Recombinant Attenuated Edwardsiella piscicida Vaccine Displaying Regulated Lysis to Confer Biological Containment and Protect Catfish against Edwardsiellosis.

We implemented a unique strategy to construct a recombinant attenuated Edwardsiella vaccine (RAEV) with a biological containment phenotype that causes regulated bacterial cell wall lysis. This process ensures that the vaccine strain is not able to persist in the environment. The murA gene is responsible for the catalysis of one of the first steps in the biosynthesis of muramic acid, which is a crucial component of the bacterial cell wall. The regulated lysis phenotype was achieved by inserting the tightly regulated araC ParaBAD cassette in place of the chromosomal murA promoter. Strains with this mutation require growth media supplemented with arabinose in order to survive. Without arabinose, they are unable to synthesize the peptidoglycan cell wall. Following the colonization of fish lymphoid tissues, the murA protein is no longer synthesized due to the lack of arabinose. Lysis is subsequently achieved in vivo, thus preventing the generation of disease symptoms and the spread of the strain into the environment. Vaccine strain χ16016 with the genotype ΔPmurA180::TT araC ParaBADmurA is attenuated and shows a higher LD50 value than that of the wild-type strain. Studies have demonstrated that χ16016 induced TLR4, TLR5, TLR8, TLR9, NOD1 and NOD2-mediated NF-κB pathways and upregulated the gene expression of various cytokines, such as il-8, il-1ß, tnf-a, il-6 and ifn-γ in catfish. We observed significant upregulation of the expression profiles of cd4, cd8 and mhc-II genes in different organs of vaccinated catfish. Vaccine strain χ16016 induced systemic and mucosal IgM titers and conferred significant protection to catfish against E. piscicida wild-type challenge. Our lysis RAEV is the first live attenuated vaccine candidate designed to be used in the aquaculture industry that displays this biological containment property.

Virulence, immunogenicity and live vaccine potential of aroA and phoP mutants of Edwardsiella piscicida in zebrafish.

Vaccination remains the most effective approach for prevention and control of infectious diseases in aquaculture. Edwardsiella piscicida is a causative agent of edwardsiellosis leading to mass mortality in a variety of fish species, leading to huge economic losses in the aquaculture industry. In this study, we have deleted the aroA and phoP genes in E. piscicida and investigated the phenotype, degrees of attenuation, immunogenicity, and ability to confer immune protection in zebrafish host. Our vaccine strain χ16028 with genotype ΔaroA11 ΔphoP12, showed significantly reduced growth, motility, biofilm formation and intracellular replication compared to the wild-type strain J118. In this regard, χ16028 exhibited retarded colonization and attenuation phenotype in zebrafish. Studies showed that χ16028 induced TLR4 and TLR5 mediated NF-kB pathway and upregulated cytokine gene expression i.e., TNF-α, IL-1ß, IL-6, IL-8 and type-I IFN in zebrafish. Zebrafish immunized by intracoelomic injection (i.c.) with χ16028 showed systemic and mucosal IgM responses and protection against the wild-type E. piscicida i.c. injection challenge. However, the protection was only 25% in zebrafish following i.c. challenge. We speculate that our vaccine strain might be very attenuated; a booster dose may trigger better immune response and increase the percentage of survival to a more significant level.

Construction and Evaluation of Recombinant Attenuated Edwardsiella piscicida Vaccine (RAEV) Vector System Encoding Ichthyophthirius multifiliis (Ich) Antigen IAG52B.

