Mannose receptor RpMR1 of Manila clam (Ruditapes philippinarum) defense against Vibrio anguillarum infection

Abstract

The mannose receptor (MR) is a member of the C-type lectin superfamily and a type I transmembrane protein that functions as a pattern recognition receptor (PRR) in immune responses. In this study, we identified 13 MR genes (RpMR1-13) in the genome of Ruditapes philippinarum and investigated their expression profiles following Vibrio anguillarum challenge. Notably, RpMR1, RpMR2, RpMR3, and RpMR4 exhibited peak expression at 72 h post-infection. We successfully purified the recombinant RpMR1 protein and demonstrated its antibacterial activity against three Gram-negative bacteria (V. splendidus, V. alginolyticus, and V. anguillarum), though it had no effect on Gram-positive bacteria. Furthermore, in vivo injection of RpMR1 significantly reduced mortality in R. philippinarum following V. anguillarum infection. To explore role of RpMR1 in immune signaling, we performed RNA interference (dsRNA-RpMR1) and observed successful gene silencing. Subsequent qRT-PCR analysis revealed that RpMR1 knockdown significantly suppressed TLR4 expression (P< 0.05) under V. anguillarum stress, confirming an interaction between RpMR1 and TLR4 in the immune response. This study provides the first functional evidence of mannose receptor-mediated immunity in mollusks, offering new insights into the molecular defense mechanisms of R. philippinarum against bacterial infection.

Keywords

Ruditapes philippinarum / Mannose receptor / Gene expression / Immune function

References

[1] BaiY, NieH, WangZ, YanX. Genome-wide identification and transcriptome-based expression profiling of Wnt gene family in Ruditapes philippinarum. Comp Biochem Physiol Part D Genomics Proteomics, 2020, 35100709

[2] BaileyTL, BodenM, BuskeFA, FrithM, GrantCE, ClementiL, RenJ. MEME SUITE: tools for motif discovery and searching. Nucleic Acids Res., 2009, 37(Web Server issue): W202-208

[3] BalderramasHA, PenitentiM, RodriguesDR, BachiegaTF, FernandesRK, IkomaMR, Dias-MelicioLA. Human neutrophils produce IL-12, IL-10, PGE2 and LTB4 in response to Paracoccidioides brasiliensis. Involvement of TLR2, mannose receptor and dectin-1. Cytokine., 2014, 67(1): 36-43

[4] BickmoreWA, van SteenselB. Genome architecture: domain organization of interphase chromosomes. Cell, 2013, 152(6): 1270-1284

[5] Cano I, Blaker E, Hartnell D, Farbos A, Moore KA, Cobb A, Santos EM. Transcriptomic Responses to Koi Herpesvirus in Isolated Blood Leukocytes from Infected Common Carp. Viruses. 2024;16 (3):380.

[6] DongP, ZhangS, GandinV, XieL, WangL, LemireAL, LiW. Cohesin prevents cross-domain gene coactivation. Nat Genet, 2024, 56(8): 1654-1664

[7] GaziU, Martinez-PomaresL. Influence of the mannose receptor in host immune responses. Immunobiology, 2009, 214(7): 554-561

[8] HoffmannJA, KafatosFC, JanewayCA, EzekowitzRA. Phylogenetic perspectives in innate immunity. Science, 1999, 284(5418): 1313-1318

[9] HuB, JinJ, GuoAY, ZhangH, LuoJ, GaoG. GSDS 2.0: an upgraded gene feature visualization server. Bioinformatics., 2015, 31(8): 1296-1297

[10] HuoW, QinL, GuoW, ZhangX, DuQ, XiaX. PvMR1, a novel C-type lectin plays a crucial role in the antibacterial immune response of Pacific white shrimp. Penaeus Vannamei Fish Shellfish Immunol, 2024, 145109346

[11] HuoW, QinL, GuoW, ZhangX, XiaX. Characteristics and functional analysis of a novel mannose receptor in Penaeus vannamei. Fish Shellfish Immunol, 2024, 15300010

[12] IbrahimDM, MundlosS. The role of 3D chromatin domains in gene regulation: a multi-facetted view on genome organization. Curr Opin Genet Dev, 2020, 61: 1-8

[13] IslamS, ZhangS, EggermontM, BrutoM, Le RouxF, DefoirdtT. The impact of the multichannel quorum sensing systems of Vibrio tasmaniensis and Vibrio crassostreae on virulence towards blue mussel (Mytilus edulis) larvae. Aquaculture, 2022, 547737414

[14] JiangK, JiangL, NieH, HuoZ, YanX. Molecular cloning and expression analysis of tyrosinases (tyr) in four shell-color strains of Manila clam Ruditapes philippinarum. PeerJ, 2020, 8e8641

[15] JiangK, NieH, LiD, YanX. New insights into the Manila clam and PAMPs interaction based on RNA-seq analysis of clam through in vitro challenges with LPS, PGN, and poly(I:C). BMC Genomics, 2020, 211531

