A homolog of teleostean signal transducer and activator of transcription 3 (STAT3) from rock bream, <i>Oplegnathus fasciatus</i>: Structural insights, transcriptional modulation, and subcellular localization

Bathige, S.D.N.K.,Thulasitha, William Shanthakumar,Umasuthan, Navaneethaiyer,Jayasinghe, J.D.H.E.,Wan, Qiang,Nam, Bo-Hye,Lee, Jehee Elsevier 2017 Veterinary immunology and immunopathology Vol.186 No.-

<P><B>Abstract</B></P> <P>Signal transducer and activator of transcription 3 (STAT3) is one of the crucial transcription factors in the Janus kinase (JAK)/STAT signaling pathway, and it was previously considered as acute phase response factor. A number of interleukins (ILs) such as IL-5, IL-6, IL-9, IL-10, IL-12, and IL-22 are known to be involved in activation of STAT3. In addition, various growth factors and pathogenic or oxidative stresses mediate the activation of a wide range of functions via STAT3. In this study, a STAT3 homolog was identified and functionally characterized from rock bream (RbSTAT3), <I>Oplegnathus fasciatus</I>. <I>In silico</I> characterization revealed that the RbSTAT3 amino acid sequence shares highly conserved common domain architectural features including N-terminal domain, coiled coil domain, DNA binding domain, linker domain, and Src homology 2 (SH2) domains. In addition, a fairly conserved transcriptional activation domain (TAD) was located at the C-terminus. Comparison of RbSTAT3 with other counterparts revealed higher identities (>90%) with fish orthologs. The genomic sequence of <I>RbSTAT3</I> was obtained from a bacterial artificial chromosome (BAC) library, and was identified as a multi-exonic gene (24 exons), as found in other vertebrates. Genomic structural comparison and phylogenetic studies have showed that the evolutionary routes of teleostean and non-teleostean vertebrates were distinct. Quantitative real time PCR (qPCR) analysis revealed that the spatial distribution of <I>RbSTAT3</I> mRNA expression was ubiquitous and highly detectable in blood, heart, and liver tissues. Transcriptional modulation of <I>RbSTAT3</I> was examined in blood and liver tissues after challenges with bacteria (<I>Edwardsiella tarda</I> and <I>Streptococcus iniae</I>), rock bream irido virus (RBIV), and immune stimulants (LPS and poly (I:C)). Significant changes in <I>RbSTAT3</I> transcription were also observed in response to tissue injury. In addition, the transcriptional up-regulation of <I>RbSTAT3</I> was detected in rock bream heart cells upon recombinant rock bream IL-10 (rRbIL-10) treatment. Subcellular localization and nuclear translocation of rock bream STAT3 following poly (I:C) treatment were also demonstrated. Taken together, the results of the current study provide important evidence for potential roles of rock bream STAT3 in the immune system and wound healing processes.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Identification of STAT3 (RbSTAT3) ortholog from rock bream. </LI> <LI> Genomic structure and putative promoter analysis of <I>RbSTAT3</I>. </LI> <LI> Expression profile (<I>In vivo</I>) of <I>RbSTAT3</I> upon pathogenic and PAMP stress, and tissue injury. </LI> <LI> Expression profile (<I>In vitro</I>) of <I>RbSTAT3</I> in response to rock bream IL-10. </LI> <LI> Subcellular localization of RbSTAT3. </LI> </UL> </P>

Molecular cloning, transcriptional profiling, and subcellular localization of signal transducer and activator of transcription 2 (STAT2) ortholog from rock bream, Oplegnathus fasciatus

Bathige, S.D.N.K.,Umasuthan, N.,Priyathilaka, T.T.,Thulasitha, W.S.,Jayasinghe, J.D.H.E.,Wan, Q.,Nam, B.H.,Lee, J. Elsevier/North-Holland 2017 Gene Vol.626 No.-

