Ze-Quan Mo

Grouper (Epinephelus coioides) IL-34/MCSF2 and MCSFR1/MCSFR2 were involved in mononuclear phagocytes activation against Cryptocaryon irritans infection

MCSF and its well-known receptor MCSFR had been well studied in humans, regulating the differentiation, proliferation, and survival of the mononuclear phagocyte system. IL-34, which is an alternative ligand of MCSF receptor, was recently identified as a novel cytokine and functionally overlaps with MCSF. However, the functional study of these receptors and their ligands in fish are largely unknown. In the present study, the cDNA of two potential grouper MCSFR ligands have been cloned, EcIL-34 (657 bp) and EcMCSF2 (804 bp), as well as an additional copy of grouper MCSFR, EcMCSFR2 (3141 bp). Sequence analysis showed that these three molecules had higher identities with other fish counterparts compared to mammals and their conserved structures and important functional residues were also analyzed. Tissue distribution analysis showed that EcIL-34 is dominant in brain, gill and spleen compared to EcMCSF2, which is dominant in head kidney, trunk kidney, skin, heart and muscle. EcMCSFR1 was dominant in the most tissues except head kidney and liver compared to EcMCSFR2. The different tissue distribution patterns of these two grouper MCSF receptors and their two ligands indicate the different mononuclear phagocyte differentiation and activation modes in different tissues. In Cryptocaryon irritans infected grouper, EcIL-34 and EcMCSFR2 were the most strongly up-regulated ligand and receptor in the infected sites, gill and skin. Their up-regulation confirmed the proliferation and activation of phagocytes in C. irritans infected sites, which would improve the antigen presentation and elicit the host local specific immune response. In C. irritans infected grouper head kidney, both ligands EcIL-34 and EcMCSF2 (especially EcMCSF2) were up-regulated, but both receptors EcMCSFR1 and EcMCSFR2 were down-regulated, which indicated that the phagocytes differentiation and proliferation may have occurred in this hemopoietic organ, and after that they migrated to the infected cites. The down-regulation of EcIL-34 and EcMCSF2 and no significant change of EcMCSFR1 and EcMCSFR2 in most time point of grouper spleen showed it was less involved in phagocytes response to C. irritans infection.

Comparative proteome analysis of two Streptococcus agalactiae strains from cultured tilapia with different virulence

Streptococcus agalactiae is a major piscine pathogen, which causes significant morbidity and mortality among numerous fish species, and results in huge economic losses to aquaculture. Many S. agalactiae strains showing different virulence characteristics have been isolated from infected tilapia in different geographical regions throughout South China in the recent years, including natural attenuated S. agalactiae strain TFJ0901 and virulent S. agalactiae strain THN0901. In the present study, survival of tilapia challenged with S. agalactiae strain TFJ0901 and THN0901 (10(7)CFU/fish) were 93.3% and 13.3%, respectively. Moreover, there are severe lesions of the examined tissues in tilapia infected with strain THN0901, but no significant histopathological changes were observed in tilapia infected with the strain TFJ0901. In order to elucidate the factors responsible for the invasive potential of S. agalactiae between two strains TFJ0901 and THN0901, a comparative proteome analysis was applied to identify the different protein expression profiles between the two strains. 506 and 508 cellular protein spots of S. agalactiae TFJ0901 and THN0901 were separated by two dimensional electrophoresis, respectively. And 34 strain-specific spots, corresponding to 27 proteins, were identified successfully by MALDI-TOF mass spectrometry. Among them, 23 proteins presented exclusively in S. agalactiae TFJ0901 or THN0901, and the other 4 proteins presented in different isomeric forms between TFJ0901 and THN0901. Most of the strain-specific proteins were just involved in metabolic pathways, while 7 of them were presumed to be responsible for the virulence differences of S. agalactiae strain TFJ0901 and THN0901, including molecular chaperone DnaJ, dihydrolipoamide dehydrogenase, thioredoxin, manganese-dependent inorganic pyrophosphatase, elongation factor Tu, bleomycin resistance protein and cell division protein DivIVA. These virulence-associated proteins may contribute to identify new diagnostic markers and help to understand the pathogenesis of S. agalactiae.