We have successfully designed and constructed a RAEV vector system with regulated-delayed attenuation in vivo attributes that synthesizes Ichthyophthirius multifiliis (Ich) protective antigen IAG52B to enable vaccination of fish susceptible to edwardsiellosis and white spot disease. The first feature of this vaccine delivery system is an Edwardsiella piscicida strain carrying genomic deletions of asdA. AsdA is an enzyme necessary for the synthesis of diaminopimelic acid (DAP), which is an essential component of the peptidoglycan layer of the cell wall of Gram-negative bacteria. asdA mutant strains have obligate growth requirements for DAP in the medium or a plasmid vector with the wild-type asdA gene enabling synthesis of DAP. This balanced-lethal plasmid vector-host system in E. piscicida enables as a second feature the synthesis of recombinant antigens to induce protective immunity against fish pathogens. Recombinant protective antigen IAG52B from the fish pathogen I. multifiliis was synthesized by RAEV strains harboring the AsdA+ plasmid pG8R8029. The third feature of this vaccine strain is a regulated-delayed attenuation in vivo phenotype that is based on the replacement of an arabinose-regulated araC ParaBAD cassette for the promoters of the fur and crp genes of E. piscicida such that the expression of these genes is dependent on arabinose provided during growth. Thus, following colonization, the Fur and Crp proteins stop being synthesized due to the lack of arabinose and attenuation is progressively achieved in vivo to prevent generation of diseases symptoms. Our vaccine strain χ16022 with the genotype ΔasdA10 ΔPfur170::TT araC ParaBADfur ΔPcrp68::TT araC ParaBADcrp contains the AsdA+ plasmid, pG8R8029, which encodes the IAG52B antigen. Vaccine strain χ16022(pG8R8029) is attenuated and induces systemic and mucosal IgM titer against E. piscicida and Ich in zebrafish. In addition, transcript levels of tnf-α, il-1ß, il-6 and il-8 were significantly increased in different tissues of vaccinated zebrafish compared to unimmunized fish. Zebrafish vaccinated with χ16022(pG8R8029) showed 60% survival upon intracoelomic (i.c.) challenge with a lethal dose of virulent E. piscicida strain J118. Our RAEV system could be used as a generalized vaccine-vector system to protect teleost fish against multiple bacterial, viral and parasitic infectious diseases.

Pathogenicity and immunogenicity of Edwardsiella piscicida ferric uptake regulator (fur) mutations in zebrafish.

Edwardsiella piscicida is the etiological agent of edwardsiellosis in fish and causes severe economic losses in global aquaculture. Vaccination would be the most effective method to prevent infectious diseases and their associated economic losses. The ferric uptake regulator (Fur) is an important transcriptional global regulator of Gram-negative bacteria. In this study, we examined the regulatory function of Fur in E. piscicida. We designed a strain that displays features of the wild-type virulent strain of E. piscicida at the time of immunization to enable strains first to effectively colonize lymphoid tissues and then to exhibit a regulated delayed attenuation in vivo to preclude inducing disease symptoms. Regulated delayed attenuation in vivo is based on the substitution of a tightly regulated araC ParaBAD cassette for the promoter of the fur gene such that expression of this gene is dependent on arabinose provided during growth. Thus, following E. piscicida mutant colonization of lymphoid tissues, the Fur protein ceases to be synthesized due to the absence of arabinose such that attenuation is gradually manifest in vivo to preclude induction of diseases symptoms. We deleted the promoter, including all sequences that interact with activator or repressor proteins, for the fur gene, and substituted the improved araC ParaBAD cassette to yield an E. piscicida strain with the ΔPfur170:TT araC ParaBADfur deletion-insertion mutation (χ16012). Compared to the wild-type strain J118, χ16012 exhibited retarded growth and enhanced siderophore production in the absence of arabinose. mRNA levels of Fur-regulated genes were analyzed in iron deplete or replete condition in wild-type and fur mutant strains. We observed zebrafish immunized with χ16012 showed better colonization and protection compared to the Δfur (χ16001). Studies showed that E. piscicida strain χ16012 is attenuated and induces systemic and mucosal IgM titer in zebrafish. In addition, we found an increase in transcript levels of tnf-α, il-1ß, il-8 and ifn-γ in different tissues of zebrafish immunized with χ16012 compared to the unimmunized group. We conclude that, E. piscicida with regulated delayed attenuation could be an effective immersion vaccine for the aquaculture industry.

Molecular cloning and characterization of LrTLR4, analysis of its inductive expression and associated down-stream signaling molecules following lipopolysaccharide stimulation and Gram-negative bacterial infection.