Signal transducer and activator of transcription 2 (STAT2) is a key element that transduces signals from the cell membrane to the nucleus via the type I interferon-signaling pathway. Although the structural and functional aspects of STAT proteins are well studied in mammals, information on teleostean STATs is very limited. In this study, a STAT paralog, which is highly homologous to the STAT2 members, was identified from a commercially important fish species called rock bream and designated as RbSTAT2. The RbSTAT2 gene was characterized at complementary DNA (cDNA) and genomic sequence levels, and was found to possess structural features common with its mammalian counterparts. The complete cDNA sequence was distributed into 24 exons in the genomic sequence. The promoter proximal region was analyzed and found to contain potential transcription factor binding sites to regulate the transcription of RbSTAT2. Phylogenetic studies and comparative genomic structure organization revealed the distinguishable evolution for fish and other vertebrate STAT2 orthologs. Transcriptional quantification was performed by SYBR Green quantitative real-time PCR (qPCR) and the ubiquitous expression of RbSTAT2 transcripts was observed in all tissues analyzed from healthy fish, with a remarkably high expression in blood cells. Significantly (P<0.05) altered transcription of RbSTAT2 was detected after immune challenge experiments with viral (rock bream irido virus; RBIV), bacterial (Edwardsiella tarda and Streptococcus iniae), and immune stimulants (poly I:C and LPS). Antiviral potential was further confirmed by WST-1 assay, by measuring the viability of rock bream heart cells treated with RBIV. In addition, results of an in vitro challenge experiment signified the influence of rock bream interleukin-10 (RbIL-10) on transcription of RbSTAT2. Subcellular localization studies by transfection of pEGFP-N1/RbSTAT2 into rock bream heart cells revealed that the RbSTAT2 was usually located in the cytoplasm and translocated near to the nucleus upon poly I:C administration. Altogether, these findings suggest that RbSTAT2 is involved in various biologically crucial mechanisms, and provides immune protection to the rock bream.

Three novel C1q domain containing proteins from the disk abalone Haliotis discus discus: Genomic organization and analysis of the transcriptional changes in response to bacterial pathogens

Bathige, S.D.N.K.,Umasuthan, N.,Jayasinghe, J.D.H.E.,Godahewa, G.I.,Park, H.C.,Lee, J. Academic Press 2016 FISH AND SHELLFISH IMMUNOLOGY Vol.56 No.-

<P>The globular C1q (gC1q) domain containing proteins, commonly referred as C1q domain containing (C1qDC) proteins, are an essential family of proteins involved in various innate immune responses. In this study, three novel C1qDC proteins were identified from the disk abalone (Haliotis discus discus) transcriptome database and designated as AbC1qDC1, AbC1qDC2, and AbC1qDC3. The cDNA sequences of AbC1qDC1, AbC1qDC2, and AbC1qD0 consisted of 807, 1305, and 660 bp open reading frames (ORFs) encoding 269, 435, and 220 amino acids (aa), respectively. Putative signal peptides and the N-terminal gC1q domain were identified in all three AbC1qDC proteins. An additional predicted motif region, known as the coiled coil region (CCR), was identified next to the signal sequence of AbC1qDC2. The genomic organization of the AbC1qDCs was determined using a bacterial artificial chromosome (BAC) library. It was found that the CDS of AbC1qDC1 was distributed among three exons, while the CDSs of AbC1qDC2 and AbC1qDC3 were distributed between two exons. Sequence analysis indicated that the AbC1qDC proteins shared <40% identity with other counterparts from different species. According to the neighbor joining phylogenetic tree, the proteins were grouped within an invertebrate group with high evolutionary distances, which suggests that they are new members of the C1qDC family. Higher expression of AbC1qDC1 and AbC1qDC2 was detected in hepatopancreas, muscle, and mantle tissues compare to the other tissues analyzed, using reverse transcription, followed by quantitative real-time PCR (qPCR) using SYBR Green, whereas AbC1qDC3 was predominantly expressed in gill tissues, followed by muscles and the hepatopancreas. The temporal expression of AbC1qDC transcripts in gills after bacterial (Vibrio parahaemolyticus and Listeria monocytogenes) and lipopolysaccharide stimulation indicated that AbC1qDCs can be strongly induced by both Gram-negative and Gram-positive bacterial species with different response profiles. The results of this study suggest that AbC1qDCs are involved in immune responses against invading bacterial pathogens. (C) 2016 Published by Elsevier Ltd.</P>