Proteomic analysis of differentially expressed proteins in the marine fish parasitic ciliate Cryptocaryon irritans

Cryptocaryoniasis is a severe disease of farmed marine fish caused by the parasitic ciliate Cryptocaryon irritans. This disease can lead to considerable economic loss, but studies on proteins linked to disease development and antigenic proteins for vaccine development have been relatively scarce to date. In this study, 53 protein spots with differential abundance, representing 12 proteins, were identified based on a pair-wise comparison among theronts, trophonts, and tomonts. Meanwhile, 33 protein spots that elicited serological responses in rabbits were identified, representing 9 proteins. In addition, 27 common antigenic protein spots reacted with grouper anti-sera, representing 10 proteins. Most of the identified proteins were involved in cytoskeletal and metabolic pathways. Among these proteins, actin and α-tubulin appeared in all three developmental stages with differences in molecular weights and isoelectric points; 4 proteins (vacuolar ATP synthase catalytic subunit α, mcm2-3-5 family protein, 26S proteasome subunit P45 family protein and dnaK protein) were highly expressed only in theronts; while protein kinase domain containing protein and heat shock protein 70 showed high levels of expression only in trophonts and tomonts, respectively. Moreover, actin was co-detected with 3 rabbit anti-sera while β-tubulin, V-type ATPase α subunit family protein, heat shock protein 70, mitochondrial-type hsp70, and dnaK proteins showed immunoreactivity with corresponding rabbit anti-sera in theronts, trophonts, and tomonts. Furthermore, β-tubulin, the metabolic-related protein enolase, NADH-ubiquinone oxidoreductase 75 kDa subunit, malate dehydrogenase, as well as polypyrimidine tract-binding protein, glutamine synthetase, protein kinase domain containing protein, TNFR/NGFR cysteine-rich region family protein, and vacuolar ATP synthase catalytic subunit α, were commonly detected by grouper anti-sera. Therefore, these findings could contribute to an understanding of the differences in gene expression and phenotypes among the different stages of parasitic infection, and might be considered as a source of candidate proteins for disease diagnosis and vaccine development.

Comparative transcriptional profile of the fish parasite Cryptocaryon irritans

BackgroundCryptocaryon irritans is an obligate ectoparasitic ciliate pathogen of marine fishes. It can infect most marine teleosts and cause heavy economic losses in aquaculture. There is currently no effective method of controlling this disease, and little information is available regarding the genes involved in its development and virulence. We aimed to investigate the distinct features of the three major life-cycle stages of C. irritans in terms of gene transcription level, and identify candidate vaccines/drug targets. We established a reference transcriptome of C. irritans by RNA-seq.MethodsThree cDNA libraries using total poly(A)+ mRNA isolated from trophonts, tomonts, and theronts was constructed and sequenced, respectively. Clean reads from the three stages were de novo assembled to generated unigene. Annotation of unigenes and transcriptomic comparison of three stages was performed.ResultsTotals of 73.15, 62.23, and 109.57 million clean reads were generated from trophont, tomont, and theront libraries, respectively. After de novo assembly, 49,104 unigenes were obtained, including 9,253 unigenes with significant similarities to proteins from other ciliates. Transcriptomic comparisons revealed that 2,470 genes were differentially expressed among the three stages, including 2,011, 1,404, and 1,797 genes that were significantly differentially expressed in tomont/theront, tomont/trophont, and theront/trophont pairwise comparisons, respectively. Based on the results of hierarchical clustering, all differentially expressed genes (DEGs) were located in five major clusters. DEGs in clusters 1 and 2 were more highly expressed in tomonts than in other stages, DEGs in cluster 3 were dominant in the tomont and trophont stages, whereas clusters 4 and 5 included genes upregulated in the theront stage. In addition, Immobilization antigens (I-antigens) and proteases have long been considered major targets for vaccine development and potential drug targets in parasites, respectively. In the present study, nine putative I-antigens transcripts and 161 protease transcripts were found in the transcriptome of C. irritans.ConclusionIt was concluded that DEGs enriched in tomonts were involved in cell division, to increase the number of theronts and ensure parasite continuity. DEGs enriched in theronts were associated with response to stimuli, whereas genes enriched in trophonts were related to nutrient accumulation and cell growth. In addition, the I-antigen and protease transcripts in our transcriptome could contribute to the development of vaccines or targeted drugs. Together, the results of the present study provide novel insights into the physiological processes of a marine parasitic ciliate.

