Structural and Functional Dissects of the JAK/STAT Signaling Pathway in Teleosts Through Molecular Delineation of STAT Family Members from Rock Bream (Oplegnathus fasciatus) Revealing Their Roles During Infection and Injury

Abstract

The rock bream, Oplegnathus fasciatus is a commercially important mariculture aquacrop widely cultured in Eastern and Southeastern Asian countries. The production of rock bream fish in the aquaculture farm was unable to fulfill the high demand due to the loss of production caused by various issues. The high vulnerability of rock bream towards bacterial and viral diseases was led to the mass mortality of rock bream accounting great losses to the aquaculture farmers. The infectious disease outbreaks are very common in fish farms due to high stocking density and intensive culture techniques. It has been identified that number of diseases such as edwardsiellosis caused by Edwardsiella tarda and streptococcosis caused by Streptococcus iniae are mostly infected bacterial diseases have affected the rock bream fish. In addition, most serious virus disease caused by rock bream irido virus (RBIV) has led great loss of rock bream production. In order to battle against enormous pathological threatens, sustainable disease management strategies are required to be introduced. Therefore, deeper understanding of the molecular and cellular mechanisms of host immunity will be greatly effect for the rock bream aquaculture farm. The immune system is a complex network of organs containing cells that recognize foreign substances in the body and eliminate them. Numbers of components or signaling molecules such as antibacterial and/or antiviral peptides, signaling molecules that can initiate the antibacterial and/or antiviral pathways have been widely studied particularly in mammals. Janus kinase/signal transducers and activators of transcription (JAK/STAT) signaling pathway is a key signaling pathway that essentially work on various immune responses. In mammals, the JAK/STAT signaling pathway has been well studied and showed its relation to the wide array of functional roles including cell differentiation, migration, proliferation, embryo development, brain development, initiation of gene expression, apoptosis, dendritic cell development, myogenic differentiation, tissue injury and other essential immune responses. Seven STATs including STAT1, STAT2, STAT3, STAT4, STAT5a, STAT5b and STAT6 have been identified and well characterized in mammals. All these STATs share common structural features with six conserved domains with different functions: N-terminal domain regulates the nuclear translocation, coiled coil domain interacts with various proteins, DNA binding domain interacts with DNA, Src homology 2 (SH2) domain plays a vital role in signal transduction via phosphotyrosins, linker domain connects both DNA binding domain and SH2 domain, and transcriptional activation domain located at the C-terminal and regulates the transcriptional responses. However, the signaling components and their associated elements of the teleostean JAK/STAT signaling cascade are poorly understood or haphazardly studied in some fish species. The study of complete JAK/STAT signaling pathway with its associated modules in teleost is a challenging task for the researchers. The major effort of this study was to characterize all the STAT members that have been already identified in mammals. Eight STAT members including two STAT1 isoforms, STAT2, STAT3, STAT4, two isoforms of STAT5 and STAT6 have identified and characterize structurally and functionally. The cDNA sequence and its corresponding amino acid sequence of all the STAT members were well characterized and compared with other counterparts from various taxonomical lineages. In addition, the genomic organization of all the STAT members except one of STAT5 isoform and STAT6 were investigated and compared with other vertebrate lineages in order to determine the evolutionary aspects. Moreover, 5-proximal regions of each gene were analyzed to predict the putative sites for transcriptional factor binding elements. Tissue specific expression of each STAT in healthy rock bream was examined using the quantitative real time PCR technique. Transcriptional modulations were kinetically assessed after bacterial (E. tarda and S. iniae), viral (RBIV) and PAMP (LPS and poly I:C) injections. In addition, transcriptional changes were monitored after tissue injury in order to understand the wound healing ability of STATs. The STAT genes transcriptions were also evaluated after rock bream interleukin-10 (RbIL-10) stimulation in rock bream heart cells. To further understand the antiviral potential of STAT proteins, STAT genes were cloned into pcDNA3.1(+) vector, transfected into heart cells and measured the cell viability by WST-1 assay, after introducing RBIV. Finally, subcellular localization and nuclear translocation upon poly I:C stimulation was determined. These comprehensive studies provide substantial understanding of the structural and functional role of STAT members in rock bream JAK/STAT signaling pathway. This dissertation was divided into eight chapters; two of them were for general introduction and, materials and methods, while other six chapters were separated based on the different STAT families (STAT1, 2, 3, 4, 5 and 6) and presented the results and discussion for each rock bream STATs. Identification and characterization of rock bream STAT1 genes was discussed in the third chapter. Using the combined transcriptomic and genomic sequence obtained from bacterial artificial chromosome (BAC) library, two