Advertisement

Parasitology Research

, Volume 112, Issue 12, pp 4113–4120 | Cite as

Analysis of differentially expressed genes of Trichinella spiralis larvae activated by bile and cultured with intestinal epithelial cells using real-time PCR

  • Ruo Dan Liu
  • Zhong Quan Wang
  • Lei Wang
  • Shao Rong Long
  • Hui Jun Ren
  • Jing Cui
Original Paper

Abstract

The activation of Trichinella spiralis muscle larvae (ML) by exposure to intestinal contents or bile and the intestinal epithelial cells (IECs) themselves are two pivotal requirements for the in vitro larval invasion of IECs. However, it is yet unknown which genes are involved in the process of larval invasion. The purpose of the present study was to analyze the differentially expressed genes of T. spiralis larvae activated by bile and cultured with IECs by using real-time polymerase chain reaction. Ten T. spiralis genes encoded the proteins produced by the larvae after co-culture with IECs were selected. Compared with untreated ML, four genes were up-regulated in both bile-activated and cell-cultured larvae, including calcium-dependent secretion activator (Csa; 2.55- and 16.04-fold, respectively), multi cystatin-like domain protein precursor (Mcd; 4.36 and 52), serine protease (Sp; 2.03 and 20.02), and intermediate filament protein ifa-1 (Ifa 1; 2 and 3.31). The expression of two genes, enolase (Eno; 1.51) and ribosomal protein S6 kinase beta-1 (Rsk; 1.49), was up-regulated only in cell-cultured larvae, not in bile-activated larvae. The expression of secreted 5′-nucleotidase (5 N; 1.42) and putative serine protease (Psp; 1.41) was up-regulated in bile-activated larvae, but was not changed or down-regulated after cultured with IECs. ATP synthase F1, beta subunit (ATPase; 0.58 and 0.51) and serine protease precursor (Spp; 0.42 and 0.65) were down-regulated in both bile-activated and cell-cultured larvae. This study provide some differentially expressed genes among the untreated (normal), bile-activated and cell-cultured larvae of T. spiralis. The up-regulated genes might be related with the larval invasion of IECs, but their exact biological functions need to be further investigated. This study will be helpful to further elucidate the molecular mechanism of the invasion of IECs by T. spiralis larvae and to better understand the interaction between parasite and host enterocytes.

Keywords

Serine Protease Parasitic Nematode Infective Larva Muscle Larva Muscle Larva 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Notes

Acknowledgements

This work was supported by the National Natural Science Foundation of China (No. 81271860, 81371843 and 30972579) and the specialized research fund for the doctoral universities of China (20124101110005).

