Abstract
Schistosoma mansoni and its vertebrate host have a complex and intimate connection in which several molecular stimuli are exchanged and affect both organisms. Human tumor necrosis factor alpha (hTNF-α), a pro-inflammatory cytokine, is known to induce large-scale gene expression changes in the parasite and to affect several parasite biological processes such as metabolism, egg laying, and worm development. Until now, the molecular mechanisms for TNF-α activity in worms are not completely understood. Here, we aimed at exploring the effect of hTNF-α on S. mansoni protein phosphorylation by 2D gel electrophoresis followed by a quantitative analysis of phosphoprotein staining and protein identification by mass spectrometry. We analyzed three biological replicates of adult male worms exposed to hTNF-α and successfully identified 32 protein spots with a statistically significant increase in phosphorylation upon in vitro exposure to hTNF-α. Among the differentially phosphorylated proteins, we found proteins involved in metabolism, such as glycolysis, galactose metabolism, urea cycle, and aldehyde metabolism, as well as proteins related to muscle contraction and to cytoskeleton remodeling. The most differentially phosphorylated protein (30-fold increase in phosphorylation) was 14-3-3, whose function is known to be modulated by phosphorylation, belonging to a signal transduction protein family that regulates a variety of processes in all eukaryotic cells. Further, 75 % of the identified proteins are known in mammals to be related to TNF-α signaling, thus suggesting that TNF-α response may be conserved in the parasite. We propose that this work opens new perspectives to be explored in the study of the molecular crosstalk between host and pathogen.
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Abbreviations
- hTNF-α:
-
Human tumor necrosis factor alpha
- SmTNFR:
-
Schistosoma mansoni tumor necrosis factor receptor homolog
- MS:
-
Mass spectrometry
- 2D-GE:
-
Two-dimensional gel electrophoresis
References
Agarwal S, Kulshreshtha P, Bambah Mukku D, Bhatnagar R (2008) Alpha-enolase binds to human plasminogen on the surface of Bacillus anthracis. Biochim Biophys Acta 1784(7-8):986–94. doi:10.1016/j.bbapap.2008.03.017
Amiri P et al (1992) Tumour necrosis factor alpha restores granulomas and induces parasite egg-laying in schistosome-infected SCID mice. Nature 356(6370):604–607
Ashburner M et al (2000) Gene Ontology: tool for the unification of biology. The Gene Ontology Consortium. Nat Genet 25(1):25–9
Avilan L et al (2011) Enolase: a key player in the metabolism and a probable virulence factor of trypanosomatid parasites-perspectives for its use as a therapeutic target. Enzyme Res 2011:932549. doi:10.4061/2011/932549
Blom N, Gammeltoft S, Brunak S (1999) Sequence and structure-based prediction of eukaryotic protein phosphorylation sites. J Mol Biol 294(5):1351–62
Bouwmeester T et al (2004) A physical and functional map of the human TNF-alpha/NF-kappa B signal transduction pathway. Nat Cell Biol 6(2):97–105. doi:10.1038/ncb1086
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–54
Braschi S, Curwen RS, Ashton PD, Verjovski-Almeida S, Wilson A (2006) The tegument surface membranes of the human blood parasite Schistosoma mansoni: a proteomic analysis after differential extraction. Proteomics 6(5):1471–1482
Bueding E (1950) Carbohydrate metabolism of Schistosoma mansoni. J Gen Physiol 33(5):475–95
Bunney TD, van Walraven HS, de Boer AH (2001) 14-3-3 protein is a regulator of the mitochondrial and chloroplast ATP synthase. Proc Natl Acad Sci U S A 98(7):4249–54. doi:10.1073/pnas.061437498
Candiano G et al (2004) Blue silver: a very sensitive colloidal Coomassie G-250 staining for proteome analysis. Electrophoresis 25(9):1327–33. doi:10.1002/elps.200305844
Cheever AW, Poindexter RW, Wynn TA (1999) Egg laying is delayed but worm fecundity is normal in SCID mice infected with Schistosoma japonicum and S. mansoni with or without recombinant tumor necrosis factor alpha treatment. Infect Immun 67(5):2201–8