isoforms homologous to the STAT1 were identified and designated as RbSTAT1 and RbSTAT1L. The complete cDNA sequences of RbSTAT1 (2741 bp) and RbSTAT1L (3014 bp) comprised full length coding sequences encoding poly peptides with 750 aa and 755 aa, respectively. Pairwise sequence analysis revealed that RbSTAT1 (96.5%) and RbSTAT1L (65.1%) shared highest percent of identity with mandarin fish and Burton's mouthbrooder, respectively. In comparison of RbSTAT1 with RbSTAT1L showed 42.4% identity. Characteristic STAT domains were identified in both RbSTAT1 genes. Phylogenetic studies revealed that RbSTAT1 belongs to the canonical STAT1 group, while RbSTAT1L was clustered in a separate group indicating as a novel STAT group. Genomic organization of both RbSTAT1 and RbSTAT1L revealed that both comprised with 23 exons disrupted by 22 introns. Several transcription factor binding sites (TFBSs) were recognized in the putative promoter proximal regions of the RbSTAT1 and RbSTAT1L. The qPCR analysis depicted the highest expression of RbSTAT1 and RbSTAT1L in blood cells, while exhibiting universal expression in other tissues with varied amount. Significantly elevated expression of RbSTAT1 and RbSTAT1L was examined in blood cells and liver tissues in response to the microbial and mitogen administrations, although some time points detected suppressed expressions. Transcriptional modulations were also detected in response to the tissue injury. In addition, significantly up-regulated expressions of both RbSTAT1s were observed in rock bream heart cells upon stimulation with rock bream IL-10. Further we have analyzed the antiviral potential in vitro, and revealed the significant effect of both RbSTAT1s against RBIV. Subcellular localization study revealed that both RbSTAT1s protein localized in cytoplasm as mammals under normal conditions. Based on the results, we can suggest that both RbSTAT1s play crucial roles in immune defense against invading microbes and wound healing process. Next chapter described about the molecular characteristics and functional insight of rock bream STAT2 (RbSTAT2) gene. A single sequence contig homologous to the STAT2 was identified from rock bream transcriptome database and revealed with 804 aa. Bioinformatic analysis revealed the presence of characteristic STAT domains in peptide sequence of RbSTAT2. The BAC screening identified a complete genomic sequence of RbSTAT2 with 24 exons. In addition, essential TFBSs were identified at the 5-proximal region of RbSTAT2 gene. Based on the phylogenetic studies and comparative analysis of genomic organization of RbSTAT2 with other vertebrate origins revealed that STAT2 gene from fish and other vertebrates were distinctly evolved. The SYBR Green qPCR analysis revealed that the ubiquitous expression of RbSTAT2 transcripts in all tissues analyzed, while depicting highest expression in blood cells. Significantly modulated expressions of RbSTAT2 mRNA were detected upon viral (rock bream irido virus; RBIV), bacterial (E. tarda and S. iniae) and immune stimulants (poly I:C and LPS) challenge. Antiviral potential was further confirmed by WST-1 assay by measuring the cell viability using rock bream heart cells with RBIV. In addition possible role of RbSTAT2 on wound healing process was affirmed by analyzing the transcriptional changes after tissue injury. In vitro challenge experiment with RbIL-10 revealed the significant impact on transcription of RbSTAT2. Subcellular localization studies revealed that RbSTAT2 protein was localized in cytoplasm as others and further confirmed the signal transduction ability by adding poly I:C. Altogether, these results provide the information about the involvement of RbSTAT2 on various biologically important mechanisms. The fifth chapter describes the molecular insights and various functional roles of rock bream STAT3 (RbSTAT3). The STAT3 is one of the key transcription factors in JAK/STAT signaling pathway involved in various biologically essential events such as pleiotropic activities including immune responses, cell proliferation, inflammation, embryo development, brain development, initiation of gene expression, and apoptosis. In the present study, an orthlog of STAT3 (RbSTAT3) was identified from rock bream transcriptome database and characterized structurally and functionally. The full length sequence of RbSTAT3 was encoded a polypeptide with 764 aa, and identified characteristic STAT domains with highly conserved aa residues except in TA domain region. Sequence comparison revealed that RbSTAT3 shared relatively higher identity of percentage (> 90%) with fish counterparts. Genomic sequence of RbSTAT3 was obtained from BAC sequencing, and identified as a multi-exonic (24 exons) gene as other vertebrates. Genomic structural comparison and phylogenetic studies evidently showed that the different evolutional route for teleostean and non-teleostean vertebrates. The qPCR analysis revealed that the spatial distribution of RbSTAT3 mRNA expression was ubiquitous and highly detected in blood, heart and liver tissues. Transcriptional modulation of RbSTAT3 was examined in blood and liver tissues upon challenges with bacteria (E. tarda and S. iniae), rock bream irido virus (RBIV) and immune stimulants (LPS and poly I:C). Significant changes in RbSTAT3 transcription were also noticed, in response to the tissue injury. Significant impact of RbIL-10 on RbSTAT3 transcription was recognized after analyzing the expression level of RbSTAT3 mRNA in heart cell stimulated with RbIL-10 protein. 