A bifunctional invertebrate-type lysozyme from the disk abalone, Haliotis discus discus: Genome organization, transcriptional profiling and biological activities of recombinant protein

Bathige, S.D.N.K.,Umasuthan, N.,Kasthuri, S.R.,Whang, I.,Lim, B.S.,Nam, B.H.,Lee, J. Pergamon Press ; Elsevier Science 2013 Developmental and comparative immunology Vol.41 No.2

Lysozyme is an important enzyme in the innate immune system that plays a vital role in fighting microbial infections. In the current study, we identified, cloned, and characterized a gene that encodes an invertebrate-type lysozyme from the disk abalone, Haliotis discus discus (abLysI). The full-length cDNA of abLysI consisted of 545bp with an open reading frame of 393bp that encodes 131 amino acids. The theoretical molecular mass of mature abLysI was 12.3kDa with an isoelectric point of 8.03. Conserved features in other homologs, such as catalytic sites for lytic activity (Glu<SUP>30</SUP> and Asp<SUP>41</SUP>), isopeptidase activity (His<SUP>107</SUP>), and ten cysteine residues were identified in abLysI. Genomic sequence analysis with respect to its cDNA showed that abLysI was organized into four exons interrupted by three introns. Several immune-related transcription factor binding sites were discovered in the putative promoter region. Homology and phylogeny analysis of abLysI depicted high identity and closer proximity, respectively, with an annelid i-type lysozyme from Hirudo medicinalis, and indicated that abLysI is a novel molluscan i-type lysozyme. Tissue-specific expressional studies revealed that abLysI is mainly transcribed in hepatopancreas followed by mantle. In addition, abLysI mRNA expression was induced following bacterial (Vibrio parahaemolyticus and Listeria monocytogenes) and viral (viral hemorrhagic septicemia virus) challenges. Recombinantly expressed abLysI [(r)abLysI] demonstrated strong lytic activity against Micrococcus lysodeikticus, isopeptidase activity, and antibacterial activity against several Gram-positive and Gram-negative bacteria. Moreover, (r)abLysI showed optimum lytic activity at pH 4.0 and 60<SUP>o</SUP>C, while exhibiting optimum isopeptidase activity at pH 7.0. Taken together, these results indicate that abLysI is potentially involved in immune responses of the disk abalone to protect it from invaders.

Molecular insights of two STAT1 variants from rock bream (Oplegnathus fasciatus) and their transcriptional regulation in response to pathogenic stress, interleukin-10, and tissue injury

Bathige, S.D.N.K.,Umasuthan, N.,Godahewa, G.I.,Thulasitha, W.S.,Jayasinghe, J.D.H.E.,Wan, Q.,Lee, J. Academic Press 2017 Fish & shellfish immunology Vol.69 No.-

