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Parasitology Research

, Volume 114, Issue 9, pp 3551–3557 | Cite as

The siRNA-mediated silencing of Trichinella spiralis nudix hydrolase results in reduction of larval infectivity

  • Zhong Quan Wang
  • Shuai Bing Zhang
  • Peng Jiang
  • Ruo Dan Liu
  • Shao Rong Long
  • Xi Zhang
  • Hui Jun Ren
  • Jing Cui
Short Communication

Abstract

Previous studies showed that Trichinella spiralis Nudix hydrolase (TsNd) bound to intestinal epithelial cells (IECs), and vaccination of mice with rTsNd or TsNd DNA produced a partial protective immunity against T. spiralis infection. In this study, three TsNd specific small interfering RNA (siRNA) were designed to silence the expression of TsNd in T. spiralis larvae. SiRNAs were delivered to the larvae by electroporation. Silencing effect of TsNd transcription and expression was determined by real-time PCR and Western blotting, respectively. The infectivity of the larvae treated with siRNA was investigated by the in vitro larval invasion of IECs and experimental infection in mice. The results showed that siRNAs were efficiently delivered into T. spiralis larvae through electroporation. Real-time PCR and Western blotting showed that transcription and expression level of TsNd gene was inhibited 73.3 and 76.7 %, respectively, after being electroporated with 2 μM of siRNA-275 for 1 day. Silencing TsNd expression inhibited significantly the larval invasion of IECs (P < 0.01) and was in a dose-dependent manner (r = −0.97941). The mice with infected larvae treated with TsNd siRNA displayed a 63.6 % reduction in intestinal adult worms and 68.8 % reduction in muscle larval burden compared with mice infected with control siRNA-treated larvae. Our results showed that silencing TsNd expression in T. spiralis significantly reduced the larval infectivity and survival in host.

Keywords

Trichinella spiralis Nudix hydrolases RNAi siRNA Infectivity 

Notes

Acknowledgement

This work was supported by the National Natural Science Foundation of China (nos. 81371843 and 81271860).

