Development of a Novel shRNA Construct pSh-IRAK-4 for Silencing of IRAK-4 Gene and Delineating TLR-Mediated Pathway in Penaeus monodon In-Vitro
Ranjeeta Kumari1, Madhusudhana Rao B2, Gulshan Kumar2,
Gireesh Babu P2, Gayatri Tripathi2, KV Rajendran2
and MKBedekar*2
1Fisheries College & Research Institute, TNJFU, Thoothukudi- 628008
2Central Institute of Fisheries Education (Deemed University) ICAR, Panch Marg, Off Yari Road, Mumbai – 400 061
*Corresponding Author: Megha Kadam Bedekar, Aquatic Environment and Health
Management Division, ICAR-CIFE, Mumbai, India.
Received:
October 03, 2022; Published: October 17, 2022
Abstract
In order to understand the TLR pathway defence mechanism of Penaeus monodon, the essential molecule involved in the transduction of Toll-pathway, Interleukin-1 (IL-1) receptor-associated kinase-4 (IRAK-4) was investigated by the mechanism of RNA interference (RNAi) by silencing of the mRNA expression of IRAK-4 gene. In this study we have developed short hairpin RNA (shRNA) construct in pSUPER vector targeting IRAK-4 gene silencing of TLR pathway in P. monodon. The silencing efficiency of pSh-IRAK-4 construct was first confirmed in-vitro in primary haemocyte culture by transfection using pSh-IRAK-4 plasmid, followed by induction with Lipopolysaccharides (LPS). Loss of expression of IRAK-4 was studied by relative gene expression in pSH-IRAK-4 group compared to LPS induced group, the maximum suppression of IRAK-4 gene in cell culture was recorded as 96% at 12h and 93.5% at 24h post LPS induction in pSh-IRAK-4 group. After confirmation of silencing efficiency of construct, the expression of TLR genes of IRAK-4 mediated pathway, was studied post LPS induction both in vitro using real-time qRT-PCR with β-actin as the internal reference gene. For this IRAK-4 downstream genes TRAF6, Dorsal and 4 Antimicrobial peptides (AMPs) (ALF, PEN, AST, Crustin) molecules were studied. Significant downregulation of mRNA expression level in downstream molecules of TLR pathway below IRAK-4 gene viz., TRAF6, Dorsal, and 4 AMPs (ALF, PEN, AST, Crustin) compared to LPS group in response to LPS stimulation was observed in pSh-IRAK-4 group (P ≤ 0.05). Taking all these results together, it is confirmed that TLR pathway is governed by central mediator kinase molecule IRAK-4, when induced by LPS ligand, NF-kB activates the downstream cascades of AMPs of the Toll pathway in P. monodon. Our result confirms the designing of a novel pSh-IRAK-4 construct and its application in efficient silencing of IRAK-4 gene in P. monodon. Plasmid-based IRAK-4 knockdown approach would provide an insight to the role of IRAK-4 in shrimp immune system.
Keywords: RNA Interference; pSUPER Vector; shRNA; IRAK-4
References
- Nie Li., et al. "Toll-like receptors, associated biological roles, and signaling networks in non-mammals”. Frontiers in Immunology 9 (2018): 1523.
- Roy Craig R and Edward S Mocarski. "Pathogen subversion of cell-intrinsic innate immunity”. Nature Immunology 11 (2007): 1179-1187.
- Hemmrich Georg., et al. "The evolution of immunity: a low-life perspective”. Trends in Immunology10 (2007): 449-454.
- Ge Hui., et al. "Molecular cloning and expression of interleukin-1 receptor-associated kinase 4, an important mediator of Toll-like receptor signal pathway, from small abalone Haliotis diversicolor”. Fish and Shellfish Immunology4-5 (2011): 1138-1146.
- Janssens Sophie and Rudi Beyaert. "Functional diversity and regulation of different interleukin-1 receptor-associated kinase (IRAK) family members”. Molecular Cell2 (2003): 293-302.
- Li Shyun., et al. "IRAK-4: a novel member of the IRAK family with the properties of an IRAK-kinase”. Proceedings of the National Academy of Sciences8 (2002): 5567-5572.
- Wang Zhulun., et al. "Crystal structures of IRAK-4 kinase in complex with inhibitors: a serine/threonine kinase with tyrosine as a gatekeeper”. Structure12 (2006): 1835-1844.
- Towb Par., et al. "Tube Is an IRAK-4 homolog in a Toll pathway adapted for development and immunity”. Journal of Innate Immunity4 (2009): 309-321.