Signal transducers and activators of transcription 1 (STAT1) is critically involved in mediating cytokine-driven signaling, and triggers the transcription of target genes to activate cellular functions. Although the structural and functional aspects of STAT members have been well described in mammals, only limited information is available for the STAT genes in teleost fishes. In the present study, two variants of STAT1 genes (RbSTAT1 and RbSTAT1L) were identified from rock bream and characterized at the cDNA and genomic sequence levels. RbSTAT1 and RbSTAT1L were found to share a common domain architecture with mammalian STAT1. Phylogenetic analysis revealed that RbSTAT1 shows a common evolutionary trajectory with other STAT1 counterparts, whereas RbSTAT1L showed a separate path, implying that it could be a novel member of the STAT family. The genomic organizations of RbSTAT1 and RbSTAT1L illustrated a similar exon-intron pattern with 23 exons in the coding sequence. Transcription factor-binding sites, which are mostly involved in the regulation of immune responses, were predicted at the putative promoter regions of the RbSTAT1 and RbSTAT1L genes. SYBR Green qPCR analysis revealed the ubiquitous expression of RbSTAT1 and RbSTAT1L transcripts in different fish tissues with the highest level observed in peripheral blood cells. Significantly modulated transcripts were noted upon viral (rock bream iridovirus [RBIV]), bacterial (Edwardsiella tarda and Streptococcus iniae), and pathogen-associated molecular pattern (lipopolysaccharide and poly I:C) stimulations. The WST-1 cell viability assay affirmed the potential antiviral capacity of RbSTAT1 and RbSTAT1L against RBIV. A possible role of RbSTAT1 and RbSTAT1L in the wound healing process was revealed according to their modulated expression in injured fish. In addition, the transcriptional regulation of RbSTAT1 and RbSTAT1L was analyzed by qPCR following stimulation with rock bream interleukin-10. Taken together, these findings suggest that the STAT1-mediated Janus kinase/STAT pathway might at least in part be involved in the regulatory mechanisms underlying the immune defensive roles against microbial pathogens and the wound healing process.

A homolog of Kunitz-type serine protease inhibitor from rock bream, Oplegnathus fasciatus: Molecular insights and transcriptional modulation in response to microbial and PAMP stimulation, and tissue injury

Bathige, S.D.N.K.,Umasuthan, N.,Godahewa, G.I.,Jayasinghe, J.D.H.E.,Whang, I.,Noh, J.K.,Lee, J. Academic Press 2015 FISH AND SHELLFISH IMMUNOLOGY Vol.46 No.2

Serine proteases and their inhibitors play vital roles in diverse biological processes. In this study, we identified and characterized cDNA coding for a Kunitz-type serine protease inhibitor (SPI), which we designated as RbKSPI, in a commercially important species, rock bream. The full-length cDNA sequence of RbKSPI consisted of 2452 bp with an open reading frame (ORF) of 1521 bp encoding a polypeptide of 507 amino acid (aa) residues. In the RbKSPI protein, MANEC, PKD, LDLa, and two Kunitz domains responsible for various functions were identified as characteristic features. Homology analysis revealed that RbKSPI shared the highest identity with the Kunitz homolog in Takifugu rubripes (77.6%). Phylogenetic analysis indicated that RbKSPI clusters with other teleostean KSPIs. In tissue-specific expression analysis, RbKSPI transcripts were detected in all the tested tissues, with the highest expression in gill tissue, followed by kidney and intestine. The mRNA expression of RbKSPI significantly increased in blood cells upon stimulation with two strains of bacteria (Edwardsiella tarda and Streptococcus iniae) and two pathogen-associated molecular patterns (PAMPs; LPS and poly I:C). Meanwhile, down-regulated expression of RbKSPI was observed in response to tissue injury. Collectively, these results suggest that the RbKSPI may be involved in essential immune defense against microbial pathogens and in the wound-healing process.

Evidences for the involvement of an invertebrate goose-type lysozyme in disk abalone immunity: Cloning, expression analysis and antimicrobial activity

Bathige, S.D.N.K.,Umasuthan, N.,Whang, I.,Lim, B.S.,Jung, H.B.,Lee, J. Academic Press 2013 Fish & shellfish immunology Vol.35 No.5