Toll-like receptors (TLRs) play key roles in innate immunity from lower to higher vertebrates. Among various TLR types, TLR4 was reported to recognize LPS in higher vertebrates resulting in the activation of down-stream signaling pathway. Except in some teleosts, function of TLR4 in most fish species including rohu (Labeo rohita) a commercially important fish species in the South-East Asian countries remained unknown. To investigate it, full-length cDNA of Labeo rohita TLR4 (LrTLR4) was cloned, and it consisted of 2729 bp, with a single ORF of 2469 bp encoding a polypeptide of 822 aa with a predicted molecular mass of 94.753 kDa. Structurally, LrTLR4 consisted of 25 LRRs (leucine rich repeat regions), one TM (trans-membrane) domain and one TIR (Toll/interleukin-1 receptor) domain, and was similar to higher vertebrate's TLR4. Phylogenetically, LrTLR4 exhibited highest (85%) identity with the common carp TLR4b amino acids sequence, and formed a separate subgroup in the phylogenetic tree. LrTLR4 was widely expressed in all tested organs/tissues, and amidst the tissues highest expression was detected in blood and the lowest in eye. In response to LPS-stimulation, LrTLR4 was induced with the activation of MyD88-dependent and TRIF-dependent signaling pathway resulting in pro-inflammatory cytokines (interleukin 6 and 8) and type I IFN gene expression. Infection of rohu with a Gram-negative fish pathogen (Aeromonas hydrophila), also activated LrTLR4. Together, these findings suggest the important role of TLR4 in LPS sensing and augmentation of innate immunity against Gram-negative bacterial infection in fish.

Immunoglobulin (Ig) D in Labeo rohita is widely expressed and differentially modulated in viral, bacterial and parasitic antigenic challenges.

Immunoglobulins (Igs) play critical roles in protecting host against diverse pathogenic invasion and diseases. Among all Ig isotypes, IgD is the most recently-evolved and enigmatic molecule detected in all vertebrates species except birds. In South-East Asia, Labeo rohita (rohu) is the leading candidate fish species for freshwater aquaculture, and this article describes about IgD gene expression in rohu following viral, bacterial and parasitic antigenic challenges. The partial cDNA (761bp) of Labeo rohita-IgD (LrIgD) was cloned and submitted in the GenBank with the accession no KT883581. Phylogenetically, LrIgD was closely related to grass carp IgD. Analysis of LrIgD gene expression in juveniles by quantitative real-time PCR (qRT-PCR) assay revealed gradual increase in IgD expression with the advancement of time. In the healthy rohu fingerlings, LrIgD expression occurred predominantly in kidney followed by liver and spleen. In response to rhabdoviral antigenic stimulation, LrIgD expression was significantly enhanced in all tested tissues. In bacterial (Aeromonas hydrophila) infection, transcripts of LrIgD increased more dramatically in liver followed by kidney and gill. In parasitic (Argulus) infection, most significant expression of IgD was noted in the skin, followed by kidney, liver, spleen and gill. These results collectively suggest the key role of IgD in the immune response of rohu during viral, bacterial and parasitic infections.

Characterization and Inductive Expression Analysis of Interferon Gamma-Related Gene in the Indian Major Carp, Rohu (Labeo rohita).

Interferon gamma (IFN-γ) is one of the key cytokines that plays a major role against viral and intracellular bacterial infection. In addition to the IFN-γ gene, teleost fish possess a second copy known as IFN-γ-related (IFN-γrel) gene. This report describes structural and functional properties of IFN-γrel gene in the Indian major carp, rohu (Labeo rohita), a commercially important freshwater fish species in the Indian subcontinent. The rohu IFN-γrel gene consisted of four exons with three intervening introns and phylogenetically closely related to grass carp. The full-length IFN-γrel cDNA comprised 927 bp nucleotides with a single open reading frame of 504 bp, encoding 167 amino acids (aa) polypeptide with a signal peptide of 24 aa. The mature rohu IFN-γrel protein was 143 aa with a predicted molecular weight of 16.85 kDa. Basal expression analysis of IFN-γrel showed its wide range of expression in all examined tissues: The highest was in the skin and the lowest was in the liver. In response to LPS, poly I:C, iE-DAP, muramyl dipeptide stimulations, and bacterial infections, IFN-γrel gene expression was significantly (p<0.05) induced in treated fish tissues as compared with their control. The IFN-γrel was expressed as recombinant protein (rIFN-γrel) and confirmed through western blot. Stimulation of peripheral blood leukocytes with rIFN-γrel protein resulted in the activation of IFN-γ receptor and marked induction of inducible nitric oxide synthase gene expression. These results together may suggest the important role of IFN-γrel as an antimicrobial cytokine in fish.

Modulation of TLR2, TLR4, TLR5, NOD1 and NOD2 receptor gene expressions and their downstream signaling molecules following thermal stress in the Indian major carp catla (Catla catla).

