C.S. Purushothaman

Molecular cloning, characterisation and expression analysis of melanoma differentiation associated gene 5 (MDA5) of green chromide, Etroplus suratensis.

Innate immune system recognises pathogen-associated molecular patterns (PAMPs) by limited number of germline encoded and non-clonally developed pathogen recognition receptors (PRRs). Retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) are important cytosolic PRRs for sensing viral RNAs. The receptor encoded by melanoma differentiation associated gene 5 (MDA5), an RLR, recognises viral RNA and enhances antiviral response in host cells. The full-length MDA5 cDNA in Etroplus suratensis was cloned and found to have 3673 nucleotides encoding a polypeptide of 978 amino acids. The deduced amino acid sequence contains four main structural domains: two CARD domains in the N-terminal region, a DExDc (DEAH/DEAD box helicase domain), HELICc (C-terminal helicase) domain and a C-terminal regulatory domain (RD). Phylogenetic analysis revealed a close relationship of E. suratensis MDA5 (EsMDA5) with MDA5 of Neolamprologus brichardi and Oreochromis niloticus, both belonging to Cichlidae family. EsMDA5 transcripts were ubiquitously expressed in all the 12 tissues tested in healthy fish. Although, transcript level was found to be the highest in muscle, high expression was also detected in the spleen, head kidney and hindgut. In poly I:C-injected fish, EsMDA5 transcripts showed peak expression in the spleen, intestine and heart at 12h post-injection (hpi). However, in gill and kidney tissues, maximum up-regulation of EsMDA5 was observed at 6 and 48 hpi, respectively. Further, liver tissue showed an increasing trend in expression profile from 6 to 48 hpi. Interferon promoter stimulator-1 (IPS-1) gene, an adaptor triggering RIG-I- and MDA5-mediated type I interferon induction, also showed up-regulated expression at initial time-points in poly I:C-injected E. suratensis. The constitutive expression and up-regulation of EsMDA5 and the IPS-1 genes in different tissues indicate that EsMDA5 may play an important role in sensing viral PAMPs in conjunction with IPS-1.

Identification, ontogeny and expression analysis of a novel laboratory of genetics and physiology 2 (LGP2) transcript in Asian seabass, Lates calcarifer

ABSTRACT LGP2 (laboratory of genetics and physiology 2) is an important member of the retinoic acid‐inducible gene I (RIG‐I)‐like receptors (RLRs), which plays a significant role in antiviral innate immunity. In this study, we have cloned the full‐length cDNA sequence of LGP2 from Asian seabass, Lates calcarifer (AsLGP2). The complete AsLGP2 cDNA sequence consisted of 2586 nucleotides encoding a putative protein of 681 amino acids with a molecular mass of 77.6 kDa. From the AsLGP2 protein, four different conserved domains were predicted: a DExDc (DEAD/DEAH box helicase domain), a bacterial type III restriction enzyme domain (RES III), a HELICc (Helicase superfamily c‐terminal domain and a RIG‐I_C‐RD (RIG‐I C‐terminal regulatory domain). The transcript of AsLGP2 could be detected in all the 11 tissues tested in healthy animals with high expression noticed in tissues facing external environment such as gill, hindgut and skin. The ontogenic expression profile of AsLGP2 implies a possible maternal transfer of this gene as it has been detected in all early embryonic developmental stages along with unfertilized eggs. Viral analogue, poly I:C, injection resulted in rapid up‐regulated expression in different tissues with the highest modulation of expression observed in kidney followed by liver and gill. A rapid response of AsLGP2 expression was also observed in the different tissues of Vibrio alginolyticus‐injected L. calcarifer, while significant change in expression was noticed following Staphylococcus aureus infection. Similarly, exposure to different pathogen‐mimicking microbial analogues such as poly I:C, LPS and PGN resulted in enhanced expression of AsLGP2 in SISK cell‐line. Taking together, these observations suggest that AsLGP2 can act as both antiviral and antibacterial cytosolic receptor and may play a significant role in embryonic and larval development in marine euryhaline teleosts like Asian seabass. HighlightsA novel LGP2 transcript identified, cloned and characterized from Asian seabass, Lates calcarifer (AsLGP2).AsLGP2 constitutively expressed in embryonic and larval developmental stages.Poly I:C positively modulates the AsLGP2 expression in vivo and in vitro.AsLGP2 responds towards bacterial infection and different bacterial ligands.AsLGP2 may act as a positive regulatory molecule with both antiviral and antibacterial functions in marine euryhaline teleost.