Lysozymes are ubiquitously distributed enzymes with hydrolytic activity against bacterial peptidoglycan and function to protect organisms from microbial pathogens. In this study, an invertebrate goose-type lysozyme, designated as abLysG, was identified in the disk abalone, Haliotis discus discus. The full-length cDNA of abLysG was 894 bp in length with an open reading frame of 789 bp encoding a polypeptide of 263 amino acids containing a signal peptide and a characteristic soluble lytic transglycosylase domain. Six cysteine residues and two catalytic residues (Glu<SUP>142</SUP> and Asp<SUP>168</SUP>) conserved among molluscs were also identified. The 3D homology structural models of abLysG and hen egg white lysozyme had similar conformations of the active sites involved in the binding of substrate. BAC sequence data revealed that the genomic structure of disk abalone g-type lysozyme comprises 7 exons with 6 intervening introns. The deduced amino acid sequence of abLysG shared 45.2-61.6% similarity with those of other molluscs and vertebrates. The TFSEARCH server predicted a variety of transcription factor-binding sites in the 5'-flanking region of the abLysG gene, some of which are involved in transcriptional regulation of the lysozyme gene. abLysG expression was detected in multiple tissues with the highest expression in mantle. Moreover, qPCR analysis of abLysG mRNA expression demonstrated significant up-regulation in gill in response to infection by live bacteria (Vibrio parahaemolyticus and Listeria monocytogenes), virus (viral hemorrhagic septicemia) and bacterial mimics (LPS and PGN). Expression of the recombinant disk abalone g-type lysozyme in Escherichia coli BL21, demonstrated its bacteriolytic activity against several Gram-negative and Gram-positive bacterial species. Collectively these data suggest that abLysG is an antimicrobial enzyme with a potential role in the disk abalone innate immune system to protect it from bacterial and viral infections.

An invertebrate signal transducer and activator of transcription 5 (STAT5) ortholog from the disk abalone, Haliotis discus discus: Genomic structure, early developmental expression, and immune responses to bacterial and viral stresses

Bathige, S.D.N.K.,Umasuthan, N.,Park, H.C.,Lee, J. Pergamon Press ; Elsevier Science 2016 DEVELOPMENTAL AND COMPARATIVE IMMUNOLOGY Vol.56 No.-

<P>Signal transducer and activator of transcription (STAT) family members are key signaling molecules that transduce cellular responses from the cell membrane to the nucleus upon Janus kinase (JAK) activation. Although seven STAT members have been reported in mammals, very limited information on STAT genes in molluscans is available. In this study, we identified and characterized a STAT paralog that is homologous to STAT5 from the disk abalone, Haliotis discus discus, and designated as AbSTAT5. Comparison of the deduced amino acid sequence for AbSTAT5 (790 amino acids) with other counterparts revealed conserved residues important for functions and typical domain regions, including the N-terminal domain, coiled-coil domain, DNA-binding domain, linker domain, and Src homology 2 (SH2) domains as mammalian counterparts. Analysis of STAT phylogeny revealed that AbSTAT5 was clustered with the molluscan subgroup in STAT5 Glade with distinct evolution. According to the genomic structure of AbSTAT5, the coding sequence was distributed into 20 exons with 19 introns. Immunologically essential transcription factor-binding sites, such as GATA-1, HNF, SP1, C/EBP, Oct-1, AP1, c-Jun, and Sox-2, were predicted at the 5'-proximal region of AbSTAT5. Expression of AbSTAT5 mRNA was detected in different stages of embryonic development and observed at considerably higher levels in the morula and late veliger stages. Tissue-specific expressional studies revealed that the highest level of AbSTAT5 transcripts was detected in hemocytes, followed by gill tissues. Temporal expressions of AbSTAT5 were analyzed upon live bacterial (Vibrio parahemolyticus and Listeria monocytogenes), viral (viral hemorrhagic septicemia virus), and pathogen-associated molecular pattern (lipopolysaccharides and Poly I:C) stimulations, and significant elevations indicated immune modulation. These results suggest that AbSTAT5 may be involved in maintaining innate immune responses from developmental to adult stages in the disk abalone. Further, this study provides a basis for structural and functional exploration of STAT members in the invertebrate JAK/STAT signaling pathway. (C) 2015 Elsevier Ltd. All rights reserved.</P>

Antibacterial activity and immune responses of a molluscan macrophage expressed gene-1 from disk abalone, Haliotis discus discus

Bathige, S.D.N.K.,Umasuthan, N.,Whang, I.,Lim, B.S.,Won, S.H.,Lee, J. Academic Press 2014 FISH AND SHELLFISH IMMUNOLOGY Vol.39 No.2