References

  1. Bolás-Fernandez F, Corral-Bezara LD (2006) TSL-1 antigens of Trichinella: an overview of their potential role in parasite invasion, survival and serodiagnosis of trichinellosis. Res Vet Sci 81:297–303PubMedCrossRefGoogle Scholar
  2. Bruce RG (1970) Structure of the esophagus of the infective juvenile and adult Trichinella spiralis. J Parasitol 56:540–549PubMedCrossRefGoogle Scholar
  3. Campbell WC (1983) Trichinella and trichinosis. Plenum, New YorkCrossRefGoogle Scholar
  4. 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:e49913. doi: 10.1371/journal.pone.0049913 PubMedCrossRefGoogle Scholar
  5. Cuttell L, Corley SW, Gray CP, Vanderlinde PB, Jackson LA, Traub RJ (2012) Real-time PCR as a surveillance tool for the detection of Trichinella infection in muscle samples from wildlife. Vet Parasitol 188:285–293. doi: 10.1016/j.vetpar.2012.03.054 PubMedCrossRefGoogle Scholar
  6. Dzik JM (2006) Molecules released by helminth parasites involved in host colonization. Acta Biochim Pol 53:33–64PubMedGoogle Scholar
  7. Gagliardo LF, McVay CS, Appleton JA (2002) Molting, ecdysis, and reproduction of Trichinella spiralis are supported in vitro by intestinal epithelial cells. Infect Immun 70:1853–1859PubMedCrossRefGoogle Scholar
  8. Gamble HR, Bessonov AS, Cuperlovic K, Gajadhar AA, van Knapen F, Noeckler K, Schenone H, Zhu X (2000) International Commission on Trichinellosis: recommendations on methods for the control of Trichinella in domestic and wild animals intended for human consumption. Vet Parasitol 93:393–408PubMedCrossRefGoogle Scholar
  9. Guenther S, Nockler K, Von N, Rosenegk M, Landgraf M, Ewers C, Wieler LH, Schierack P (2008) Detection of Trichinella spiralis, T. britovi and T. pseudospiralis in muscle tissue with real-time PCR. J Microbiol Methods 75:287–292PubMedCrossRefGoogle Scholar
  10. Han CX, Liu HX, Zhao DM (2006) The quantification of prion gene expression in sheep using real-time RT-PCR. Virus Genes 33:359–364PubMedCrossRefGoogle Scholar
  11. Hartmann S, Lucius R (2003) Modulation of host immune responses by nematode cystatins. Int J Parasitol 33:1291–302PubMedCrossRefGoogle Scholar
  12. Hedstrom L (2002) Serine protease mechanism and specificity. Chem Rev 102:4501–4524. doi: 10.1021/cr000033x PubMedCrossRefGoogle Scholar
  13. Jiang P, Wang ZQ, Cui J, Zhang X (2012) Comparison of artificial digestion and Baermann's methods for detection of Trichinella spiralis pre-encapsulated larvae in muscles with low-level infections. Foodborne Pathog Dis 9:27–31. doi: 10.1089/fpd.2011.0985 PubMedCrossRefGoogle Scholar
  14. Kang SA, Cho MK, Park MK, Kim DH, Hong YC, Lee YS, Cha HJ, Ock MS, Yu HS (2012) Alteration of helper T-cell related cytokine production in splenocytes during Trichinella spiralis infection. Vet Parasitol 186:319–327. doi: 10.1016/j.vetpar.2011.12.002 PubMedCrossRefGoogle Scholar
  15. Kapel CM, Gamble HR (2000) Infectivity, persistence, and antibody response to domestic and sylvatic Trichinella spp. in experimentally infected pigs. Int J Parasitol 30:215–21PubMedCrossRefGoogle Scholar
  16. Kenneth JL, Thomas DS (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCt CT method. Methods 25:402–408CrossRefGoogle Scholar
  17. Li F, Cui J, Wang ZQ, Jiang P (2010) Sensitivity and optimization of artificial digestion in the inspection of meat for Trichinella spiralis. Foodborne Pathog Dis 7:879–885PubMedCrossRefGoogle Scholar
  18. Li CK, Seth R, Gray T, Bayston R, Mahida YR, Wakelin D (1998) Production of proinflammatory cytokines and inflammatory mediators in human intestinal epithelial cells after invasion by Trichinella spiralis. Infect Immun 66:2200–2206PubMedGoogle Scholar
  19. Man Warren T, Gagliardo L, Geyer J, McVay C, Pearce-Kelling S, Appleton J (1997) Invasion of intestinal epithelia in vitro by the parasitic nematode Trichinella spiralis. Infect Immun 65(11):4806–4812Google Scholar
  20. Murray J, Manoury B, Balic A, Watts C, Maizels RM (2005) Bm-CPI- 2, a cystatin from Brugia malayi nematode parasites, differs from Caenorhabditis elegans cystatins in a specific site mediating inhibition of the antigen-processing enzyme AEP. Mol Biochem Parasitol 139:197–203PubMedCrossRefGoogle Scholar
  21. Nagano I, Wu Z, Takahashi Y (2009) Functional genes and proteins of Trichinella spp. Parasitol Res 104:197–207. doi: 10.1007/s00436-008-1248-1 PubMedCrossRefGoogle Scholar