Toll-like receptors (TLRs) and nucleotide binding and oligomerization domain (NOD) receptors are pattern recognition receptors (PRRs) that recognize pathogen-associated molecular patterns (PAMPs) and play crucial role in innate immunity. In addition to PAMPs, PRRs recognize endogenous molecules released from damaged tissue or dead cells [damage-associated molecular patterns (DAMPs)] and activate signaling cascades to induce inflammatory processes. In the aquatic environment, large variation in seasonal and diurnal water temperature causes heat and cold stresses in fish, resulting in tissue injury and mortality of fish. In the Indian subcontinent, catla (Catla catla) is an economically important freshwater fish species and is prone to thermal stresses. To investigate the response of pattern recognition receptors in thermal stress, we analyzed TLRs (TLR2, TLR4 and TLR5) and NOD (NOD1 and NOD2) receptors gene expression in catla following heat and cold stress. Analysis of tissue samples (gill, liver, kidney and blood) of the thermal stressed and control fish by quantitative real-time PCR (qRT-PCR) assay revealed significant (p < 0.05) induction of TLR2, TLR4 and NOD2 gene expression in majority of the tested tissues of the treated fish as compared to the control. The expression of TLR5 and NOD1 gene was also induced in the heat and cold stressed fish, but mostly restricted in the blood. The downstream signaling molecule of TLR and NOD signaling pathway viz., MyD88 (myeloid differentiation primary response gene 88) and RICK (receptor interacting serine-threonine protein kinase-2) was also induced in the thermal stressed fish suggesting the engagement of TLR and NOD signaling pathway during thermal stress.

LRRsearch: An asynchronous server-based application for the prediction of leucine-rich repeat motifs and an integrative database of NOD-like receptors.

The leucine-rich repeat (LRR) motifs of the nucleotide-binding oligomerization domain like receptors (NLRs) play key roles in recognizing and binding various pathogen associated molecular patterns (PAMPs) resulting in the activation of downstream signaling and innate immunity. Therefore, identification of LRR motifs is very important to study ligand-receptor interaction. To date, available resources pose restrictions including both false negative and false positive prediction of LRR motifs from the primary protein sequence as their algorithms are relied either only on sequence based comparison or alignment techniques or are over biased for a particular LRR containing protein family. Therefore, to minimize the error (≤5%) and to identify a maximum number of LRR motifs in the wide range of proteins, we have developed "LRRsearch" web-server using position specific scoring matrix (PSSM) of 11 residue LRR-HCS (highly conserved segment) which are frequently observed motifs in the most divergent classes of LRR containing proteins. A data library of 421 proteins, distributed among five known NLR families has also been integrated with the "LRRsearch" for the rich user experience. The access to the "LRRsearch" program is freely available at http://www.lrrsearch.com/.

Toll-like receptor 22 in Labeo rohita: molecular cloning, characterization, 3D modeling, and expression analysis following ligands stimulation and bacterial infection.

Toll-like receptors (TLRs) are a class of innate immune receptors that sense pathogens or their molecular signatures and activate signaling cascades to induce a quick and non-specific immune response in the host. Among various types of TLRs, TLR22 is exclusively present in teleosts and amphibians and is expected to play the distinctive role in innate immunity. This report describes molecular cloning, three-dimensional (3D) modeling, and expression analysis of TLR22 in rohu (Labeo rohita), the most commercially important freshwater fish species in the Indian subcontinent. The open reading frame (ORF) of rohu TLR22 (LrTLR22) comprised of 2,838 nucleotides (nt), encoding 946 amino acid (aa) residues with the molecular mass of ∼ 107.6 kDa. The secondary structure of deduced LrTLR22 exhibited the presence of signal peptide (1-22 aa), 18 leucine-rich repeat (LRR) regions (79-736 aa), and TIR domain (792-935 aa). The 3D model of LrTLR22-LRR regions together elucidated the horse-shoe-shaped structure having parallel ß-strands at the concave surface and few α-helices at the convex surface. The TIR domain structure revealed alternate presence of five α-helices and ß-sheets. Phylogenetically, LrTLR22 was closely related to common carp and exhibited significant similarity (92.2 %) and identity (86.1 %) in their amino acids. In rohu, TLR22 was constitutively expressed in all embryonic developmental stages, and tissue-specific analysis illustrated its expression in all examined tissues, highest was in liver and lowest in brain. In vivo modulation of TLR22 gene expression was analyzed by quantitative real-time PCR (qRT-PCR) assay following stimulation with lipopolysaccharide (LPS), synthetic double stranded RNA (polyinosinic-polycytidylic acid), and bacterial (Aeromonas hydrophila) RNA. Among these ligands, bacterial RNA most significantly (p < 0.05) induced TLR22 gene expression in most of the tested tissues. In A. hydrophila infection, induction of TLR22 gene expression was also observed in majority of the tested tissues. Together, these data suggested that in addition to sensing other microbial signatures, TLR22 can recognize bacterial RNA and may play the important role in augmenting innate immunity in fish.