Chung JJ, Okamoto Y, Coblitz B, Li M, Qiu Y, Shikano S (2009) PI3K/Akt signalling-mediated protein surface expression sensed by 14-3-3 interacting motif. FEBS J 276(19):5547–58. doi:10.1111/j.1742-4658.2009.07241.x
Colley DG, Bustinduy AL, Secor WE, King CH (2014) Human schistosomiasis. Lancet 383(9936):2253–64. doi:10.1016/S0140-6736(13)61949-2
Curwen RS, Ashton PD, Johnston DA, Wilson RA (2004) The Schistosoma mansoni soluble proteome: a comparison across four life-cycle stages. Mol Biochem Parasitol 138(1):57–66. doi:10.1016/j.molbiopara.2004.06.016
Davies SJ et al (2004) Involvement of TNF in limiting liver pathology and promoting parasite survival during schistosome infection. Int J Parasitol 34(1):27–36
De Corte V, Gettemans J, Vandekerckhove J (1997) Phosphatidylinositol 4,5-bisphosphate specifically stimulates PP60(c-src) catalyzed phosphorylation of gelsolin and related actin-binding proteins. FEBS Lett 401(2-3):191–6
DeMarco R, Verjovski-Almeida S (2009) Schistosomes—proteomics studies for potential novel vaccines and drug targets. Drug Discov Today 14(9-10):472–8. doi:10.1016/j.drudis.2009.01.011
Dong S et al (2007) 14-3-3 Integrates prosurvival signals mediated by the AKT and MAPK pathways in ZNF198-FGFR1-transformed hematopoietic cells. Blood 110(1):360–9. doi:10.1182/blood-2006-12-065615
El Ridi R, Tallima H, Mahana N, Dalton JP (2010) Innate immunogenicity and in vitro protective potential of Schistosoma mansoni lung schistosomula excretory—secretory candidate vaccine antigens. Microbes Infect/Institut Pasteur 12(10):700–9. doi:10.1016/j.micinf.2010.04.012
Fan J et al (2011) Tyrosine phosphorylation of lactate dehydrogenase A is important for NADH/NAD(+) redox homeostasis in cancer cells. Mol Cell Biol 31(24):4938–50. doi:10.1128/MCB.06120-11
Freitas TC, Pearce EJ (2010) Growth factors and chemotactic factors from parasitic helminths: molecular evidence for roles in host-parasite interactions versus parasite development. Int J Parasitol 40(7):761–73. doi:10.1016/j.ijpara.2010.02.013
Fu H, Subramanian RR, Masters SC (2000) 14-3-3 proteins: structure, function, and regulation. Annu Rev Pharmacol Toxicol 40:617–47. doi:10.1146/annurev.pharmtox.40.1.617
Hahnel S, Lu Z, Wilson RA, Grevelding CG, Quack T (2013) Whole-organ isolation approach as a basis for tissue-specific analyses in Schistosoma mansoni. PLoS Negl Trop Dis 7(7):e2336. doi:10.1371/journal.pntd.0002336
Haseeb MA, Solomon WB, Palma JF (1996) Schistosoma mansoni: effect of recombinant tumor necrosis factor alpha on fecundity and [14C]-tyrosine uptake in females maintained in vitro. Comp Biochem Physiol C Pharmacol Toxicol Endocrinol 115(3):265–9
Hojlund K et al (2003) Proteome analysis reveals phosphorylation of ATP synthase beta -subunit in human skeletal muscle and proteins with potential roles in type 2 diabetes. J Biol Chem 278(12):10436–42. doi:10.1074/jbc.M212881200
Hynes GM, Willison KR (2000) Individual subunits of the eukaryotic cytosolic chaperonin mediate interactions with binding sites located on subdomains of beta-actin. J Biol Chem 275(25):18985–94. doi:10.1074/jbc.M910297199
Kim J et al (2011) Proteomic analysis of phosphotyrosyl proteins in human embryonic stem cell-derived neural stem cells. Neurosci Lett 499(3):158–63. doi:10.1016/j.neulet.2011.05.039
Koukouritaki SB, Vardaki EA, Papakonstanti EA, Lianos E, Stournaras C, Emmanouel DS (1999) TNF-alpha induces actin cytoskeleton reorganization in glomerular epithelial cells involving tyrosine phosphorylation of paxillin and focal adhesion kinase. Mol Med 5(6):382–92
Liang P, MacRae TH (1997) Molecular chaperones and the cytoskeleton. J Cell Sci 110(Pt 13):1431–40
Luo R, Zhou C, Lin J, Yang D, Shi Y, Cheng G (2012) Identification of in vivo protein phosphorylation sites in human pathogen Schistosoma japonicum by a phosphoproteomic approach. J Proteome 75(3):868–77. doi:10.1016/j.jprot.2011.10.003
Maule AG, Marks NJ (2006) Parasitic flatworms: molecular biology, biochemistry, immunology and physiology. CABI, Centre for Agriculture and Biosciences International, Oxfordshire
