface of phagocytes, and could be utilized as a potential molecular marker to differentiate phagocytes from non-phagocytic haemocytes. The results, collectively, provide knowledge on the functional characters of oyster phagocytes, which would contribute to deep investigation of cell typing and cellular immunity in bivalves.

Ocean acidification stimulates alkali signal pathway: A bicarbonate sensing soluble adenylyl cyclase from oyster Crassostrea gigas mediates physiological changes induced by CO2 exposure

Ocean acidification (OA) has been demonstrated to have severe effects on marine organisms, especially marine calcifiers. However, the impacts of OA on the physiology of marine calcifiers and the underlying mechanisms remain unclear. Soluble adenylyl cyclase (sAC) is an acid-base sensor in response to [HCO3-] and an intracellular source of cyclic AMP (cAMP). In the present study, an ortholog of sAC was identified from pacific oyster Crassostrea gigas (designated as CgsAC) and the catalytic region of CgsAC was cloned and expressed. Similar to the native CgsAC from gill tissues, the recombinant CgsAC protein (rCgsAC) exhibited [HCO3-] mediated cAMP-forming activity, which could be inhibited by a small molecule KH7. After 16days of CO2 exposure (pH=7.50), the mRNA transcripts of CgsAC increased in muscle, mantle, hepatopancreas, gill, male gonad and haemocytes, and two truncated CgsAC forms of 45kD and 20kD were produced. Cytosolic CgsAC could be translocated from the cytoplasm and nuclei to the membrane in response to CO2 exposure. Besides, CO2 exposure could increase the production of cAMP and intracellular pH of haemocytes, which was regulated by CgsAC (p<0.05), suggesting the existence of a [HCO3-]/CgsAC/cAMP signal pathway in oyster. The elevated CO2 could induce an increase of ROS level (p<0.05) and a decrease of phagocytic rate of haemocytes (p<0.05), which could be inhibited by KH7. The results collectively suggest that CgsAC is an important acid-base sensor in oyster and the [HCO3-]/CgsAC/cAMP signal pathway might be responsible for intracellular alkalization effects on oxidative phosphorylation and innate immunity under CO2 exposure. The changes of intracellular pH, ROS, and phagocytosis mediated by CgsAC might help us to further understand the effects of ocean acidification on marine calcifiers.

Soluble adenylyl cyclase mediates mitochondrial pathway of apoptosis and ATP metabolism in oyster Crassostrea gigas exposed to elevated CO2

Abstract Ocean acidification (OA) has deleterious impacts on immune response and energy homeostasis status of Mollusca. In the present study, the apoptosis ratio of hemocytes and the adenosine triphosphate (ATP) allocation in gill tissues were determined after Pacific oysters Crassostrea gigas were exposed to elevated CO2 environment (pH = 7.50) for 16 days.The apoptosis ratio in CO2 exposure group (35.2%) was significantly higher (p < 0.05) than that in the control group, and the increased apoptosis ratio induced by elevated CO2 could be significantly inhibited (p < 0.05) by KH7, a specific inhibitor of a bicarbonate sensor soluble adenylyl cyclase (sAC). After CO2 exposure, sAC in oyster (CgsAC) was found to be clustered with mitochondria in the cytoplasm, and the pro‐caspase‐3 was cleaved into two small fragments. Moreover, the activities of caspase‐3 and caspase‐9 also increased post CO2 exposure and these increases could be inhibited by KH7. However, the activities of caspase‐8 did not change significantly compared with that in the control group. After CO2 exposure, the ATP content in the gill increased significantly (p < 0.05) and such increase could also be inhibited by KH7. The ATP content in purified gill mitochondria decreased significantly (p < 0.05) after CO2 exposure, which was also inhibited by KH7. These results implied that the elevated CO2 could activate the mitochondria‐CgsAC pathway of apoptosis and ATP metabolism in oyster, and this pathway played essential roles in maintaining the homeostasis and the balance of energy metabolism in response to OA. HighlightsCgsAC mediates the increase of apoptosis rate caused by CO2 exposure.The signal of CgsAC was clustered with mitochondria after CO2 exposure.CgsAC mediates the increase of Caspase‐3 and caspase‐9 activity caused by CO2 exposure.CgsAC mediates the increase of ATP in gill tissue caused by CO2 exposure.CgsAC mediates the decrease of ATP in gill mitochondria caused by CO2 exposure.