NOD1 and NOD2 receptors in mrigal (Cirrhinus mrigala): inductive expression and downstream signalling in ligand stimulation and bacterial infections.

Nucleotide binding and oligomerization domain (NOD)1 and NOD2 are important cytoplasmic pattern recognition receptors (PRRs) and key members of the NOD-like receptor (NLR) family. They sense a wide range of bacteria or their products and play a key role in inducing innate immunity. This report describes the role of NOD1 and NOD2 receptors signalling in innate immunity in the Indian major carp, mrigal (Cirrhinus mrigala). Tissue-specific expression analysis of NOD1 and NOD2 genes by quantitative real-time PCR (qRT-PCR) revealed their wide distribution in various organs/tissues. In the untreated fish, the highest expression of NOD1 and NOD2 was detected in liver and blood, respectively. Stimulation with NOD1- and NOD2-specific ligands, i.e. iE-DAP and MDP, activated NOD1 and NOD2 receptor signalling in vivo and in vitro resulting in significant (p less than 0.05) induction of downstream signalling molecule RICK, and the effector molecules IL-1 beta, IL-8 and IFN- gamma in the treated group as compared to their controls. In response to both Gram-positive and Gram-negative bacterial infections, NOD1 and NOD2 receptors signalling were activated and IL-1 beta, IL-8 and IFN- gamma were induced. These findings highlight the important role of NOD receptors in eliciting innate immune response during the pathogenic invasion to the fish.

Elucidation of novel structural scaffold in rohu TLR2 and its binding site analysis with peptidoglycan, lipoteichoic acid and zymosan ligands, and downstream MyD88 adaptor protein.

Toll-like receptors (TLRs) play key roles in sensing wide array of microbial signatures and induction of innate immunity. TLR2 in fish resembles higher eukaryotes by sensing peptidoglycan (PGN) and lipoteichoic acid (LTA) of bacterial cell wall and zymosan of yeasts. However, in fish TLR2, no study yet describes the ligand binding motifs in the leucine rich repeat regions (LRRs) of the extracellular domain (ECD) and important amino acids in TLR2-TIR (toll/interleukin-1 receptor) domain that could be engaged in transmitting downstream signaling. We predicted these in a commercially important freshwater fish species rohu (Labeo rohita) by constructing 3D models of TLR2-ECD, TLR2-TIR, and MyD88-TIR by comparative modeling followed by 40 ns (nanosecond) molecular dynamics simulation (MDS) for TLR2-ECD and 20 ns MDS for TLR2-TIR and MyD88-TIR. Protein (TLR2-ECD)-ligands (PGN, LTA, and zymosan) docking in rohu by AutoDock4.0, FlexX2.1, and GOLD4.1 anticipated LRR16-19, LRR12-14, and LRR20-CT as the most important ligand binding motifs. Protein (TLR2-TIR)-protein (MyD88-TIR) interaction by HADDOCK and ZDOCK predicted BB loop, α B-helix, α C-helix, and CD loop in TLR2-TIR and BB loop, α B-helix, and CD loop in MyD88-TIR as the critical binding domains. This study provides ligands recognition and downstream signaling.

Activation of nucleotide-binding oligomerization domain 1 (NOD1) receptor signaling in Labeo rohita by iE-DAP and identification of ligand-binding key motifs in NOD1 by molecular modeling and docking.