McGonigle S, Loschiavo M, Pearce EJ (2002) 14-3-3 proteins in Schistosoma mansoni; identification of a second epsilon isoform. Int J Parasitol 32(6):685–93
Mouveaux T et al (2014) Nuclear glycolytic enzyme enolase of Toxoplasma gondii functions as a transcriptional regulator. PLoS One 9(8):e105820. doi:10.1371/journal.pone.0105820
Nettelblad FA, Engstrom L (1987) The kinetic effects of in vitro phosphorylation of rabbit muscle enolase by protein kinase C. A possible new kind of enzyme regulation. FEBS Lett 214(2):249–52
Oliveira KC et al (2009) Identification of the Schistosoma mansoni TNF-alpha receptor gene and the effect of human TNF-alpha on the parasite gene expression profile. PLoS Negl Trop Dis 3(12):e556. doi:10.1371/journal.pntd.0000556
Pancholi V (2001) Multifunctional alpha-enolase: its role in diseases. Cell Mol Life Sci: CMLS 58(7):902–20
Perez-Sanchez R, Valero ML, Ramajo-Hernandez A, Siles-Lucas M, Ramajo-Martin V, Oleaga A (2008) A proteomic approach to the identification of tegumental proteins of male and female Schistosoma bovis worms. Mol Biochem Parasitol 161(2):112–23. doi:10.1016/j.molbiopara.2008.06.011
Perkins DN, Pappin DJ, Creasy DM, Cottrell JS (1999) Probability-based protein identification by searching sequence databases using mass spectrometry data. Electrophoresis 20(18):3551–67. doi:10.1002/(SICI)1522-2683(19991201)20:18<3551::AID-ELPS3551>3.0.CO;2-2
Piast M, Kustrzeba-Wojcicka I, Matusiewicz M, Banas T (2005) Molecular evolution of enolase. Acta Biochim Pol 52(2):507–13
Protasio AV et al (2012) A systematically improved high quality genome and transcriptome of the human blood fluke Schistosoma mansoni. PLoS Negl Trop Dis 6(1):e1455. doi:10.1371/journal.pntd.0001455
Qian CY et al (2011) Kinetics of circulating antigen 14-3-3 in sera of rabbits firstly infected with Schistosoma japonicum and treated with/without praziquantel. Parasitol Res 108(2):493–5. doi:10.1007/s00436-010-2112-7
Qian CY et al (2012) Characterization of IgG responses of rabbits to Sj14-3-3 protein after experimental infection with Schistosoma japonicum. Parasitol Res 111(5):2209–11. doi:10.1007/s00436-012-2973-z
Ribeiro-dos-Santos G, Verjovski-Almeida S, Leite LC (2006) Schistosomiasis—a century searching for chemotherapeutic drugs. Parasitol Res 99(5):505–21. doi:10.1007/s00436-006-0175-2
Roy S et al (1998) 14-3-3 facilitates Ras-dependent Raf-1 activation in vitro and in vivo. Mol Cell Biol 18(7):3947–55
Schechtman D et al (2001) Expression and immunolocalization of the 14-3-3 protein of Schistosoma mansoni. Parasitology 123(Pt 6):573–82
Schmelzle K, White FM (2006) Phosphoproteomic approaches to elucidate cellular signaling networks. Curr Opin Biotechnol 17(4):406–14. doi:10.1016/j.copbio.2006.06.004
Spiess C, Meyer AS, Reissmann S, Frydman J (2004) Mechanism of the eukaryotic chaperonin: protein folding in the chamber of secrets. Trends Cell Biol 14(11):598–604. doi:10.1016/j.tcb.2004.09.015
Supek F, Bosnjak M, Skunca N, Smuc T (2011) REVIGO summarizes and visualizes long lists of gene ontology terms. PLoS One 6(7):e21800. doi:10.1371/journal.pone.0021800
Szklarczyk D et al (2015) STRING v10: protein-protein interaction networks, integrated over the tree of life. Nucleic Acids Res 43(Database issue):D447–52. doi:10.1093/nar/gku1003
Thandavarayan RA et al (2008) 14-3-3 protein regulates Ask1 signaling and protects against diabetic cardiomyopathy. Biochem Pharmacol 75(9):1797–806. doi:10.1016/j.bcp.2008.02.003
Tzivion G, Avruch J (2002) 14-3-3 proteins: active cofactors in cellular regulation by serine/threonine phosphorylation. J Biol Chem 277(5):3061–4. doi:10.1074/jbc.R100059200
Tzivion G, Shen YH, Zhu J (2001) 14-3-3 proteins; bringing new definitions to scaffolding. Oncogene 20(44):6331–8. doi:10.1038/sj.onc.1204777
Van Der Hoeven PC, Van Der Wal JC, Ruurs P, Van Blitterswijk WJ (2000) Protein kinase C activation by acidic proteins including 14-3-3. Biochem J 347(Pt 3):781–5