The membrane-attack complex/perforin (MACPF) domain-containing proteins play an important role in the innate immune response against invading microbial pathogens. In the current study, a member of the MACPF domain-containing proteins, macrophage expressed gene-1 (MPEG1) encoding 730 amino acids with the theoretical molecular mass of 79.6 kDa and an isoelectric point (pI) of 6.49 was characterized from disk abalone Haliotis discus discus (AbMPEG1). We found that the characteristic MACPF domain (Val<SUP>131</SUP>-Tyr<SUP>348</SUP>) and transmembrane segment (Ala<SUP>669</SUP>-Ile<SUP>691</SUP>) of AbMPEG1 are located in the N- and C-terminal ends of the protein, respectively. Ortholog comparison revealed that AbMPEG1 has the highest sequence identity with its pink abalone counterpart, while sequences identities of greater than 90% were observed with MPEG1 members from other abalone species. Likewise, the furin cleavage site KRRRK was highly conserved in all abalone species, but not in other species investigated. We identified an intron-less genomic sequence within disk abalone AbMPEG1, which was similar to other mammalian, avian, and reptilian counterparts. Transcription factor binding sites, which are important for immune responses, were identified in the 5'-flanking region of AbMPEG1. qPCR revealed AbMPEG1 transcripts are present in every tissues examined, with the highest expression level occurring in mantle tissue. Significant up-regulation of AbMPEG1 transcript levels was observed in hemocytes and gill tissues following challenges with pathogens (Vibrio parahemolyticus, Listeria monocytogenes and viral hemorrhagic septicemia virus) as well as pathogen-associated molecular patterns (PAMPs: lipopolysaccharides and poly I:C immunostimulant). Finally, the antibacterial activity of the MACPF domain was characterized against Gram-negative and -positive bacteria using a recombinant peptide. Taken together, these results indicate that the biological significance of the AbMPEG1 gene includes a role in protecting disk abalone through the ability of AbMPEG1 to initiate an innate immune response upon pathogen invasion.

Two variants of selenium-dependent glutathione peroxidase from the disk abalone Haliotis discus discus: Molecular characterization and immune responses to bacterial and viral stresses

Bathige, S.D.N.K.,Umasuthan, N.,Godahewa, G.I.,Thulasitha, W.S.,Whang, I.,Won, S.H.,Kim, C.,Lee, J. Academic Press 2015 FISH AND SHELLFISH IMMUNOLOGY Vol.45 No.2

Glutathione peroxidase (GPx) is an essential member of the antioxidant systems of living organisms and may be involved in immune defense against pathogenic invasion. In the current study, two selenium-dependent glutathione peroxidases (AbSeGPxs) that shared 54.3% identity were identified from the disk abalone Haliotis discus discus. The open reading frames (ORFs) of AbSeGPx-a and AbSeGPx-b coded for 222 and 220 amino acids, respectively, with a characteristic selenocysteine residue encoded by an opal stop codon (TGA). The conserved selenocysteine insertion sequence (SECIS) element was predicted in the 3' untranslated region (UTR) of both isoforms, and they were found to form two stem-loop structures. Amino acid comparison and phylogenetic studies revealed that the AbSeGPxs were closely related to those in other mollusk species and were evolutionarily distinct from those of other taxonomic groups. The SYBR Green qPCR was employed in investigating the transcripts of AbSeGPxs. The expression of AbSeGPxs mRNA was examined in different embryonic developmental stages and differential expression patterns for AbSeGPx-a and AbSeGPx-b were noted. Meanwhile, the highest expression of AbSeGPxs was detected in the hepatopancreas of healthy adult animals. Next, transcriptional levels were profiled in hemocytes of adults to determine the immune responses of AbSeGPxs to microbial infections. The results revealed the significant up-regulation of AbSeGPx-a in a time-dependent manner after bacterial (Listeria monocytogenes and Vibrio parahaemolyticus) and viral (viral hemorrhagic septicemia virus) infections. Consequently, these findings indicate that AbSeGPx-a and AbSeGPx-b might be involved in the embryonic development of disk abalone and the regulation of immune defense system of adult animals.