The nucleotide-binding oligomerization domain 1 (NOD1) receptor recognizes various pattern-associated structures of microbes through its leucine-rich repeat (LRR) domain and activates signaling cascades to induce innate immunity. This report describes the activation of NOD1 receptor signaling by gamma-D-glutamyl-meso-diaminopimelic acid (or γ-D-Glu-mDAP [iE-DAP]) in a commercially important fish species, rohu (Labeo rohita). It also described critical motifs in the NOD1-LRR domain that could be involved in binding iE-DAP, lipopolysaccharide (LPS), and polyinosinic:polycytidylic acid (poly I:C). The activation of NOD1 receptor signaling was studied by injecting iE-DAP, and analysis of tissue samples for NOD1 and receptor-interacting serine/threonine kinase (RICK) expression was done by quantitative real-time polymerase chain reaction (qRT-PCR) assay. To identify ligand-binding motifs in NOD1, the 3D model of NOD1-LRR was generated, followed by a 6-ns molecular dynamics simulation. Molecular docking of LPS with NOD1-LRR was executed at the Hex and PatchDock servers, and iE-DAP and poly I:C in the AutoDock 4.2, FlexX 2.1, Glide 5.5, and GOLD 4.1 programs. The results of qRT-PCR revealed significant (p < 0.05) upregulation of NOD1 and RICK expression. Molecular docking revealed that the amino acid residues at LRR1-2, LRR3-7, and LRR8-9 could be involved in poly I:C, LPS, and iE-DAP binding, respectively. In fish, this is the first report describing the 3D structure of NOD1-LRR and its critical ligand-binding motifs.

Molecular cloning and characterization of Toll-like receptor 3, and inductive expression analysis of type I IFN, Mx and pro-inflammatory cytokines in the Indian carp, rohu (Labeo rohita).

Toll-like receptors (TLRs) are one of the key components of innate or non-specific immunity. Among various types of TLRs, TLR3 recognizes dsRNA, the genetic material or replicative intermediate of many RNA viruses and triggers TIR-domain-containing adapter-inducing interferon-ß dependent signalling pathway to induce type I interferon (IFN) and pro-inflammatory cytokines. In this study, we cloned and characterized full-length TLR3 cDNA in rohu (Labeo rohita), that comprised of 2,619 bp nucleotides encoding a putative protein of 873 amino acid with the estimated molecular mass of 98.57 kDa. The constitutive expression of TLR3 gene was detected in all embryonic developmental stages and in various organs/tissues of rohu fingerlings. In vivo tissue specific modulation of TLR3, type I IFN, Mx (myxovirus-resistant protein) and pro-inflammatory cytokines (TNF-α and IL-1ß) gene expression were analysed by quantitative real-time PCR following intravenous injection of polyinosinic-polycytidylic acid (poly I:C), a synthetic analogue of viral dsRNA. A significant relationship of TLR3 induction, and type I IFN, Mx, IL-1ß and TNF-α gene expression were observed in majority of the treated fish tissues, as compared to their control. Together, these data highlight the important role of TLR3 in recognizing dsRNA, and in augmenting the innate immunity in fish in response to viral infections.

Identification of MDP (muramyl dipeptide)-binding key domains in NOD2 (nucleotide-binding and oligomerization domain-2) receptor of Labeo rohita.

In lower eukaryotes-like fish, innate immunity contributed by various pattern recognition receptor (PRR) plays an essential role in protection against diseases. Nucleotide-binding and oligomerization domain (NOD)-2 is a cytoplasmic PRR that recognizes MDP (muramyl dipeptide) of the Gram positive and Gram negative bacteria as ligand and activates signalling to induce innate immunity. Hypothesizing a similar NOD2 signalling pathway of higher eukaryotes, the peripheral blood leucocytes (PBLs) of rohu (Labeo rohita) was stimulated with MDP. The data of quantitative real-time PCR (qRT-PCR) revealed MDP-mediated inductive expression of NOD2 and its down-stream molecule RICK/RIP2 (receptor-interacting serine-threonine protein kinase-2). This observation suggested the existence of MDP-binding sites in rohu NOD2 (rNOD2). To investigate it, 3D model of ligand-binding leucine-rich repeat (LRR) region of rNOD2 (rNOD2-LRR) was constructed following ab initio and threading approaches in I-TASSER web server. Structural refinement of the model was performed by energy minimization, and MD (molecular dynamics) simulation was performed in GROMACS (Groningen Machine for Chemical Simulations). The refined model of rNOD2-LRR was validated through SAVES, ProSA, ProQ, WHAT IF and MolProbity servers, and molecular docking with MDP was carried out in GOLD 4.1. The result of docking identified LRR3-7 comprising Lys820, Phe821, Asn822, Arg847, Gly849, Trp877, Trp901 and Trp931 as MDP-binding critical amino acids in rNOD2. This is the first study in fish to provide an insight into the 3D structure of NOD2-LRR region and its important motifs that are expected to be engaged in MDP binding and innate immunity.