The versatile functions of LRR-only proteins in mollusk Chlamys farreri

Leucine-rich repeat (LRR)-only proteins are involved in the innate immune responses as they mediate protein-ligand interactions. In the present study, three novel LRR-only proteins, CfLRRop-4, CfLRRop-5 and CfLRRop-6, were identified and characterized from Zhikong scallop Chlamys farreri. They all contained LRR motifs with consensus signature sequences of LxxLxLxxNxL or LxxLxLxxCxxL. All the mRNA transcripts of three CfLRRops were high abundant in hepatopancreas, gills and gonads, and their mRNA transcripts in hemocytes could respond to the stimulations of different microbes, including Vibrio anguillarum, Micrococcus luteus and Pichia pastoris. These three CfLRRops exhibited similar ligand binding and recognition characteristics as Toll-like receptors (TLRs) and NOD-like receptors (NLRs). The immune effectors, including tumor necrosis factor α, superoxide dismutase, catalase and lysozyme, varied significantly after the scallops were stimulated by recombinant LRR-only proteins. All these results indicated that LRR-only proteins are functionally differentiated and exhibit different immunomodulation activities on various downstream immune effectors.

A shell-formation related carbonic anhydrase in Crassostrea gigas modulates intracellular calcium against CO2 exposure: Implication for impacts of ocean acidification on mollusk calcification

Ocean acidification (OA) could decrease the shells and skeletons formation of mollusk by reducing the availability of carbonate ions at calcification sites. Carbonic anhydrases (CAs) convert CO2 to HCO3- and play important roles in biomineralization process from invertebrate to vertebrate. In the present study, a CA (designated as CgCA) was identified and characterized in Pacific oyster C. gigas. The cDNA of CgCA was of 927bp encoding a predicted polypeptide of 308 amino acids with a signal peptide and a CA catalytic function domain. The mRNA transcripts of CgCA were constitutively expressed in all tested tissues with the highest levels in mantle and hemocytes. During the early development period, the mRNA transcripts of CgCA could be detected in all the stages with the highest level in D-veliger larvae. Elevated CO2 increased the mRNA transcripts of CgCA in muscle, mantle, hepatopancreas, gill and hemocytes significantly (p<0.05) and induced the translocation of CgCA in hemocytes and mantle. Moreover, elevated CO2 also caused the decrease of intracellular Ca2+ in hemocytes (p<0.05). The inhibition of CA by acetazolamide and suppression of CgCA gene via RNA interference could increase the intracellular Ca2+ in hemocytes (p<0.05). Besides, the decrease of intracellular Ca2+ content caused by Ca2+ reagent ionomycin could affect localization of CgCA in mantle tissue. The results indicated CgCA played essential roles in calcification and elevated CO2 accelerated the mutual modulation between calcium and CgCA, implying reduced calcification rate and dissolved shells under OA.

The immunomodulation of a maternal translationally controlled tumor protein (TCTP) in Zhikong scallop Chlamys farreri

