Functional Study Of C-type Lectin And TOLL Receptor In Innate Immunity Of Macrobrachium Rosenbergii

Immune system of vertebrates including ininnate and adaptive immunity. Invertebrates lack an adaptive immune system and rely on innate immunity to protect them against pathogens and opportunisticmicrobial infections.In this paper,Macrobrachium rosenbergiiwas used as the research, and its pattern recognition and immune-related signal pathways were studied.(1)C-type lectins (CTLs) are pattern-recognition proteins that play an important role in innate immunity of vertebrates and invertebrates.In this study, a lectin cDNA named MrLecwas cloned and characterized from giant freshwater prawns (Macrobrachiun rosenbergii). The full-length cDNA of MrLec was 1431 bp,which contained an open reading frame of 1041 bp that encoded a protein with 346 amino acids. MrLecwas found to contain a typical signal peptide of 18 amino acids and a single carbohydrate-recognition domain with 121 amino acids.The phylogenetic analysis showed that MrLecwas grouped with vertebratesand had 57%identity with C-type lectin 3 from Marsupenaeus japonicas. Tissue expression analysis showed that MrLecwas ubiquitously distributed at a high level in the intestine,with lower expression levels in the hemocytes, heart, hepatopancreas, gill and stomach. Vibrio parahaemolyticus infection induced the upregulation of MrLec in the gills and intestine. For the white spot syndrome virus (WSSV)challenge, MrLec in gills was upregulated at 24, 36 and 48 h. In intestine, MrLec also went up at 36 and 48 h WSSV challenge. Recombinant MrLec can agglutinate (Ca2+-dependent) and bind both Gramnegative and Gram-positive bacteria. rMrLec could attach to lipopolysaccharide and peptidoglycan in a dose-dependent mannerand can promote the clearance of the bacteria in vivo. These results indicated possible MrLec involvement in the immune response of giant freshwater prawns.(2)Toll receptors are evolutionary ancient families of pattern recognition receptors with crucial roles in invertebrate innate immune response.In this study, we identified a Toll receptor (MrToll) from giant freshwater prawns(Macrobrachium rosenbergii).The full-length cDNA of MrToll is 4257 bp,which encodes a putative protein of 1367 amino acids. MrToll contains 17 LRR domains, a transmembrane domain,and a TIR domain. Phylogenetic analysis showed that MrToll was grouped with Drosophila Toll7 and other arthropod Tolls. The transcripts of MrTollare mainly distributed in the heart, hepatopancreas,gills,stomach,and intestine. A low level of MrToll expression can be detected in hemocytes and the lymphoid organ. MrToll expression in gills was gradually upregulated to the highest level from 24 h to 48 h during the white spot syndrome virus (WSSV) challenge. The expression levels of the crustin (Cru) genes Cru3 and Cru7 in gills were relatively lower than those of Cru2 and Cru4. The expression levels of Cru3 and Cru7 were inhibitedafter the RNA interference of MrToll in gills during the WSSV challenge.The anti-lipopolysaccharide factor (ALF) genes ALF2, ALF3, ALF4, and ALF5 were also regulated by MrToll in gills during the virus challenge. These findings suggest that MrToll may contribute to the innate immune defense of M.rosenbergii against WSSV.