[16] JiangK, YinZ, ZhangY, XuQ, YuY, CongW, YanX. Genome-wide investigation and expression analysis of MACPF gene family reveals its immune role in response to bacterial challenge of Manila clam. Genomics, 2021, 113(3): 1136-1145

[17] JiangK, NieH, YinZ, YanX, LiQ. Apextrin from Ruditapes philippinarum functions as pattern recognition receptor and modulates NF-κB pathway. Int J Biol Macromol, 2022, 214: 33-44

[18] KumarS, DikshitM. Nitric Oxide: Regulation and Function in Neutrophil Immune Responses. Antioxid Redox Signal, 2024, 40(16–18): 998-1024

[19] Le RouxF, WegnerKM, PolzMF. Oysters and Vibrios as a Model for Disease Dynamics in Wild Animals. Trends Microbiol, 2016, 24(7): 568-580

[20] LiJ, JiangH, WenW, ZhengJ, XuG. The dendritic cell mannose receptor mediates allergen internalization and maturation involving notch 1 signalling. Clin Exp Immunol, 2010, 162(2): 251-261

[21] LiD, NieH, DongS, HuoZ, YanX. Molecular cloning and expression analysis of C-type lectin (RpCTL) in Manila clam Ruditapes philippinarum after lipopolysaccharide challenge. Fish Shellfish Immunol, 2019, 86: 981-993

[22] LiD, NieH, JahanK, YanX. Expression analyses of C-type lectins (CTLs) in Manila clam under cold stress provide insights for its potential function in cold resistance of Ruditapes philippinarum. Comp Biochem Physiol C Toxicol Pharmacol, 2020, 230108708

[23] LiD, NieH, JiangK, LiN, HuoZ, YanX. Molecular characterization and expression analysis of fibrinogen related protein (FREP) genes of Manila clam (Ruditapes philippinarum) after lipopolysaccharides challenge. Comp Biochem Physiol C Toxicol Pharmacol, 2020, 228108672

[24] LittleTJ, HultmarkD, ReadAF. Invertebrate immunity and the limits of mechanistic immunology. Nat Immunol, 2005, 6(7): 651-654

[25] LouresFV, AraujoEF, FeriottiC, BazanSB, CalichVL. TLR-4 cooperates with Dectin-1 and mannose receptor to expand Th17 and Tc17 cells induced by Paracoccidioides brasiliensis stimulated dendritic cells. Front Microbiol, 2015, 6261

[26] ManX, PanXT, ZhangHW, WangY, LiXC, ZhangXW. A mannose receptor is involved in the anti-Vibrio defense of red swamp crayfish. Fish Shellfish Immunol, 2018, 82: 258-266

[27] Martinez-PomaresL. The mannose receptor. J Leukoc Biol, 2012, 92(6): 1177-1186

[28] NamwongP, WangS, KongQ, MouH, MaL, SrisapoomeP. Transcriptome analysis and pattern recognition receptors (PRRs) identification in different tissues of adult Pacific oysters infected with Vibrio parahaemolyticus. Aquaculture, 2022, 562738824

[29] NiD, SongL, WuL, ChangY, YuY, QiuL, WangL. Molecular cloning and mRNA expression of peptidoglycan recognition protein (PGRP) gene in bay scallop (Argopecten irradians, Lamarck 1819). Dev Comp Immunol, 2007, 31(6): 548-558

[30] NieH, JiangL, HuoZ, LiuL, YangF, YanX. Transcriptomic responses to low temperature stress in the Manila clam. Ruditapes Philippinarum Fish Shellfish Immunol, 2016, 55: 358-366

[31] Nunes-AlvesC. Inflammasomes: new LPS receptors discovered. Nat Rev Immunol, 2014, 149582

[32] OuyangA, WangH, SuJ, LiuX. Mannose Receptor Mediates the Activation of Chitooligosaccharides on Blunt Snout Bream (Megalobrama amblycephala) Macrophages. Front Immunol, 2021, 12686846

[33] RenY, LiuH, FuS, DongW, PanB, BuW. Transcriptome-wide identification and characterization of toll-like receptors response to Vibrio anguillarum infection in Manila clam (Ruditapes philippinarum). Fish Shellfish Immunol, 2021, 111: 49-58

[34] SchmittgenTD, LivakKJ. Analyzing real-time PCR data by the comparative C(T) method. Nat Protoc, 2008, 3(6): 1101-1108

[35] SchuetteV, EmbgenbroichM, UlasT, WelzM, Schulte-SchreppingJ, DraffehnAM, QuastT. Mannose receptor induces T-cell tolerance via inhibition of CD45 and up-regulation of CTLA-4. Proc Natl Acad Sci U S A, 2016, 113(38): 10649-10654

[36] Soares-da-SilvaIM, RibeiroJ, ValongoC, PintoR, VilanovaM, BleherR, MachadoJ. Cytometric, morphologic and enzymatic characterisation of haemocytes in Anodonta cygnea. Comp Biochem Physiol A Mol Integr Physiol, 2002, 132(3): 541-553