ABSTRACT Translationally controlled tumor protein (TCTP) is initially described as a highly conserved protein implicated in cell growth, and it is subsequently confirmed to play important roles in mediating the innate immune response, especially the inflammatory. In the present study, the full‐length cDNA sequence of a TCTP from Zhikong scallop Chlamys farreri (designed as CfTCTP) was cloned by rapid amplification of cDNA ends (RACE) technique based on the expression sequence tag (EST) analysis. It was of 1230 bp with an open reading frame (ORF) of 543 bp encoding a polypeptide of 180 amino acids. The deduced amino acid sequence contained a conserved TCTP signature sequence (from I47 to E58) and it shared 26.1%–48.9% similarities with previously identified TCTPs. CfTCTP was clustered with the TCTP from Argopectehs irradias in the phylogenetic tree and was designated into a single branch of mollusk with TCTP from Ruditapes philippinarum. The mRNA transcripts of CfTCTP were constitutively expressed in all the tested tissues, including haemocytes, muscle, mantle, gill, hepatopancreas, kidney and gonad, with the highest expression level in hepatopancreas. The mRNA expression level of CfTCTP in oocytes and fertilized eggs kept at a higher level, and was down‐regulated from 2‐cell embryos to the lowest level in gastrula. Then it was up‐regulated in trochophore and dropped down in the late veliger larvae to the similar level as that in oocytes. After pathogen‐associated molecular patterns (PAMPs) stimulation, the expression of CfTCTP mRNA in haemocytes was increased at 3 or 6 h, and fall down to the normal level at 24 h. The recombinant protein of CfTCTP could induce the release of histamine from BT‐549 cells. All these results indicated that CfTCTP was a pro‐inflammatory factor and it could be maternally transferred from female gonad to oocytes and offspring, and play pivotal role in the embryonic development and immune protection of scallops. HIGHLIGHTSA TCTP was identified from Zhikong scallop Chlamys farreri.CfTCTP was maternally transferred to oocytes and began to synthesis from trochophore.CfTCTP took part in the response to different challenges in the early phase of infection in scallops.rCfTCTP mediated histamine‐releasing from BT‐549 cells.

Two short peptidoglycan recognition proteins from Crassostrea gigas with similar structure exhibited different PAMP binding activity.

ABSTRACT Peptidoglycan recognition protein (PGRP) is an essential molecule in innate immunity for both invertebrates and vertebrates, owing to its prominent ability in specifically recognizing bacterial peptidoglycan (PGN) and eliminating the invading bacteria. In the present study, the full length cDNA of two PGRP genes, CgPGRPS2 and CgPGRPS4, were cloned from oyster Crassostrea gigas. Their amino acid sequences both contained one signal peptide, one typical PGRP/amidase domain with conserved catalytic residues responsible for amidase activity (55H, 90Y, 164H, 172C in CgPGRPS2, and 98H, 133Y, 207H, 215C in CgPGRPS4), and specific PGN recognition (84R, 85W, 104R, 109V in CgPGRPS2, and 127G, 128W, 147R, 152V in CgPGRPS4), and they shared 55.9% sequence similarity. The mRNA transcripts of CgPGRPS2 and CgPGRPS4 were constitutively expressed in all the examined tissues, including haemocytes, hepatopancreas, mantle, gonad, heart, adductor muscle and gill, with the highest expression level in adductor muscle and hepatopancreas, respectively. Both CgPGRPS2 and CgPGRPS4 proteins were mainly localized in the cytoplasma. The recombinant protein of CgPGRPS2 (rCgPGRPS2) could bind lipopolysaccharide (LPS), PGN and mannan (Man), as well as various microorganisms including Gram‐negative bacteria Escherichia coli, Vibrio anguillarum, Gram‐positive bacteria Staphylococcus aureus and fungi Yarrowia lipolytica. The recombinant protein of CgPGRPS4 (rCgPGRPS4) exhibited higher binding affinity to PGN, lower binding affinity to LPS, while no binding activity to Man and Y. lipolytica. The results indicated that CgPGRPS2 and CgPGRPS4 could function as pattern recognition receptors (PRR) in the innate immune response of oyster, and they exhibited a certain degree of functional differentiation in recognition of Man. HIGHLIGHTSCgPGRPS2 and CgPGRPS4 were identified as short type PGRP genes from Pacific oyster.CgPGRPS2 and CgPGRPS4 showed highest mRNA expression in adductor muscle and hepatopancreas, respectively.CgPGRPS2 and CgPGRPS4 proteins were mainly localized in cytoplasma of oyster haemocytes.rCgPGRPS2 could bind LPS, PGN, Man, E. coli, V. anguillarum, S. aureus and Y. lipolytica.rCgPGRPS4 could bind PGN, LPS, but not Man and Y. lipolytica.