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.
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References
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):174–182
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), e49913
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 6:355
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):1171–1177
Cui J, Wang ZQ (2011) An epidemiological overview of swine trichinellosis in China. Vet J 190(3):323–328
Cui J, Wang ZQ, Xu BL (2011) The epidemiology of human trichinellosis in China during 2004–2009. Acta Trop 118(1):1–5
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):1098–1106
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):32318–32324
Dupouy-Camet J (2000) Trichinellosis: a worldwide zoonosis. Vet Parasitol 93(3–4):191–200
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):806–811
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):393–408
Hussein AS, Kichenin K, Selkirk ME (2002) Suppression of secreted acetylcholinesterase expression in Nippostrongylus brasiliensis by RNA interference. Mol Biochem Parasitol 122(1):91–94
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):1015–1023
Liu P et al (2015a) Protective immunity against Trichinella spiralis infection induced by TsNd vaccine in mice. Parasit Vectors 8(1):185
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 153:29–38
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):4113–4120
Liu X et al (2015c) The molecular characterization and RNAi silencing of SjZFP1 in Schistosoma japonicum. Parasitol Res 114(3):903–911
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 7:600
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):36852–36863
McLennan AG (2006) The Nudix hydrolase superfamily. Cell Mol Life Sci 63(2):123–143
Mitreva M et al (2011) The draft genome of the parasitic nematode Trichinella spiralis. Nat Genet 43(3):228–235
Moskwa B (1999) Trichinella spiralis: in vitro cytotoxicity of peritoneal cells against synchronous newborn larvae of different age. Parasitol Res 85(1):59–63
Murrell KD, Pozio E (2011) Worldwide occurrence and impact of human trichinellosis, 1986–2009. Emerg Infect Dis 17(12):2194–2202
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 10:392
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):248
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), e67570
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):1857–1863
Schmittgen TD, Livak KJ (2008) Analyzing real-time PCR data by the comparative C (T) method. Nat Protoc 3:1101–1108
Struwe WB, Warren CE (2010) High-throughput RNAi screening for N-glycosylation dependent loci in Caenorhabditis elegans. Methods Enzymol 480:477–493
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):148–154
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 7:40
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):1451–1458
Yang Y et al (2012) RNAi silencing of type V collagen in Schistosoma japonicum affects parasite morphology, spawning, and hatching. Parasitol Res 111(3):1251–1257
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):567–572
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This work was supported by the National Natural Science Foundation of China (nos. 81371843 and 81271860).
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Wang, Z.Q., Zhang, S.B., Jiang, P. et al. The siRNA-mediated silencing of Trichinella spiralis nudix hydrolase results in reduction of larval infectivity. Parasitol Res 114, 3551–3557 (2015). https://doi.org/10.1007/s00436-015-4650-5
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DOI: https://doi.org/10.1007/s00436-015-4650-5