- Suzuki Nobutaka., et al. "IRAK-4 as the central TIR signaling mediator in innate immunity”. Trends in Immunology10 (2002a): 503-506.
- Swantek JL., et al. "IL-1 receptor-associated kinase modulates host responsiveness to endotoxin”. Journal of Immunology 164 (2000): 4301-4306.
- Takeda Kiyoshi and Shizuo Akira. "Toll-like receptors in innate immunity”. International Immunology1 (2005): 1-14.
- Umasuthan Navaneethaiyer., et al. "Insights into molecular profiles and genomic evolution of an IRAK4 homolog from rock bream (Oplegnathus fasciatus): immunogen-and pathogen-induced transcriptional expression”. Fish and Shellfish Immunology2 (2015): 436-448.
- Liu Yingying., et al. "Lipopolysaccharide-induced gene expression of interleukin-1 receptor-associated kinase 4 and interleukin-1β in rough skin sculpin (Trachidermus fasciatus)”. Fish and Shellfish Immunology4 (2012): 690-698.
- Wang Guo-Dong., et al. "Molecular cloning and responsive expression of macrophage expressed gene from small abalone Haliotis diversicolor supertexta”. Fish and Shellfish Immunology3 (2008): 346-359.
- Suzuki Nobutaka., et al. "Severe impairment of interleukin-1 and Toll-like receptor signalling in mice lacking IRAK-4”. Nature 6882 (2002b): 750-754.
- Dunne Aisling., et al. "IRAK1 and IRAK4 promote phosphorylation, ubiquitination, and degradation of MyD88 adaptor-like (Mal)”. The Journal of Biological Chemistry47 (2016): 24802.
- Deepika A., et al. "Toll-pathway in tiger shrimp (Penaeus monodon) responds to white spot syndrome virus infection: evidence through molecular characterisation and expression profiles of MyD88, TRAF6 and TLR genes”. Fish and Shellfish Immunology2 (2014): 441-454.
- Hannon Gregory J and John J Rossi. "Unlocking the potential of the human genome with RNA interference”. Nature 7006 (2004): 371-378.
- Tomari Yukihide and Phillip D Zamore. "Perspective: machines for RNAi”. Genes and Development5 (2005): 517-529.
- Kwang Jimmy. "Oral vaccination of baculovirus-expressed VP28 displays enhanced protection against white spot syndrome virus in Penaeus monodon”. PloS one11 (2011): e26428.
- Rajeshkumar S., et al. "Oral delivery of DNA construct using chitosan nanoparticles to protect the shrimp from white spot syndrome virus (WSSV)”. Fish and Shellfish Immunology3 (2009): 429-437.
- Sun Piera S., et al. "Evaluation of methods for DNA delivery into shrimp zygotes of Penaeus (Litopenaeus) vannamei”. Aquaculture1-4 (2005): 19-26.
- Taxman Debra J., et al. "Short hairpin RNA (shRNA): design, delivery, and assessment of gene knockdown”. RNA Therapeutics. Humana Press, (2010): 139-156.
- Shekhar Mudagandur S and Yuanan Lu. "Application of nucleic-acid-based therapeutics for viral infections in shrimp aquaculture”. Marine Biotechnology1 (2009): 1-9.
- Su Jianguo., et al. "A key gene of the RNA interference pathway in the black tiger shrimp, Penaeus monodon: identification and functional characterisation of Dicer-1”. Fish and Shellfish Immunology2 (2008): 223-233.
- de Fougerolles A. "VORNLOCHER HP MARAGANORE J”. Lieberman Journal (2007): 443-453.
- Sambrook J and D W Russell. "Molecular cloning: a laboratory manual”. New York, Cold Spring Harbor Lab. Press (2001).
- Schmittgen Thomas D and Kenneth J Livak. "Analyzing real-time PCR data by the comparative CT method”. Nature Protocols6 (2008): 1101-1108.
- Sreedharan K., et al. "Ontogeny and expression analysis of tube (interleukin-1 receptor-associated kinase-4 homolog) from Penaeus monodon in response to white spot syndrome virus infection and on exposure to ligands”. Agriculture Gene 3 (2017): 21-31.
- Yu Dongmei., et al. "Construction and identification of a vector expressing RNA interference aimed at the human cyclinD1 gene and its expression in vitro”. Chinese Journal of Clinical Oncology5 (2007): 338-342.