Characterization and expression analysis of grouper (Epinephelus coioides) co-stimulatory molecules CD83 and CD80/86 post Cryptocaryon irritans infection

Abstract Co‐stimulatory molecules (CD83, CD80 and CD86), belong to immunoglobulin superfamily, are type I membrane glycoprotein, which express on antigen presenting cells and provide the second signal for the activation of T lymphocytes. In the present study, we cloned the groupers CD83 (675 bp) and CD80/86 (876 bp). Homology analysis showed that both EcCD83 and EcCD80/86 shares the highest amino acid similarity (51% and 47%) for the overall sequence with puffer fish (Takifugu rubripes). Some conserved features and important functional residues in mammalian CD83, CD80 and CD86 were also identified from these molecules of teleosts including grouper, suggesting the function of both molecules may be conserved among vertebrates. In transfected HEK293T cells, both molecules localized on the membrane surface. Tissue distribution analysis showed both EcCD83 and EcCD80/86 mRNAs were mainly expressed in immune organs, and EcCD80/86 was extremely higher expressed in mucosal immune tissues including skin and gill than systematic immune organs, which indicates these co‐stimulatory molecules may prime T cell activation in local mucosal tissues. In Cryptocaryon irritans infected groupers, the expression level of EcCD83 and EcCD80/86 were both seen significant up‐regulation in the skin at most tested time points. HighlightsCD83 and CD80/86 genes were identified from the orange‐spotted grouper.EcCD80/86 was extremely high expressed in skin and gill.EcCD83 and EcCD80/86 were significantly up‐regulated in C. irritans infected skin.

Two novel p38 MAPKs identified from Epinephelus coioides and their expression pattern in response to Cryptocaryon irritans infection

Abstract P38 mitogen‐activated protein kinases (MAPKs) are one of the most important central regulatory proteins response to extra environmental stresses. In this study, two novel p38 MAPKs, Ec‐P38&ggr; and Ec‐P38&dgr;, were identified from Epinephelus coioides, an economically important cultured fish in China and Southeast Asian counties. Both of Ec‐p38&ggr; and Ec‐p38&dgr; sequences contain a serine/threonine protein kinase (S_TKc) domain and a highly conserved Thr‐Gly‐Tyr (TGY) motif. Analysis of phylogenetic relationships illustrated that p38 amino acid sequences were conserved between different species indicating that the functions may be similar. The four subtypes of p38 (&agr;, &bgr;, &ggr;, and &dgr;) mRNA can be detected in all thirteen tissues examined, but the expression level is different in these tissues. The expression patterns of the four Ec‐p38 subtypes in E. coioides were also detected response to Cryptocaryon irritans infection, one of the most important protozoan pathogens of marine fish. The expression of four p38 subtypes was up‐regulated in the tissues examined, with the highest expressions of Ec‐p38&agr; (5.2 times) and Ec‐p38&dgr; (4.2 times) occurring in the skin, while Ec‐p38&bgr; (24.8 times) and &ggr; (16.6 times) occurred in the spleen. There was no significantly correlation between the expression of Ec‐p38&ggr;/Ec‐p38&dgr; and the expression of nuclear factor kappaB (NF‐kB). The results indicated the sequences and the characters of Ec‐p38&ggr; and Ec‐p38&dgr; were conserved, the p38 subtypes showed tissue‐specific expression patterns in healthy grouper, and their expressions were significantly up‐regulated post C. irritans infection, suggesting these p38 MAPKs may play important roles in these tissues during pathogen‐caused inflammation. HighlightsEc‐P38&ggr; and Ec‐P38&dgr; genes were identified from Epinephelus coioides.Ec‐P38&ggr; and Ec‐P38&dgr; were expressed in all thirteen tissues examined.Ec‐P38&ggr; and Ec‐P38&dgr; were significantly up‐regulated response to Cryptocaryon irritans infection.

Characterization and expression patterns of ERK1 and ERK2 from Epinephelus coioides against Cryptocaryon irritans infection

