Acta Scientific Microbiology (ISSN: 2581-3226)

Research Article Volume 5 Issue 11

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


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


  1. Nie Li., et al. "Toll-like receptors, associated biological roles, and signaling networks in non-mammals”. Frontiers in Immunology 9 (2018): 1523.
  2. Roy Craig R and Edward S Mocarski. "Pathogen subversion of cell-intrinsic innate immunity”. Nature Immunology 11 (2007): 1179-1187.
  3. Hemmrich Georg., et al. "The evolution of immunity: a low-life perspective”. Trends in Immunology10 (2007): 449-454.
  4. 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.
  5. Janssens Sophie and Rudi Beyaert. "Functional diversity and regulation of different interleukin-1 receptor-associated kinase (IRAK) family members”. Molecular Cell2 (2003): 293-302.
  6. 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.
  7. 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.
  8. 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.
  9. Suzuki Nobutaka., et al. "IRAK-4 as the central TIR signaling mediator in innate immunity”. Trends in Immunology10 (2002a): 503-506.
  10. Swantek JL., et al. "IL-1 receptor-associated kinase modulates host responsiveness to endotoxin”. Journal of Immunology 164 (2000): 4301-4306.
  11. Takeda Kiyoshi and Shizuo Akira. "Toll-like receptors in innate immunity”. International Immunology1 (2005): 1-14.
  12. 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.
  13. 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.
  14. 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.
  15. Suzuki Nobutaka., et al. "Severe impairment of interleukin-1 and Toll-like receptor signalling in mice lacking IRAK-4”. Nature 6882 (2002b): 750-754.
  16. Dunne Aisling., et al. "IRAK1 and IRAK4 promote phosphorylation, ubiquitination, and degradation of MyD88 adaptor-like (Mal)”. The Journal of Biological Chemistry47 (2016): 24802.
  17. 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.
  18. Hannon Gregory J and John J Rossi. "Unlocking the potential of the human genome with RNA interference”. Nature 7006 (2004): 371-378.
  19. Tomari Yukihide and Phillip D Zamore. "Perspective: machines for RNAi”. Genes and Development5 (2005): 517-529.
  20. Kwang Jimmy. "Oral vaccination of baculovirus-expressed VP28 displays enhanced protection against white spot syndrome virus in Penaeus monodon”. PloS one11 (2011): e26428.
  21. 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.
  22. Sun Piera S., et al. "Evaluation of methods for DNA delivery into shrimp zygotes of Penaeus (Litopenaeus) vannamei”. Aquaculture1-4 (2005): 19-26.
  23. Taxman Debra J., et al. "Short hairpin RNA (shRNA): design, delivery, and assessment of gene knockdown”. RNA Therapeutics. Humana Press, (2010): 139-156.
  24. Shekhar Mudagandur S and Yuanan Lu. "Application of nucleic-acid-based therapeutics for viral infections in shrimp aquaculture”. Marine Biotechnology1 (2009): 1-9.
  25. 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.
  26. de Fougerolles A. "VORNLOCHER HP MARAGANORE J”. Lieberman Journal (2007): 443-453.
  27. Sambrook J and D W Russell. "Molecular cloning: a laboratory manual”. New York, Cold Spring Harbor Lab. Press (2001).
  28. Schmittgen Thomas D and Kenneth J Livak. "Analyzing real-time PCR data by the comparative CT method”. Nature Protocols6 (2008): 1101-1108.
  29. 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.
  30. 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.
  31. Chen Weizao., et al. "RNA silencing: A remarkable parallel to protein-based immune systems in vertebrates?”. FEBS Letters11 (2005): 2267-2272.
  32. Chen Xiuhui., et al. "RNA interference-based therapy and its delivery systems”. Cancer and Metastasis Reviews 1 (2018): 107-124.
  33. Downward, Julian. "RNA interference”. Bmj7450 (2004): 1245-1248.
  34. Cottrell Tricia R and Tamara L Doering. "Silence of the strands: RNA interference in eukaryotic pathogens”. TRENDS in Microbiology1 (2003): 37-43.
  35. Lee Y., et al. "microRNA maturation: Stepwise processing and subcellular localization”. EMBO Journal 21 (2002): 4663-4670.
  36. Robalino Javier., et al. "Induction of antiviral immunity by double-stranded RNA in a marine invertebrate”. Journal of Virology19 (2004): 10442-10448.
  37. 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.
  38. Xu Jianyang., et al. "Silencing shrimp white spot syndrome virus (WSSV) genes by siRNA”. Antiviral Research 2 (2007): 126-131.
  39. Elbashir Sayda M., et al. "RNA interference is mediated by 21-and 22-nucleotide RNAs”. Genes and Development2 (2001): 188-200.
  40. 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.
  41. 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.
  42. 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.
  43. 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.
  44. Brummelkamp T R., et al. "A system for stable expression of short interfering RNAs in mammalian cells”. Science 296 (2002): 550-553.
  45. 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.
  46. 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.
  47. 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.
  48. Bennett Joshua and Daniel T Starczynowski. "IRAK1 and IRAK4 as emerging therapeutic targets in hematologic malignancies”. Current Opinion in Hematology1 (2022): 8.
  49. 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.
  50. 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.
  51. Zheng Cuihong., et al. "Gene silencing efficiency of shRNA expression vectors targeting Cx43 in vitro”. Frontiers of Medicine in China2 (2009): 130-135.
  52. 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.
  53. 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.
  54. 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.
  55. Das Rekha., et al. "Captive maturation studies in Penaeus monodon by GIH silencing using constitutively expressed long hairpin RNA”. Aquaculture 448 (2015): 512-520.
  56. 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).
  57. Somboonwiwat Kunlaya., et al. "Differentially expressed genes in hemocytes of Vibrio harveyi-challenged shrimp Penaeus monodon”. BMB Reports1 (2006): 26-36.
  58. 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.
  59. 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.
  60. 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.
  61. 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.
  62. Takeda Kiyoshi and Shizuo Akira. "TLR signaling pathways”. Seminars in Immunology1 (2004).
  63. Seki Ekihiro and David A Brenner. "Toll‐like receptors and adaptor molecules in liver disease: update”. Hepatology 1 (2008): 322-335.
  64. 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.
  65. 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.
  66. 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.
  67. 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.
  68. Picard Capucine., et al. "Pyogenic bacterial infections in humans with IRAK-4 deficiency”. Science 5615 (2003): 2076-2079.
  69. 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.
  70. 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.
  71. 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.
  72. Söderhäll Irene., et al. "An ancient role for a prokineticin domain in invertebrate hematopoiesis”. The Journal of Immunology 10 (2005): 6153-6160.


Citation: Ranjeeta Kumari., et al. “Silencing of IRAK-4 Gene and Delineating TLR-Mediated Pathway in Penaeus monodon In-Vitro". Acta Scientific Microbiology 5.11 (2022): 24-37.


Copyright: © 2022 Megha Kadam Bedekar., et al.. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.


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