- Chen Weizao., et al. "RNA silencing: A remarkable parallel to protein-based immune systems in vertebrates?”. FEBS Letters11 (2005): 2267-2272.
- Chen Xiuhui., et al. "RNA interference-based therapy and its delivery systems”. Cancer and Metastasis Reviews 1 (2018): 107-124.
- Downward, Julian. "RNA interference”. Bmj7450 (2004): 1245-1248.
- Cottrell Tricia R and Tamara L Doering. "Silence of the strands: RNA interference in eukaryotic pathogens”. TRENDS in Microbiology1 (2003): 37-43.
- Lee Y., et al. "microRNA maturation: Stepwise processing and subcellular localization”. EMBO Journal 21 (2002): 4663-4670.
- Robalino Javier., et al. "Induction of antiviral immunity by double-stranded RNA in a marine invertebrate”. Journal of Virology19 (2004): 10442-10448.
- Wu Wenlin., et al. "Antiviral phagocytosis is regulated by a novel Rab-dependent complex in shrimp Penaeus japonicus”. The Journal of Proteome Research01 (2008): 424-431.
- Xu Jianyang., et al. "Silencing shrimp white spot syndrome virus (WSSV) genes by siRNA”. Antiviral Research 2 (2007): 126-131.
- Elbashir Sayda M., et al. "RNA interference is mediated by 21-and 22-nucleotide RNAs”. Genes and Development2 (2001): 188-200.
- Miyagishi Makoto., et al. "Optimization of an siRNA‐expression system with an improved hairpin and its significant suppressive effects in mammalian cells”. The Journal of Gene Medicine: A Cross‐disciplinary Journal for Research on the Science of Gene Transfer and its Clinical Applications7 (2004): 715-723.
- Rubinson Douglas A., et al. "Corrigendum: A lentivirus-based system to functionally silence genes in primary mammalian cells, stem cells and transgenic mice by RNA interference”. Nature Genetics6 (2007): 803-804.
- Mu Chuang., et al. "Long non-coding RNAs (lncRNAs) of sea cucumber: large-scale prediction, expression profiling, non-coding network construction, and lncRNA-microRNA-gene interaction analysis of lncRNAs in Apostichopus japonicus and Holothuria glaberrima during LPS challenge and radial organ complex regeneration”. Marine Biotechnology4 (2016): 485-499.
- Yu JY., et al. "RNA interference by expression of short-interfering RNAs and hairpin RNAs in mammalian cells”. Proceedings of the National Academy of Sciences of the United States of America 99 (2002): 6047-6052.
- Brummelkamp T R., et al. "A system for stable expression of short interfering RNAs in mammalian cells”. Science 296 (2002): 550-553.
- Lye Elizabeth., et al. "IRAK‐4 kinase activity is required for IRAK‐4‐dependent innate and adaptive immune responses”. European Journal of Immunology3 (2008): 870-876.
- Zhang Linlin., et al. "Transcriptome analysis reveals a rich gene set related to innate immunity in the Eastern oyster (Crassostrea virginica)”. Marine Biotechnology1 (2014): 17-33.
- Suzuki Nobutaka., et al. "IL-1 receptor-associated kinase 4 is essential for IL-18-mediated NK and Th1 cell responses”. The Journal of Immunology8 (2003): 4031-4035.
- Bennett Joshua and Daniel T Starczynowski. "IRAK1 and IRAK4 as emerging therapeutic targets in hematologic malignancies”. Current Opinion in Hematology1 (2022): 8.
- Einav Yulia., et al. "shRNA-mediated RNA interference as a tool for genetic synthetic lethality screening in mouse embryo fibroblasts”. FEBS Letters1 (2005): 199-202.
- Gao Yulong., et al. "Effective inhibition of infectious bursal disease virus replication in vitro by DNA vector-based RNA interference”. Antiviral Research2 (2008): 87-94.
- Zheng Cuihong., et al. "Gene silencing efficiency of shRNA expression vectors targeting Cx43 in vitro”. Frontiers of Medicine in China2 (2009): 130-135.
- Hou Fujun., et al. "RNAi knock-down of shrimp Litopenaeus vannamei Toll gene and immune deficiency gene reveals their difference in regulating antimicrobial peptides transcription”. Developmental and Comparative Immunology2 (2014): 255-260.
- Wang KH., et al. "RNAi knock-down of the Litopenaeus vannamei Toll gene (LvToll) significantly increases mortality and reduces bacterial clearance after challenge with Vibrio harveyi”. Developmental and Comparative Immunology 1 (2010): 49-58.