Report of leucine-rich repeats (LRRs) from Scylla serrata: Ontogeny, molecular cloning, characterization and expression analysis following ligand stimulation, and upon bacterial and viral infections.

Leucine-rich repeat (LRR) proteins are present in all living organisms, and their participation in signal transduction and defense mechanisms has been elucidated in humans and mosquitoes. LRRs possibly involve in protein-protein interactions also and show differential expression pattern upon challenge with pathogens. In the present study, a new LRR gene was identified in mud crab, Scylla serrata. LRR gene mRNA levels in different developmental stages and various tissues of S. serrata were analysed. Further, the response of the gene against different ligands, Gram-negative bacterium, and white spot syndrome virus (WSSV) was investigated in vitro and in vivo. Full-length cDNA sequence of S. serrata LRR (SsLRR) was found to be 2290 nucleotide long with an open reading frame of 1893bp. SsLRR encodes for a protein containing 630 deduced amino acids with 17 conserved LRR domains and exhibits significant similarity with crustacean LRRs so that these could be clustered into a branch in the phylogenetic tree. SsLRR mRNA transcripts were detected in all the developmental stages (egg, Zoea1-5, megalopa and crab instar), haemocytes and various tissues such as, stomach, gill, muscle, hepatopancreas, hematopoietic organ, heart, epithelial layer and testis by reverse-transcriptase PCR. SsLRR transcripts in cultured haemocytes showed a 2-fold increase in expression at 1.5 and 12h upon Poly I:C induction. WSSV challenge resulted in significant early up-regulation at 3h in-vitro and late up-regulation at 72h in-vivo. Peptidoglycan (PGN)-induction resulted in marginal up-regulation of SsLRR at timepoints, 6, 12 and 24h (fold change below 1.5) and no significant change in the expression at early timepoints. LPS-stimulation, on the other hand, showed either down-regulation or normal level of expression at all timepoints. However, a delayed 5-fold up-regulation was observed in vivo against Vibrio parahaemolyticus infection at 72hpi. The constitutive expression of the LRR gene in all the early life-stages, and its response to various ligands and to viral challenge suggest the possible role of the LRR in immune defense in mud crab. The result provides additional information which would help in future studies in understanding the innate immune pathways in crustaceans.

Molecular characterisation, ontogeny and expression analysis of melanoma differentiation-associated factor 5 (MDA5) from Asian seabass, Lates calcarifer

ABSTRACT MDA5 is the pivotal member of the retinoic acid‐inducible gene I (RIG‐I)‐like receptors (RLRs) and is reported to play a crucial role in type I IFN‐mediated responses against pathogen‐associated molecular patterns (PAMPs), especially nucleic acids. In this study, we have identified and cloned the full‐length cDNA sequence of MDA5, which comprises 3398 nucleotides and encodes for a putative protein of 978 AA length, in Asian seabass, Lates calcarifer. From the putative amino acid sequence of AsMDA5, four different conserved domains could be predicted: two N‐terminal CARD domains, a DExDc domain, a HELICc domain and a C‐terminal RIG‐1_C‐RD domain. The mRNA transcript of AsMDA5 could be detected in all the 11 tissues tested in healthy animals with the highest expression in heart followed by gill and skin. The ontogenetic expression profile showed constitutive expression in developmental stages starting from unfertilized eggs, which implies the possibility of maternally acquired immunity of RLRs in offspring. The viral analogue poly I:C could modulate the AsMDA5 expression both in vivo and in vitro. In all the tissues, AsMDA5 expression was found to be highly regulated following injection with poly I:C with the highest expression observed in kidney. The expression level of AsMDA5 was found to be modulated at different time‐points following challenge with Gram‐negative bacterium, Vibrio alginolyticus, and Gram‐positive bacterium, Staphylococcus aureus. Similarly, noticeable change in AsMDA5 expression was detected in SISK cell line induced with either LPS or PGN. The observations made in this study suggest that in euryhaline marine teleosts like Asian seabass, MDA5 gene serves as one of the pivotal receptor for the detection of viral and bacterial PAMP, and might play an important antimicrobial role during early embryonic development. HighlightsFull‐length cDNA sequence of MDA5 was identified, cloned and characterized from Asian seabass, Lates calcarifer (AsMDA5).AsMDA5 constitutively expressed in embryonic and larval developmental stages.Viral analogue, poly I:C positively modulates the AsMDA5 expression in vivo and in vitro.AsMDA5 responds towards bacterial pathogens and different bacterial ligands.AsMDA5 may be the pivotal RLR molecule with both antiviral and antibacterial functions in marine euryhaline teleost.