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 evidences for potential roles for rock bream STAT3 in immune system and wound healing process. Bioinformatic analysis and functional evidences of rock bream STAT4 gene are described in chapter 6. A member of STAT4 was identified from rock bream and designated as RbSTAT4. The cDNA sequence of RbSTAT4 was comprised with a 2205 bp ORF encoding 735 amino acids. As typical features, conserved 6 domains were identified. Phylogenetic study revealed the closest relationship with mandarinfish, and distinct evolution between fish and other vertebrates. Genomic sequence obtained from BAC screening was comprised with 24 exons interrupted by 23 exons. Immunologically crucial TFBSs were predicted at the putative promoter region of RbSTAT4. The SYBR Green qPCR analysis revealed that the expression of RbSTAT4 transcripts was highly detected in gill tissues followed by spleen and liver. Upon stimulations with LPS, poly I:C, E. tarda and RBIV, significant elevations were examined in gill, head kidney and spleen particularly in middle phase of the experiment. According to the results of this study, the expression of RbSTAT4 in healthy fish showed in gill tissues. However, the expressions of other STAT members were highly detected in blood cells. Therefore, these results suggest that RbSTAT4 is mainly functioned in gill tissues. Based on these evidences, we can hypothesize that RbSTAT4 might play possible immune roles against bacterial and viral stresses in rock bream. The next chapter describes the structural and functional characterization of rock bream STAT5 genes. Here, two different isoforms homologous to the STAT5 were identified from rock bream transcriptome database and designated as RbSTAT5-1 and RbSTAT5-2. Sequence analysis of these sequences revealed that both RbSTAT5-1 and RbSTAT5-2 were comprised with 786 amino acids in their protein sequence. In comparison of these protein sequences with other counterparts revealed the conserved residues important for functions and, typical domain regions including N-terminal domain, coiled coil domain, DNA binding domain, linker domain, Src homology 2 (SH2) domain and trans activation domain (TAD) as mammalian counterparts. As other STAT genes, a distinct evolutional path for fish and other vertebrates was observed based on the phylogenetic tree constructed by NJ method. BAC sequence analysis was able to identify the complete genomic sequence only for RbSTAT5-1, even though the genomic sequence of RbSTAT5-2 needed to be established. According to the genomic structure of RbSTAT5-1, the coding sequence was distributed into eighteen exons with seventeen introns. Further, the 5-proximal region of RbSTAT5-1 was analyzed to predict the possible TFBSs, and detected number of sites which were corresponding to bind the immunologically essential transcription factors such as ICSBP, ISGF-3, GATA-1, HNF-3, SP1, C/EBP, Oct-1, AP1, c-Jun, IRF-1 and NF-B. tissue specific expression analysis performed by qPCR revealed the highest expression of both RbSTAT5-1 and RbSTAT5-2 in blood cells. Transcriptional modulations of RbSTAT5s were examined upon challenge with live bacterial, viral and PAMPs. In addition, significant changes of RbSTAT5s were observed in response to the tissue injury. These results provide important information for the capability of RbSTAT5s in immune responses against microbes and wound healing process. Last chapter of this dissertation describes the discovery of structural and functional features of rock bream STAT6 genes. In this study a full length sequence of STAT6 gene was identified from rock bream sequence database and designated as RbSTAT6. The cDNA sequence of RbSTAT6 (4683 bp) comprised an ORF of 2313 bp that encodes 771 amino acids. Bioinformatics analysis of RbSTAT6 revealed its structural conservation as other STAT6 counterparts. Mainly, six different domain regions including N-terminal domain, coiled coil domain, DNA binding domain, linker domain, Src homology 2 (SH2) domain and transactivation domain (TAD), and some residues important for function were conserved well. In comparison of RbSTAT6 with other counterparts revealed that the percentage of identity was relatively low. As other STATs, the teleostean and non-teleostean STAT6 members evolved distinctly according to the molecular phylogenetic tree. Quantification of transcription level of RbSTAT6 in different tissues of healthy fish showed highest expression in blood cells followed by liver tissue as other STAT members of rock bream except RbSTAT4. Transcriptional modulations of RbSTAT6 were detected in blood cells and liver tissues upon viral, bacterial and PAMP injections. In addition, significantly altered transcription of RbSTAT6 was detected in blood cells and liver tissue in response to injury. Meanwhile, transcriptional changes were kinetically analyzed in rock bream heart cells after treatment with rock bream IL-10, and results revealed the significantly elevated expression of RbSTAT6 at middle phase experiment. Taken together these results implied the potential role of RbSTAT6 in host immune responses and wound healing in rock bream. As concluding remarks, the present study describes the all STAT members for the first time in a single fish species. This approach in comprehensive study of STAT family members in JAK/STAT signaling cascade of rock bream will provide the enormous information about the structural features and functional roles specifically in response to the host immunity and tissue injury. Further, this information regarding all STAT genes may contribute to establish the teleostean JAK/STAT signaling pathway.