Vaughan RA, Garcia-Smith R, Trujillo KA, Bisoffi M (2013) Tumor necrosis factor alpha increases aerobic glycolysis and reduces oxidative metabolism in prostate epithelial cells. Prostate 73(14):1538–46. doi:10.1002/pros.22703
Wajant H, Pfizenmaier K, Scheurich P (2003) Tumor necrosis factor signaling. Cell Death Differ 10(1):45–65. doi:10.1038/sj.cdd.4401189
Wang X et al (2012) Identification and molecular characterization of a novel signaling molecule 14-3-3 epsilon in Clonorchis sinensis excretory/secretory products. Parasitol Res 110(4):1411–20. doi:10.1007/s00436-011-2642-7
Watanabe T, Hosoya H, Yonemura S (2007) Regulation of myosin II dynamics by phosphorylation and dephosphorylation of its light chain in epithelial cells. Mol Biol Cell 18(2):605–16. doi:10.1091/mbc.E06-07-0590
Woodcock JM, Murphy J, Stomski FC, Berndt MC, Lopez AF (2003) The dimeric versus monomeric status of 14-3-3zeta is controlled by phosphorylation of Ser58 at the dimer interface. J Biol Chem 278(38):36323–7. doi:10.1074/jbc.M304689200
Wygrecka M et al (2009) Enolase-1 promotes plasminogen-mediated recruitment of monocytes to the acutely inflamed lung. Blood 113(22):5588–98. doi:10.1182/blood-2008-08-170837
Xing H, Zhang S, Weinheimer C, Kovacs A, Muslin AJ (2000) 14-3-3 proteins block apoptosis and differentially regulate MAPK cascades. EMBO J 19(3):349–58. doi:10.1093/emboj/19.3.349
Xiong ZJ, Storey KB (2012) Regulation of liver lactate dehydrogenase by reversible phosphorylation in response to anoxia in a freshwater turtle. Comp Biochem Physiol B Biochem Mol Biol 163(2):221–8. doi:10.1016/j.cbpb.2012.06.001
Yang LL et al (2009) Schistosoma japonicum: proteomics analysis of differentially expressed proteins from ultraviolet-attenuated cercariae compared to normal cercariae. Parasitol Res 105(1):237–48. doi:10.1007/s00436-009-1387-z
Yokota S, Yanagi H, Yura T, Kubota H (2001) Cytosolic chaperonin-containing t-complex polypeptide 1 changes the content of a particular subunit species concomitant with substrate binding and folding activities during the cell cycle. Eur J Biochem/FEBS 268(17):4664–73
Zhang B, Liu JY (2013) Mass spectrometric identification of in vivo phosphorylation sites of differentially expressed proteins in elongating cotton fiber cells. PLoS One 8(3):e58758. doi:10.1371/journal.pone.0058758
Zuo S et al (2010) 14-3-3 epsilon dynamically interacts with key components of mitogen-activated protein kinase signal module for selective modulation of the TNF-alpha-induced time course-dependent NF-kappaB activity. J Proteome Res 9(7):3465–78. doi:10.1021/pr9011377
Acknowledgments
We thank Núcleo de Enteroparasitas/Centro de Parasitologia e Micologia at Instituto Adolfo Lutz for supplying S. mansoni-infected hamsters. This work was supported by Fundação de Amparo a Pesquisa do Estado de São Paulo (FAPESP) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq). MLPC, KCO, and JMCB received fellowships from FAPESP. DS and SVA received established investigator fellowship awards from CNPq.
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Katia C. Oliveira and Mariana L. P. Carvalho contributed equally to this work.
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Supplementary Fig. 1
SDS-PAGE with total protein extract of male adult worms treated with hTNF-α (T) or the respective control exposed to vehicle (C). Three biological replicate assays (1 to 3) are shown. M is the molecular weight markers sample (TIFF 1959 kb)
Supplementary Fig. 2
Model of interaction of SmTNFR and differentially phosphorylated proteins (TIF 295 kb)
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(XLSX 24 kb)
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Oliveira, K.C., Carvalho, M.L.P., Bonatto, J.M.C. et al. Human TNF-α induces differential protein phosphorylation in Schistosoma mansoni adult male worms. Parasitol Res 115, 817–828 (2016). https://doi.org/10.1007/s00436-015-4812-5
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DOI: https://doi.org/10.1007/s00436-015-4812-5