Structural insights of Labeo catla (catla) myxovirus resistance protein,GTP binding recognition and constitutive expression induced with Poly I:C.

The Myxovirus resistance (Mx) proteins are critical effectors belonging to the super-family of guanidine triphosphatase, often stimulated by type I interferon (IFN) and mediates antiviral responses to restrict the replication of numerous viral genes in fishes. In teleosts, Mx proteins display diverse and complicated antiviral activity in different species. The present investigation seeks to characterize the Mx gene from Labeo catla upon induction by double-stranded (ds) RNA, polyinosinic-polycytidylic acid, (poly I: C). Molecular modeling and all-atoms molecular dynamics (MD) simulations were employed to understand the architecture of the GTPase domain and its plausible mode of GTP recognition in Mx protein. The full-length L. catla Mx (LcMx) gene sequence (1821 bp nucleotides) encodes an open reading frame of 606 amino acids. Domain search indicated conserved tripartite domain architecture of LcMx and forms a major cluster with the Mx from other teleosts. The positively charged Arginine and polar Glutamine residues from helix 3 and 4 of stalk region LcMx aid in homo-oligomerization. MD simulation portrayed the role of conserved critical residues aid in GTP recognition by the GTPase domain which perfectly corroborates with experimental findings and prior MD studies. After injection of poly I:C, the temporal mRNA profile showed that LcMx expression was significantly elevated in the spleen, brain, kidney, liver, muscle, heart, intestine, and gill tissues. Collectively, these results suggest that the elevated expression of the major innate immune defense gene Mx was able to inhibit the poly I: C mediated virulence in fish.Communicated by Ramaswamy H. Sarma.

Co-Prevalence of Virulence and Pathogenic Potential in Multiple Antibiotic Resistant Aeromonas spp. from Diseased Fishes with In Silico Insight on the Virulent Protein Network.

Aeromonas species exhibit widespread presence in food, poultry, and aquaculture. They are major multi-drug-resistant fish pathogens. This study aims to identify Aeromonas species harbouring virulence genes aerolysin, flagellin, and lipase from diseased fishes of Assam wetlands with association with antibiotic resistance and in vivo pathogenicity. One hundred and thirty-four Aeromonas strains were isolated and thirty representative species identified using genus-specific 16S rRNA gene amplification. A. veronii was most prevalent (53.7%) followed by A. hydrophila (40.2%), A. caviae (4.47%), and A. dhakensis (1.49%). Ninety percent (90%) of strains harboured at least one of the studied virulence genes: aerA (73.3%), lip (46.6%), and flaA (26.6%). The highest multiple antibiotic resistance (MAR) index 0.8 corresponded to A. hydrophila DBTNE1 (MZ723069), containing all the studied genes. The lowest LD50 values (1.6 × 106 CFU/fish) corresponded to isolates having both aerA and lip. ß-lactams showed utmost resistance and lowest for aminoglycosides. There was a significant (p < 0.05) Pearson chi-square test of association between the occurrence of virulence and antibiotic resistance. The in silico protein−protein interaction revealed important drug targets, such as σ28 transcription factor, aminoacyl-tRNA synthetase, and diacylglycerol kinase, with significant (p < 0.05) enrichment. This study suggests that fish-isolate Aeromonas strains represent potential threat to aquaculture with subsequent risk of transferring antibiotic resistance to human pathogens.

Immunogenic Effects of Dietary Terminalia arjuna Bark Powder in Labeo rohita, a Fish Model: Elucidated by an Integrated Biomarker Response Approach.

Utilizing agro-industrial waste and herbal products to create a circular bioeconomy is becoming increasingly popular. Terminalia arjuna is a significant ethnomedicinal plant that has not yet been exploited in animal feed. In the present study, nutritional Terminalia arjuna bark powder-based fish feed was created and supplied to a candidate fish species Labeo rohita at varied levels: 0% (0 g/kg), 0.5% (5 g/kg), 1% (10 g/kg), and 1.5% (15 g/kg). These treatment groups are denoted as CT, T1, T2, and T3, respectively. Utilizing a contemporary comprehensive biomarker response strategy, the study clarified the genomic influence of dietary herb inclusion. In response to bacterial infection, the immunogenic genes, STAT 1 (signal transducer and activator of transcription 1), ISG 15 (interferon stimulating gene), and Mx "myxovirus resistance gene", were shown to be elevated. The results of densitometry demonstrated a dose-dependent increase in STAT 1 and ISG 15, with Mx exhibiting maximal values at 1 g/kg TABP (Terminalia arjuna bark powder-based feed). This is the first study to identify TABP as an immunomodulator in fish and established the IBR (Integrated Bio-marker Response) as a reliable marker in evaluating the impact of multiple drivers in a holistic manner. Thus, the present study cleared the path for TABP to be utilized as an effective feed additive which enhances the specific adaptive immune system of the fish for the production of the Green fish product for a sustainable circular bioeconomy.

Molecular characterization, constitutive expression and GTP binding mechanism of Cirrhinus mrigala (Hamilton, 1822) Myxovirus resistance (Mx) protein.