  22. Nagano I, Wu Z, Asano K, Takahashi Y (2011) Molecular cloning and characterization of transgelin-like proteins mainly transcribed in newborn larvae of Trichinella spp. Vet Parasitol 178:134–142PubMedCrossRefGoogle Scholar
  23. Newlands GF, Skuce PJ, Knox DP, Smith WD (2001) Cloning and expression of cystatin, a potent cysteine protease inhibitor from the gut of Haemonchus contortus. Parasitology 122:371–378PubMedCrossRefGoogle Scholar
  24. Ren HJ, Cui J, Wang ZQ, Liu RD (2011) Norma l mouse intestinal epithelial cells as a model for the in vitro invasion of Trichinella spiralis infective larvae. PLoS One 6:e27010PubMedCrossRefGoogle Scholar
  25. Ren HJ, Cui J, Yang W, Liu RD, Wang ZQ (2013a) Identification of differentially expressed genes in Trichinella spiralis larvae after exposure to host intestine milieu. PLoS One 8:e67570. doi: 10.1371/journal.pone.0067570 PubMedCrossRefGoogle Scholar
  26. Ren HJ, Liu RD, Wang ZQ, Cui J (2013b) Construction of a Trichinella spiralis phage display library and use for identifying the host enterocyte–parasite interactions. Parasitol Res 112:1857–1863. doi: 10.1007/s00436-013-3339-x PubMedCrossRefGoogle Scholar
  27. Robinson MW, Connolly B (2005) Proteomic analysis of the excretory–secretory proteins of the Trichinella spiralis L1 larva, a nematode parasite of skeletal muscle. Proteomics 5:4525–4532PubMedCrossRefGoogle Scholar
  28. Romaris F, North SJ, Gagliardo LF, Butcher BA, Ghosh K, Beiting DP, Panico M, Arasu P, Dell A, Morris HR, Appleton JA (2002) A putative serine protease among the excretory–secretory glycoproteins of L1 Trichinella spiralis. Mol Biochem Parasitol 122:149–160PubMedCrossRefGoogle Scholar
  29. Schmittgen TD, Livak KJ (2008) Analyzing real-time PCR data by the comparative C (T) method. Nat Protoc 3:1101–1108PubMedCrossRefGoogle Scholar
  30. Stewart GL, Despommier DD, Burnham J, Raines KM (1987) Trichinella spiralis: behavior, structure, and biochemistry of larvae following exposure to components of the host enteric environment. Exp Parasitol 63:195–204PubMedCrossRefGoogle Scholar
  31. Theodoropoulos G, Petrakos G (2010) Trichinella spiralis: differential effect of host bile on the in vitro invasion of infective larvae into epithelial cells. Exp Parasitol 126:441–444PubMedCrossRefGoogle Scholar
  32. Theodoropoulos G, Prokou M, Georgiadou V, Petrakos M, Webster P, Kapel CM (2005) Effects of raw biles and their non-protein fractions from fox, pig, sheep and chicken on the survival of Trichinella spp. in vitro. Vet Parasitol 132:63–67PubMedCrossRefGoogle Scholar
  33. Wang N, Stamenović D (2000) Contribution of intermediate filaments to cell stiffness, stiffening, and growth. Am J Physiol Cell Physiol 279:C188–194PubMedGoogle Scholar
  34. Wang L, Cui J, Wang SW, Wang ZQ (2010) Observation of the in vitro invasion of intestinal epithelial cells by Trichinella spiralis larvae and their development in those cells. J Pathog Biol 5:901–903Google Scholar
  35. Wang SW, Wang ZQ, Cui J (2011) Protein change of intestinal epithelial cells induced in vitro by Trichinella spiralis infective larvae. Parasitol Res 108:593–599. doi: 10.1007/s00436-010-2102-9 PubMedCrossRefGoogle Scholar
  36. Wang ZQ, Wang L, Cui J (2012) Proteomic analysis of Trichinella spiralis proteins in intestinal epithelial cells after culture with their larvae by shotgun LC-MS/MS approach. J Proteomics 75:2375–2383. doi: 10.1016/j.jprot.2012.02.005 PubMedCrossRefGoogle Scholar
  37. Wang L, Wang ZQ, Cui J (2013a) Proteomic analysis of the changed proteins of Trichinella spiralis infective larvae after co-culture in vitro with intestinal epithelial cells. Vet Parasitol 194:160–163. doi: 10.1016/j.vetpar.2013.01.045 CrossRefGoogle Scholar
  38. Wang L, Wang ZQ, Cui J (2013b) Protein changes of Trichinella spiralis muscle larvae in vitro induced by bovine bile. Vet Parasitol 194:164–167. doi: 10.1016/j.vetpar.2013.01. 046 CrossRefGoogle Scholar
  39. Whelan JA, Russell NB, Whelan MA (2003) A method for the absolute quantification of cDNA using real-time PCR. J Immunol Methods 278:261–269PubMedCrossRefGoogle Scholar
  40. Yang Y, Jian W, Qin W (2010) Molecular cloning and phylogenetic analysis of small GTPase protein Tscdc42 from Trichinella spiralis. Parasitol Res 106:801–808. doi: 10.1007/s00436-010-1735-z PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2013

Authors and Affiliations

  1. 1.Department of ParasitologyMedical College, Zhengzhou UniversityZhengzhouChina

Personalised recommendations