[37] StahlPD, EzekowitzRA. The mannose receptor is a pattern recognition receptor involved in host defense. Curr Opin Immunol, 1998, 10(1): 50-55

[38] TanAM, ChenHC, PochardP, EisenbarthSC, HerrickCA, BottomlyHK. TLR4 signaling in stromal cells is critical for the initiation of allergic Th2 responses to inhaled antigen. J Immunol, 2010, 184(7): 3535-3544

[39] TiscarPG, MoscaF. Defense mechanisms in farmed marine molluscs. Vet Res Commun, 2004, 28(Suppl 1): 57-62

[40] WakiT, ShimokawaJ, WatanabeS, YoshinagaT, OgawaK. Experimental challenges of wild Manila clams with Perkinsus species isolated from naturally infected wild Manila clams. J Invertebr Pathol, 2012, 111(1): 50-55

[41] WangL, LiuL, ZhouY, ZhaoX, XiM, WeiS, FangR. Molecular cloning and expression analysis of mannose receptor C type 1 in grass carp (Ctenopharyngodon idella). Dev Comp Immunol, 2014, 43(1): 54-58

[42] WangM, ChenY, ZhangY, ZhangL, LuX, ChenZ. Mannan-binding lectin directly interacts with Toll-like receptor 4 and suppresses lipopolysaccharide-induced inflammatory cytokine secretion from THP-1 cells. Cell Mol Immunol, 2011, 8(3): 265-275

[43] Wang X, Liu Y, Li Z, Gao X, Dong J, Zhang J, Zhang L. Genome-Wide Identification and Expression Profile Analysis of the Phospholipase C Gene Family in Wheat (Triticum aestivum L.). Plants (Basel). 2020;9 (7):885.

[44] XaplanteriP, LagoumintzisG, DimitracopoulosG, PaliogianniF. Synergistic regulation of Pseudomonas aeruginosa-induced cytokine production in human monocytes by mannose receptor and TLR2. Eur J Immunol, 2009, 39(3): 730-740

[45] YanX, NieH, HuoZ, DingJ, LiZ, YanL, JiangL. Clam Genome Sequence Clarifies the Molecular Basis of Its Benthic Adaptation and Extraordinary Shell Color Diversity. iScience., 2019, 19: 1225-1237

[46] YinZ, LinZ, LiuY, YanX, NieH. Genome-wide investigation and expression analysis of TLR gene family reveals its immune role in Vibrio tolerance challenge of Manila clam. Fish Shellfish Immunol Rep, 2022, 3100072

[47] YinZ, NieH, JiangK, YanX. Molecular Mechanisms Underlying Vibrio Tolerance in Ruditapes philippinarum Revealed by Comparative Transcriptome Profiling. Front Immunol, 2022, 13879337

[48] YuF, ChenJ, LinJ, ZhongZ, LuY, ZengX, LeiX. TLR4 involved in immune response against Vibrio Parahaemolyticus by MyD88-dependent pathway in Crassostrea hongkongensis. Fish Shellfish Immunol, 2023, 134108591

[49] YuJ, NieH, YinZ, YanX. Transcriptomic analysis provides molecular insights into immune response of Ruditapes philippinarum under Vibrio parahaemolyticus challenge. Aquaculture, 2023, 569739365

[50] ZhangJ, TachadoSD, PatelN, ZhuJ, ImrichA, ManfruelliP, CushionM. Negative regulatory role of mannose receptors on human alveolar macrophage proinflammatory cytokine release in vitro. J Leukoc Biol, 2005, 78(3): 665-674

[51] ZhangM, LiuY, SongC, NingJ, CuiZ. Characterization and functional analysis of a novel mannose-binding lectin from the swimming crab Portunus trituberculatus. Fish Shellfish Immunol, 2019, 89: 448-457

[52] ZhangM, LuZ, TangM, PanG, ZhaoL, QinZ, LinL. Mannose receptor of Epinephelus coioides exerts antiviral activity against red-spotted grouper nervous necrosis virus and regulates apoptosis and inflammation. Aquaculture, 2021, 546737264

[53] ZhangY, NieH, YinZ, YanX. Comparative transcriptomic analysis revealed dynamic changes of distinct classes of genes during development of the Manila clam (Ruditapes philippinarum). BMC Genomics, 2022, 231676

[54] ZhangJ, LiuK, GongX, ZhangN, ZengY, RenW, HuangA. Transcriptome analysis of the hepatopancreas from the Litopenaeus vannamei infected with different flagellum types of Vibrio alginolyticus strains. Front Cell Infect Microbiol, 2023, 131265917

[55] ZhengF, AsimM, LanJ, ZhaoL, WeiS, ChenN, LiuX. Molecular Cloning and Functional Characterization of Mannose Receptor in Zebra Fish (Danio rerio) during Infection with Aeromonas sobria. Int J Mol Sci, 2015, 16(5): 10997-11012

Funding

Outstanding Young Scientific and Technological Talents Foundation of Dalian(2024RJ014)

RIGHTS & PERMISSIONS

The Author(s)