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.

ABSTRACT

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 IC 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.