Structural insights of rohu TLR3, its binding site analysis with fish reovirus dsRNA, poly I:C and zebrafish TRIF.

In response to double stranded RNA (dsRNA) viruses, toll-like receptor 3 (TLR3) in fish activates signaling like human, and induces innate immunity. This suggested the existence of dsRNA binding domains in fish TLR3 as reported in higher vertebrates. In in silico analysis, leucine rich repeat (LRR) regions (4-6, 13-14, 20-22), and LRR (8-15, 17-24) were identified as key domains in rohu TLR3 as poly I:C and dsRNA of fish reovirus (AGCRV,VHSV and IHNV) binding regions. 3D-models of rohu TLR3-TIR and zebrafish TRIF were generated by homology and ab initio modeling respectively, and their interacting domains were predicted. This is the first report of TLR3 modeling in fish.

Molecular cloning and characterization of nucleotide binding and oligomerization domain-1 (NOD1) receptor in the Indian Major Carp, rohu (Labeo rohita), and analysis of its inductive expression and down-stream signalling molecules following ligands exposure and Gram-negative bacterial infections.

Nucleotide binding and oligomerization domain-1 (NOD1) is a cytoplasmic pattern recognition receptor (PRR), and is a member of the NOD-like receptor (NLR) family. It senses a wide range of bacteria and viruses or their products, and plays a key role in inducing innate immunity. In this report, NOD1 gene was cloned and characterized in rohu (Labeo rohita), a fish species of highest commercial importance in the Indian subcontinent. The full-length rohu NOD1 (rNOD1) cDNA comprised of 3168 bp with a single open reading frame (ORF) of 2814 bp, encoding a polypeptide of 937 amino acids (aa) with an estimated molecular mass of 106.13 kDa. Structurally, it comprised of one caspase recruitment domain (CARD) at N-terminal, seven leucine rich repeat (LRR) regions at C-terminal and one NACHT domain in between N and C-terminals. Phylogenetically, rNOD1 was closely related to grass carp NOD1 (gcNOD1), and exhibited significant similarity (95.8%) and identity (91.0%) in their amino acids. Ontogenic expression analysis of rNOD1 and its associated down-stream signaling molecule RICK (receptor interacting serine­threonine kinase) by quantitative real-time PCR (qRT-PCR) revealed their constitutive expression in all embryonic developmental stages. Basal expression analysis of rNOD1 showed its wide range of expression in all examined tissues, highest was in spleen and the lowest was in blood. Inductive expression of rNOD1 was observed following LPS and poly I:C exposure, and Aeromonas hydrophila, Edwardsiella tarda and Shigella flexneri infections. Expression of RICK in various organs was significantly enhanced by ligands exposure and bacterial infections, and was correlated with the inductive expression of rNOD1. Together, these findings highlighted the important role of NOD1 in fish in response to pathogenic invasion.

3D modeling and molecular dynamics simulation of an immune-regulatory cytokine, interleukin-10, from the Indian major carp, Catla catla.

Interleukin-10 (IL-10) is a pleiotropic immune-regulatory cytokine that is expressed in various species of fish and higher vertebrates, and is activated during infection. In spite of its important role, IL-10 has not been well characterized either functionally or structurally in fish. To analyze its properties and function, we constructed a 3D model of IL-10 in the Indian major carp, the catla (Catla catla), which is a highly preferred fish species and the most commercially important one in the Indian subcontinent. The catla IL-10 model was constructed by comparative modeling using human IL-10 (2ILK) as the template, and a 5 ns molecular dynamics (MD) simulation was carried out to characterize its structural and dynamical features, which was validated by the SAVES, WHAT IF and MolProbity servers. Analysis using the VAST server revealed a comparatively low level of homology between catla and human IL-10 amino acids at the N-terminal (22.7%) compared to the C-terminal (38.29%). Six conserved domains (A-F) were predicted in catla that threaded well with human IL-10, but their putative interaction sites varied significantly. The amino acid residues in helices A and F differed in length between catla and human IL-10, which may lead to the differences in the IL-10/IL-10R complexes of these two species. The existence of two highly conserved amino acid residues (Cys5 and Cys10) in fish IL-10 but not in higher vertebrate (including human) IL-10 was analyzed in this 3D model. CastP, cons-PPISP and InterProSurf server identified several binding pockets with various probe radii, but Cys5 and Cys10 did not form any significant bonds relating to structural stabilization or protein-protein interactions.