References

  1. Badger JL, Wass CA, Kim KS (2000) Identification of Escherichia coli K1 genes contributing to human brain microvascular endothelial cell invasion by differential fluorescence induction. Mol Microbiol 36(1):174–182CrossRefPubMedGoogle Scholar
  2. Chen X, Yang Y, Yang J, Zhang Z, Zhu X (2012) RNAi-mediated silencing of paramyosin expression in Trichinella spiralis results in impaired viability of the parasite. PLoS One 7(11), e49913PubMedCentralCrossRefPubMedGoogle Scholar
  3. Cui J et al (2013a) Proteomic analysis of surface proteins of Trichinella spiralis muscle larvae by two-dimensional gel electrophoresis and mass spectrometry. Parasit Vectors 6:355PubMedCentralCrossRefPubMedGoogle Scholar
  4. Cui J, Ren HJ, Liu RD, Wang L, Zhang ZF, Wang ZQ (2013b) Phage-displayed specific polypeptide antigens induce significant protective immunity against Trichinella spiralis infection in BALB/c mice. Vaccine 31(8):1171–1177CrossRefPubMedGoogle Scholar
  5. Cui J, Wang ZQ (2011) An epidemiological overview of swine trichinellosis in China. Vet J 190(3):323–328CrossRefPubMedGoogle Scholar
  6. Cui J, Wang ZQ, Xu BL (2011) The epidemiology of human trichinellosis in China during 2004–2009. Acta Trop 118(1):1–5CrossRefPubMedGoogle Scholar
  7. Dinh PT, Brown CR, Elling AA (2014) RNA Interference of Effector Gene Mc16D10L Confers Resistance Against Meloidogyne chitwoodi in Arabidopsis and Potato. Phytopathology 104(10):1098–1106CrossRefPubMedGoogle Scholar
  8. Dunn CA, O'Handley SF, Frick DN, Bessman MJ (1999) Studies on the ADP-ribose pyrophosphatase subfamily of the nudix hydrolases and tentative identification of trgB, a gene associated with tellurite resistance. J Biol Chem 274(45):32318–32324CrossRefPubMedGoogle Scholar
  9. Dupouy-Camet J (2000) Trichinellosis: a worldwide zoonosis. Vet Parasitol 93(3–4):191–200CrossRefPubMedGoogle Scholar
  10. Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC (1998) Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391(6669):806–811CrossRefPubMedGoogle Scholar
  11. Gamble HR et al (2000) International Commission on Trichinellosis: recommendations on methods for the control of Trichinella in domestic and wild animals intended for human consumption. Vet Parasitol 93(3–4):393–408CrossRefPubMedGoogle Scholar
  12. Hussein AS, Kichenin K, Selkirk ME (2002) Suppression of secreted acetylcholinesterase expression in Nippostrongylus brasiliensis by RNA interference. Mol Biochem Parasitol 122(1):91–94CrossRefPubMedGoogle Scholar
  13. Kang LW, Gabelli SB, Cunningham JE, O'Handley SF, Amzel LM (2003) Structure and mechanism of MT-ADPRase, a nudix hydrolase from Mycobacterium tuberculosis. Structure 11(8):1015–1023CrossRefPubMedGoogle Scholar
  14. Liu P et al (2015a) Protective immunity against Trichinella spiralis infection induced by TsNd vaccine in mice. Parasit Vectors 8(1):185PubMedCentralCrossRefPubMedGoogle Scholar
  15. Liu P et al (2015b) Oral vaccination of mice with Trichinella spiralis nudix hydrolase DNA vaccine delivered by attenuated Salmonella elicited protective immunity. Exp Parasitol 153:29–38CrossRefPubMedGoogle Scholar
  16. Liu RD, Wang ZQ, Wang L, Long SR, Ren HJ, Cui J (2013) Analysis of differentially expressed genes of Trichinella spiralis larvae activated by bile and cultured with intestinal epithelial cells using real-time PCR. Parasitol Res 112(12):4113–4120CrossRefPubMedGoogle Scholar
  17. Liu X et al (2015c) The molecular characterization and RNAi silencing of SjZFP1 in Schistosoma japonicum. Parasitol Res 114(3):903–911CrossRefPubMedGoogle Scholar
  18. Long SR et al (2014) Molecular identification of Trichinella spiralis nudix hydrolase and its induced protective immunity against trichinellosis in BALB/c mice. Parasit Vectors 7:600PubMedCentralCrossRefPubMedGoogle Scholar
  19. Luo Y et al (2011) InvA protein is a Nudix hydrolase required for infection by pathogenic Leptospira in cell lines and animals. J Biol Chem 286(42):36852–36863PubMedCentralCrossRefPubMedGoogle Scholar
  20. McLennan AG (2006) The Nudix hydrolase superfamily. Cell Mol Life Sci 63(2):123–143CrossRefPubMedGoogle Scholar
  21. Mitreva M et al (2011) The draft genome of the parasitic nematode Trichinella spiralis. Nat Genet 43(3):228–235PubMedCentralCrossRefPubMedGoogle Scholar
  22. Moskwa B (1999) Trichinella spiralis: in vitro cytotoxicity of peritoneal cells against synchronous newborn larvae of different age. Parasitol Res 85(1):59–63CrossRefPubMedGoogle Scholar
  23. Murrell KD, Pozio E (2011) Worldwide occurrence and impact of human trichinellosis, 1986–2009. Emerg Infect Dis 17(12):2194–2202PubMedCentralCrossRefPubMedGoogle Scholar
  24. Naito Y, Yoshimura J, Morishita S, Ui-Tei K (2009) siDirect 2.0: updated software for designing functional siRNA with reduced seed-dependent off-target effect. BMC Bioinformatics 10:392PubMedCentralCrossRefPubMedGoogle Scholar
  25. Ocadiz-Ruiz R, Fonseca W, Martinez MB, Ocadiz-Quintanar R, Orozco E, Rodriguez MA (2013) Effect of the silencing of the Ehcp112 gene on the in vitro virulence of Entamoeba histolytica. Parasit Vectors 6(1):248PubMedCentralCrossRefPubMedGoogle Scholar
  26. Ren HJ, Cui J, Yang W, Liu RD, Wang ZQ (2013a) Identification of differentially expressed genes of Trichinella spiralis larvae after exposure to host intestine milieu. PLoS One 8(6), e67570PubMedCentralCrossRefPubMedGoogle Scholar
  27. Ren HJ, Liu RD, Wang ZQ, Cui J (2013b) Construction and use of a Trichinella spiralis phage display library to identify the interactions between parasite and host enterocytes. Parasitol Res 112(5):1857–1863CrossRefPubMedGoogle Scholar
  28. Schmittgen TD, Livak KJ (2008) Analyzing real-time PCR data by the comparative C (T) method. Nat Protoc 3:1101–1108CrossRefPubMedGoogle Scholar
  29. Struwe WB, Warren CE (2010) High-throughput RNAi screening for N-glycosylation dependent loci in Caenorhabditis elegans. Methods Enzymol 480:477–493CrossRefPubMedGoogle Scholar
  30. Wang B, Wang ZQ, Jin J, Ren HJ, Liu LN, Cui J (2013) Cloning, expression and characterization of a Trichinella spiralis serine protease gene encoding a 35.5 kDa protein. Exp Parasitol 134(2):148–154CrossRefPubMedGoogle Scholar
  31. Wang L, Cui J, Hu DD, Liu RD, Wang ZQ (2014a) Identification of early diagnostic antigens from major excretory-secretory proteins of Trichinella spiralis muscle larvae using immunoproteomics. Parasit Vectors 7:40PubMedCentralCrossRefPubMedGoogle Scholar
  32. Wang X, Chen W, Tian Y, Huang Y, Li X, Yu X (2014b) RNAi-mediated silencing of enolase confirms its biological importance in Clonorchis sinensis. Parasitol Res 113(4):1451–1458CrossRefPubMedGoogle Scholar
  33. Yang Y et al (2012) RNAi silencing of type V collagen in Schistosoma japonicum affects parasite morphology, spawning, and hatching. Parasitol Res 111(3):1251–1257CrossRefPubMedGoogle Scholar
  34. Zou X, Jin YM, Liu PP, Wu QJ, Liu JM, Lin JJ (2011) RNAi silencing of calcium-regulated heat-stable protein of 24 kDa in Schistosoma japonicum affects parasite growth. Parasitol Res 108(3):567–572CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2015

Authors and Affiliations

  • Zhong Quan Wang
    • 1
  • Shuai Bing Zhang
    • 1
  • Peng Jiang
    • 1
  • Ruo Dan Liu
    • 1
  • Shao Rong Long
    • 1
  • Xi Zhang
    • 1
  • Hui Jun Ren
    • 1
  • Jing Cui
    • 1
  1. 1.Department of Parasitology, Medical CollegeZhengzhou UniversityZhengzhouChina

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