Myxovirus resistance (Mx) proteins represents the subclass of the dynamin superfamily of large Guanosine triphosphates (GTPases), play esential role in intracellular vesicle trafficking, endocytosis, organelle homeostasis and mitochondria distribution. These proteins are key players of the vertebrate immune system, induced by type-I and type-III interferons (IFN) of infected host and inhibit viral replication by sequestering its nucleoprotein. In the present study, we report the sequencing and characterization of Cirrhinus mrigala Mx protein (CmMx) for the first time and observed its constitutive expression in different tissues for a period of fourteen days. The synthetic peptide, LSGVALPRGTGI, was dissolved in PBS and injected into a rabbit and the antibody raised against CmMx was used to study the level of its expression. The full length of the CmMx cDNA is 2244 bp with a molecular mass of 70.9 kDa and a predicted isoelectric point of 8.25. The 627 amino acids polypeptide formed of three main functional domains: N-terminal GTPase domain (GD), a middle domain (MD) and GTPase effector domain (GED) with carboxy terminal leucine zipper motif. The 3D models of CmMx protein was modeled based on available close structural homologs and further validated through molecular dynamics (MD) simulations. MD study revealed the importance of G-domain responsible for recognition of GTP, which perfectly corroborate with earlier studies. MM/PBSA binding free energy analysis displayed that van der Waals and electrostatic energy were the key driving force behind molecular recognition of GTP by CmMx protein. The results from this study will illuminate more lights into the ongoing research on myxovirus resistance protein and its role in inhibition of viral replication in other eukaryotic system as well.

Molecular cloning, GTP recognition mechanism and tissue-specific expression profiling of myxovirus resistance (Mx) protein in Labeo rohita (Hamilton) after Poly I:C induction.

The myxovirus resistance (Mx) proteins belong to interferon-induced dynamin GTPase and play pivotal role in the inhibition of replication of numerous viruses. These antiviral proteins are released in usual or diseased condition to prevent the viral attack and to carry regular cellular activities like endocytosis and trafficking of nucleoproteins into the nucleus. The invasion of virus up-regulates the expression of Mx transcripts and double-stranded RNA mimic like polyinosinic polycytidyilic acid (Poly I:C). To understand the tissue-specific expression profiling and mechanism of GTP recognition of Mx protein from Labeo rohita (rohu), the full-length gene was cloned, sequenced and characterized through various Bioinformatics tools for the first time. The Mx cDNA was comprised of 2297 bp, and the open reading frame of 1938 bp encodes polypeptide of 631 amino acids. The coding sequence of Mx protein possess the signature motif of dynamin superfamily, LPRG(S/K)GIVTR, the tripartite guanosine-5/triphosphate (GTP)-binding motif (GXXXSGKS/T, DXXG and T/NKXD) and the leucine zipper motifs at the C-terminal end, well conserved in all interferon-induced Mx protein in vertebrates. Western blotting confirmed the molecular weight of Mx protein to be 72 kDa. After the intraperitoneal challenge of L. rohita with a Poly I:C, up-regulation of Mx protein was observed in brain, spleen, liver, kidney, intestine, heart, muscle, and gill. Ontogeny study displayed pronounced expression of Mx protein in all stages of the developmental of Rohu after Poly I:C induction. However a persistent expression of Mx transcript was also observed in Rohu egg as well as milt without induction with Poly I:C. Higher expression of Mx gene was observed on 96 h where it was 6.4 folds higher than the control. The computational modelling of Mx protein portrayed the tripartite N-terminal G-domain that binds to GTP, the bundle-signaling element (BSE) which interconnects the G-domain to the elongated stalk domain and C-terminal helical stalk domain. In agreement with the experimental studies, a series of conserved residues viz., Gln52, Ser53, Ser54, Leu68, Pro69, Gly71, Gly73, Thr76, Asp151, Gly154, Thr220, Lys221, Val251, Cys253, Arg254, and Gly255 were computed to be indispensable for tight anchoring of GTP within binding cavity of G-domain. The binding free energy calculation study depicted that the van der Waals and electrostatic terms contributs significantly to molecular recognition of GTP. Collectively, our study provides mechanistic insights into the tissue-specific expression profiling and GTP binding mechanism of Mx protein from Labeo rohita, which is expected to drive further research on several cellular events including viral resistance and endocytosis in the near future.

Construction, De-Novo Assembly and Analysis of Transcriptome for Identification of Reproduction-Related Genes and Pathways from Rohu, Labeo rohita (Hamilton).

Rohu is a leading candidate species for freshwater aquaculture in South-East Asia. Unlike common carp the monsoon breeding habit of rohu restricts its seed production beyond season indicating strong genetic control over spawning. Genetic information is limited in this regard. The problem is exacerbated by the lack of genomic-resources. We identified 182 reproduction-related genes previously by Sanger-sequencing which were less to address the issue of seasonal spawning behaviour of this important carp. Therefore, the present work was taken up to generate transcriptome profile by mRNAseq. 16 GB, 72 bp paired end (PE) data was generated from the pooled-RNA of twelve-tissues from pre-spawning rohu using IlluminaGA-II-platform. There were 64.97 million high-quality reads producing 62,283 contigs and 88,612 numbers of transcripts using velvet and oases programs, respectively. Gene ontology annotation identified 940 reproduction-related genes consisting of 184 mainly associated with reproduction, 223 related to hormone-activity and receptor-binding, 178 receptor-activity and 355 embryonic-development related-proteins. The important reproduction-relevant pathways found in KEGG analysis were GnRH-signaling, oocyte-meiosis, steroid-biosynthesis, steroid-hormone biosynthesis, progesterone-mediated oocyte-maturation, retinol-metabolism, neuroactive-ligand-receptor interaction, neurotrophin-signaling and photo-transduction. Twenty nine simple sequence repeat containing sequences were also found out of which 12 repeat loci were polymorphic with mean expected-&-observed heterozygosity of 0.471 and 0.983 respectively. Quantitative RT-PCR analyses of 13-known and 6-unknown transcripts revealed differences in expression level between preparatory and post-spawning phase. These transcriptomic sequences have significantly increased the genetic-&-genomic resources for reproduction-research in Labeo rohita.