Molecular characterization of toll-like receptor 2 (TLR2), analysis of its inductive expression and associated down-stream signaling molecules following ligands exposure and bacterial infection in the Indian major carp, rohu (Labeo rohita).

Toll-like receptors (TLRs) are one of the key components of innate immunity. Among various TLR types, TLR2 is involved in recognizing specific microbial structures such as peptidoglycan (PGN), lipoteichoic acid (LTA), zymosan etc., and after binding them it triggers myeloid differentiation primary response gene 88 (MyD88)-dependent signaling pathway to induce various cytokines. In this report, TLR2 gene was cloned and characterized in rohu (Labeo rohita), which is highly commercially important fish species in the farming-industry of Indian subcontinent. Full-length rohu TLR2 (rTLR2) cDNA comprised of 2691 bp with a single open reading frame (ORF) of 2379 bp encoding a polypeptide of 792 amino acids (aa) with an estimated molecular mass of 90.74 kDa. Structurally, it comprised of one leucine-rich repeat region (LRR) each at N-terminal (LRR-NT; 44-55 aa) and C-terminal (LRR-CT; 574-590 aa), 21 LRRs in between C and N-terminal, one trans-membrane (TM) domain (595-612 aa), and one TIR domain (645-790 aa). Phylogenetically, rohu TLR2 was closely related to common carp and exhibited significant similarity (93.1%) and identity (88.1%) in their amino acids. During embryogenesis, rTLR2 expression was detected as early as ∼7 h post fertilization indicating its importance in embryonic innate immune defense system in fish. Basal expression analysis of rTLR2 showed its constitutive expression in all the tissues examined, highest was in the spleen and the lowest was in the eye. Inductive expression of TLR2 was observed following zymosan, PGN and LTA exposure and Streptococcus uberis and Edwardsiella tarda infections. Expression of immunoregulatory cytokine interleukin (IL)-8, in various organs was significantly enhanced by ligands exposure and bacterial infections, and was correlated with inductive expression of TLR2. In vitro studies showed that PGN treatment induced TLR2, MyD88 and TRAF6 (TNF receptor associated factor 6) expression, NF-κB (nuclear factor kappa B) activation and IL-8 expression. Blocking NF-κB resulted in down-regulation of PGN mediated IL-8 expression indicating the involvement of NF-κB in IL-8 induction. Together, these findings highlighted the important role of TLR2 in immune surveillance of various organs, and in augmenting innate immunity in fish in response to pathogenic invasion. This study will be helpful in developing preventive measures against infectious diseases in fish.

Induction of toll-like receptor (TLR) 2, and MyD88-dependent TLR- signaling in response to ligand stimulation and bacterial infections in the Indian major carp, mrigal (Cirrhinus mrigala).

Toll-like receptor 2 (TLR2) is a member of TLR family. It recognizes a wide range of bacteria and their products, and is involved in inducing innate immune responses. In this article, we reported inductive expression of TLR2 and myeloid differentiation primary response gene 88 (MyD88)-dependent signaling in the Indian major carp, mrigal (Cirrhinus mrigala) which is highly commercially important fish species in the Indian subcontinent. Ontogeny analysis of TLR2, MyD88 and TRAF6 (TNF receptor associated factor 6) genes by quantitative real-time PCR (qRT-PCR) revealed constitutive expression of these genes in all embryonic developmental stages, indicating their involvement in embryonic innate immune defense system in fish. Tissue specific expression analysis of these genes by qRT-PCR showed their wide distribution in various organs and tissues. Highest expression of TLR2 was in gill, MyD88 in liver and TRAF6 was in kidney. Inductive expression of TLR2, MyD88 and TRAF6 genes were observed following peptidoglycan (PGN)-treatment, and Streptococcus uberis and Aeromonas hydrophila infections. Expression of interleukin (IL)-8 and TNF-α in various organs were significantly enhanced by PGN-treatment and bacterial infections, and were closely associated with TLR2 induction. These findings together highlighted the contribution of TLR2 in augmenting innate immunity in fish, and indicated it's important role in immune surveillance of various organs during pathogenic invasion. This study will enrich the information in understanding the innate immune mechanism in fish, and will be helpful in developing preventive measures against infectious diseases in fish.