- Sun Yujia., et al. "Dual RNA-seq reveals the effect of the flgM gene of Pseudomonas plecoglossicida on the immune response of Epinephelus coioides”. Fish and Shellfish Immunology 87 (2019): 515-523.
- Das Rekha., et al. "Captive maturation studies in Penaeus monodon by GIH silencing using constitutively expressed long hairpin RNA”. Aquaculture 448 (2015): 512-520.
- Krishnan P. “Silencing Wssv Genes in Penaeus monodon Using DNA Vector Based RNAi Constructs: Thesis Submitted in Partial Fulfillment of the Requirements for the Degree of Ph. D. (Fish Biotechnology)”. Diss. Central Institute of Fisheries Education, (2008).
- Somboonwiwat Kunlaya., et al. "Differentially expressed genes in hemocytes of Vibrio harveyi-challenged shrimp Penaeus monodon”. BMB Reports1 (2006): 26-36.
- De Lorgeril Julien., et al. "A relationship between antimicrobial peptide gene expression and capacity of a selected shrimp line to survive a Vibrio infection”. Molecular Immunology12 (2008): 3438-3445.
- Wang Pei-Hui., et al. "The shrimp NF-κB pathway is activated by white spot syndrome virus (WSSV) 449 to facilitate the expression of WSSV069 (ie1), WSSV303 and WSSV371”. PloS one 9 (2011): e24773.
- Watthanasurorot Apiruck., et al. "A mammalian like interleukin-1 receptor-associated kinase 4 (IRAK-4), a TIR signaling mediator in intestinal innate immunity of black tiger shrimp (Penaeus monodon)”. Biochemical and Biophysical Research Communications1 (2012): 623-629.
- Ellett Justin D., et al. "Toll‐like receptor 4 is a key mediator of murine steatotic liver warm ischemia/reperfusion injury”. Liver Transplantation9 (2009): 1101-1109.
- Takeda Kiyoshi and Shizuo Akira. "TLR signaling pathways”. Seminars in Immunology1 (2004).
- Seki Ekihiro and David A Brenner. "Toll‐like receptors and adaptor molecules in liver disease: update”. Hepatology 1 (2008): 322-335.
- Medvedev Andrei E., et al. "Cutting edge: expression of IL-1 receptor-associated kinase-4 (IRAK-4) proteins with mutations identified in a patient with recurrent bacterial infections alters normal IRAK-4 interaction with components of the IL-1 receptor complex”. The Journal of Immunology11 (2005): 6587-6591.
- Somboonwiwat Kunlaya., et al. "Localization of anti-lipopolysaccharide factor (ALFPm3) in tissues of the black tiger shrimp, Penaeus monodon, and characterization of its binding properties”. Developmental and Comparative Immunology10 (2008): 1170-1176.
- Ponprateep Sirikwan., et al. "Gene silencing reveals a crucial role for anti-lipopolysaccharide factors from Penaeus monodon in the protection against microbial infections”. Fish and Shellfish Immunology1 (2012): 26-34.
- Shanthi S and B Vaseeharan. "cDNA cloning, characterization and expression analysis of a novel antimicrobial peptide gene penaeidin-3 (Fi-Pen3) from the haemocytes of Indian white shrimp Fenneropenaeus indicus”. Microbiological Research3 (2012): 127-134.
- Picard Capucine., et al. "Pyogenic bacterial infections in humans with IRAK-4 deficiency”. Science 5615 (2003): 2076-2079.
- Koziczak-Holbro Magdalena., et al. "IRAK-4 kinase activity is required for interleukin-1 (IL-1) receptor-and toll-like receptor 7-mediated signaling and gene expression”. Journal of Biological Chemistry18 (2007): 13552-13560.
- Dechamma Mundanda Muthappa., et al. "Expression of Toll-like receptors (TLR), in lymphoid organ of black tiger shrimp (Penaeus monodon) in response to Vibrio harveyi infection”. Aquaculture Reports 1 (2015): 1-4.
- Amparyup Piti., et al. "Two prophenoloxidases are important for the survival of Vibrio harveyi challenged shrimp Penaeus monodon”. Developmental and Comparative Immunology 2 (2009): 247-256.
- Söderhäll Irene., et al. "An ancient role for a prokineticin domain in invertebrate hematopoiesis”. The Journal of Immunology 10 (2005): 6153-6160.
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