Toll-like receptor (TLR) 22, a non-mammalian TLR in Asian seabass, Lates calcarifer: Characterisation, ontogeny and inductive expression upon exposure with bacteria and ligands

&NA; Toll‐like receptor (TLR) 22 is a non‐mammalian TLR found mostly in teleosts and characterized initially as a cell surface surveillance receptor for detecting extracellular long dsRNA. In the current study, the full‐length cDNA sequence consisting of 3312 nucleotides encoding for 960 amino acids in Asian seabass (Lates calcarifer) TLR22 (AsTLR22) was identified. From the putative protein sequence, signature TLR domains such as 18 LRR domains, two transmembrane domains, a single LRR_CT domain and an intracellular TIR domain could be predicted. Phylogenetic analysis showed that AsTLR22 is clustered with other teleost TLR22 and is distinctly different from the other TLR groups. The transcript of AsTLR22 was ubiquitously expressed in all the tissues tested of healthy juveniles with the highest expression in gill followed by hindgut, spleen and skin. The AsTLR22 mRNA transcript was also detected in all the developmental stages as early as unfertilized eggs with higher expression in later stages such as neurula and early embryo. The dsRNA viral analogue, poly (I:C) and Gram‐negative bacterium, Vibrio alginolyticus, were found to modulate the AsTLR22 expression in different tissues with the highest expression in kidney and liver. Gram‐positive bacterium, Staphylococcus aureus, was also found to regulate the AsTLR22 expression at certain time‐points with the highest expression in gill. Similarly, noticeable change in AsTLR22 expression was detected in SISK cell line induced with different ligands such as poly (I:C), LPS and PGN. The findings indicate that AsTLR22 responds in transcript level towards bacteria‐borne PAMPs and extracellular dsRNA in the euryhaline teleost Asian seabass. Further, this might act as an important pathogen surveillance receptor during early developmental stages. HighlightsThe TLR22 transcript was identified and characterized in Asian seabass, Lates calcarifer (AsTLR22).AsTLR22 constitutively expressed in embryonic and larval developmental stages.Viral analogue, poly I:C positively modulates the AsTLR22 expression in vivo and in vitro.AsTLR22 responds towards bacterial challenge and different bacterial ligands.

Nucleotide-binding oligomerization domain-containing protein 1 (NOD1) in Asian seabass, Lates calcarifer: Cloning, ontogeny and expression analysis following bacterial infection or ligand stimulation

ABSTRACT NOD1 (Nucleotide‐binding oligomerization domain‐containing protein 1) is one of the most prominent intracellular Nod‐like receptors (NLRs), responsible for detecting different microbial components and products arising from tissue injury. Here, we have identified and cloned NOD1 transcript in the Asian seabass, Lates calcarifer (AsNOD1), which consists of 3749 nucleotides and encodes for a predicted putative protein of 900 AA. The AsNOD1 possesses the typical structure of NLR family, consisting of N‐terminal CARD domain, centrally located NACHT domain and C‐terminal LRRs. The AsNOD1 showed ubiquitous tissue expression in 11 different tissues of healthy animals tested with high levels of expression in hindgut and gill. From the ontogenetic expression profile of AsNOD1, it is quite evident that this gene might follow a maternally‐transferred trend in euryhaline teleosts, as it is highly abundant in embryonic developmental stages. The constitutive immunomodulation of AsNOD1 in terms of expression level was clearly evident in the different tissues of Asian seabass‐injected either with Vibrio alginolyticus or poly I:C. However, injection with Staphylococcus aureus did not elicit similar immunomodulation except for the up‐regulation noticed at few time‐points in some tissues. SISK‐cell line induced with different ligands such as poly I:C, LPS and PGN also showed up‐regulation of AsNOD1 in certain time‐points in vitro. Based on the results obtained in the present study, it can be inferred that the AsNOD1 might play an immunoregulatory role upon exposure to different bacterial as well as viral PAMPs and also might be an important component of innate immune element during embryonic and larval development in the euryhaline teleost Asian seabass. HIGHLIGHTSAsian seabass NOD1 (AsNOD1) was identified, cloned and characterized.AsNOD1 was constitutively expressed in developmental stages.In healthy animals, AsNOD1 was highly expressed in hindgut and gill.V. aliginolyticus, poly I:C and bacterial ligands modulate the AsNOD1 expression both in vivo and in vitro.AsNOD1 might have both antiviral and antibacterial functions in marine euryhaline teleost.