ABSTRACT Mitogen‐activated protein kinases (MAPKs), a group of serine‐threonine protein kinases, play a crucial role in immunoreaction response to extra environmental stresses. In this study, two novel MAPKs, Ec‐ERK1 and Ec‐ERK2, were identified from Epinephelus coioides. Both Ec‐ERK1 and Ec‐ERK2 sequences contain a highly conserved Thr‐Glu‐Tyr (TEY) motif, an HRD domain, and an ATP binding loop containing GXGXXG. An analysis of phylogenetic relationships demonstrated that ERK amino acid sequences were conserved between different species indicating that the functions may be similar. Ec‐ERK1 and Ec‐ERK2 mRNA can be detected in all thirteen tissues examined, but the expression level is different in these tissues. The expression patterns of these two genes in E. coioides were also detected against Cryptocaryon irritans infection, which is capable of killing large numbers of fish in a short time and has a serious impact on aquaculture. The expression was up‐regulated in most of the tissues examined, with the highest expressions of Ec‐ERK1 (3.9 times) occurring in the head kidney and Ec‐ERK2 (3.5 times) occurring in the spleen. There was no significant correlation between the expression of Ec‐ERK1/Ec‐ERK2 and the expression of nuclear factor kappaB (NF‐kB). The results indicated the sequences and the characters of Ec‐ERK1/ERK2 were conserved, Ec‐ERK1/ERK2 showed tissue‐specific expression patterns in healthy grouper, and their expressions were significantly varied post C. irritans infection, suggesting Ec‐ERK1/ERK2 may play important roles in these tissues during pathogen‐caused inflammation. HIGHLIGHTSEc‐ERK1 and Ec‐ERK2 genes were identified from Epinephelus coioides.Ec‐ERK1 and Ec‐ERK2 were expressed in all thirteen tissues examined.Ec‐ERK1 and Ec‐ERK2 in most tissues were significantly up‐regulated response to Cryptocaryon irritans infection.

Molecular characterization and function analysis of grouper (Epinephelus coioides) Bruton's tyrosine kinase BTK

ABSTRACT Brutons tyrosine kinase (BTK) is a Tec‐family tyrosine kinase and plays a crucial role in B cell antigen receptor (BCR) signal pathway. Mutations in humans and mice BTK gene results in X‐linked agammaglobulinemia (XLA) and X‐linked immunodeficiency (XLD), respectively. To study the function of BTK in teleost, we cloned a BTK gene from orange‐spotted grouper. Homology analysis showed that the grouper BTK (EcBTK) had a high amino acid identity with other vertebrates (63%–92%) and shared the highest amino acid identity with ballan wrasse Labrus bergylta BTK. EcBTK comprises a Brutons tyrosine kinase pleckstrin homology (PH) domain, a Tec homology (TH) domain, a Src homology 3 (SH3) domain, a Src homology 2 (SH2) domain and a Protein Kinases, catalytic (PKc) domain. Tissue distribution analysis showed that EcBTK was mainly expressed in immune organs. EcBTK was uniform distributed throughout the cytoplasm of transfected HEK293T cells and overexpression of EcBTK slightly down‐regulates NF‐&kgr;B activity. Ibrutinib treatment can reduce the phosphorylation level of groupers BTK. In groupers infected with Cryptocaryon irritans, up‐regulation of EcBTK were not seen in the early stage of infected skin and gill until days 14–21. The phosphorylation level of grouper BTK was significantly increased in infected skin and gill. HighlightsBTK cDNA sequences was firstly identified from grouper.EcBTK was significant up‐regulation in C. irritans infected gill and skin.Ibrutinib treatment can reduce the phosphorylation level of groupers BTK.

Identification and functional analysis of grouper (Epinephelus coioides) B-cell linker protein BLNK

&NA; B‐cell linker protein (BLNK) is an adaptor protein that plays a crucial role in the B cell antigen receptor (BCR) signal pathway. To investigate the function of BLNK in teleost fish, we cloned a BLNK ortholog gene from the orange‐spotted grouper (Epinephelus coioides). Homology analysis showed that the grouper BLNK (EcBLNK) had a 34%–77% amino acid identity in comparison to other vertebrates and shared the highest amino acid identity with BLNK from the Asian seabass Lates calcarifer. EcBLNK comprises an N‐terminal SAM domain and a C‐terminal B‐cell linker SH2 domain. Ten tyrosine residues were well conserved between teleost fish and mammals. Tissue distribution analysis showed that EcBLNK was expressed mainly in immune organs and expression was at the highest level in head kidney. Co‐localization of EcBLNK and EcCD79a was observed in transfected HEK293T cells. Overexpression of EcBLNK did not activate nuclear factor kappa‐light‐chain‐enhancer of activated B cells. The protein level of EcBLNK in grouper head kidney leukocytes was increased by stimulation with lipopolysaccharide. In groupers infected with Cryptocaryon irritans, EcBLNK was regulated in the infected sites and the systemic organ which suggests that EcBLNK was activated in the immune response to parasite infection. HighlightsBLNK cDNA sequences were identified from grouper.EcBLNK was significant regulated in C. irritans infected gill and skin.EcBLNK in grouper HKLs was increased by the stimulation of LPS.Overexpression of EcBLNK did not activate NF‐&kgr;B.