Lysophosphatidylcholine triggers cell differentiation in the protozoan parasite Herpetomonas samuelpessoai through the CK2 pathway

Abstract

Background

Protozoa are distantly related to vertebrates but present some features of higher eukaryotes, making them good model systems for studying the evolution of basic processes such as the cell cycle. Herpetomonas samuelpessoai is a trypanosomatid parasite isolated from the hemipteran insect Zelus leucogrammus. Lysophosphatidylcholine (LPC) is implicated in the transmission and establishment of Chagas disease, whose etiological agent is Trypanosoma cruzi. LPC is synthesized by T. cruzi and its vectors, the hemipteran Rhodnius prolixus and Triatoma infestans. Platelet-activating factor (PAF), a phospholipid with potent and diverse physiological and pathophysiological actions, is a powerful inducer of cell differentiation in Herpetomonas muscarum muscarum and T. cruzi. The enzyme phospholipase A2 (PLA2) catalyzes the hydrolysis of the 2-ester bond of 3-sn-phosphoglyceride, transforming phosphatidylcholine (PC) into LPC.

Methods

In this study, we evaluated cellular differentiation, PLA2 activity and protein kinase CK2 activity of H. samuelpessoai in the absence and in the presence of LPC and PAF. Results: We demonstrate that both PC and LPC promoted a twofold increase in the cellular differentiation of H. samuelpessoai, through CK2, with a concomitant inhibition of its cell growth. Intrinsic PLA2 most likely directs this process by converting PC into LPC.

Conclusions

Our results suggest that the actions of LPC on H. samuelpessoai occur upon binding to a putative PAF receptor and that the protein kinase CK2 plays a major role in this process.

Graphic abstract

Cartoon depicting a model for the synthesis and functions of LPC in Herpetomonas samuelpessoai, based upon our results regarding the role of LPC on the cell biology of Trypanosoma cruzi [28,29,30,31,32]. N nucleus, k kinetoplast, PC phosphatidylcholine, LPC lysophosphatidylcholine, PLA2 phospholipase A2, PAFR putative PAF receptor in trypanosomatids [65], CK2 protein kinase CK2 [16].

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References

  1. 1.

    Imhof S, Roditi I (2015) The social life of African trypanosomes. Trends Parasitol 31:490–498

    PubMed  Google Scholar 

  2. 2.

    Hoare CA, Wallace FG (1966) Developmental stages of trypanosomatid flagellates: a new terminology. Nature 212:1385–1386

    Google Scholar 

  3. 3.

    Wallace FG (1977) Development stages of trypanosomatids flagellates: a new terminology revisited. Protozoology 3:51–56

    Google Scholar 

  4. 4.

    Pacheco RS, Marzochi MC, Pires MQ, Brito CM, De Madeira M, Barbosa-Santos EG (1998) Parasite genotypically related to a monoxenous trypanosomatid of dog’s flea causing opportunistic infection in an HIV positive patient. Mem Inst Oswaldo Cruz 93:531–537

    CAS  PubMed  Google Scholar 

  5. 5.

    Ranque PH, Nourrit J, Nicoli RM (1974) Systematic studies on Trypanosoma. The evolutionary stages of Trypanosoma. Bull Soc Pathol Éxot 67:377–387

    CAS  Google Scholar 

  6. 6.

    Janovy J Jr, Lee KW, Brumbaugh JA (1974) The differentiation of Herpetomonas megaseliae: ultrastructural observations. J Protozool 21:53–59

    PubMed  Google Scholar 

  7. 7.

    McGhee RB, Cosgrove WB (1980) Biology and physiology of the lower Trypanosomatidae. Microbiol Rev 44(1):140–173

    CAS  PubMed  PubMed Central  Google Scholar 

  8. 8.

    Dias FA, Vasconcellos LR, Romeiro A, Attias M, Souto-Padrón TC, Lopes AH (2014) Transovum transmission of trypanosomatid cysts in the Milkweed bug,Oncopeltus fasciatus. PLoS One 9(9):e108746. https://doi.org/10.1371/journal.pone.0108746

    CAS  Article  PubMed Central  Google Scholar 

  9. 9.

    Borghesan TC, Ferreira RC, Takata CSA (2013) Molecular phylogenetic redefinition of Herpetomonas (Kinetoplastea, Trypanosomatidae), a genus of insect parasites associated with flies. Protist 164:129–152

    PubMed  Google Scholar 

  10. 10.

    Silvester E, McWilliam KR, Matthews KR (2017) The cytological events and molecular control of life cycle development of Trypanosoma brucei in the mammalian bloodstream. Pathogens 6:29

    PubMed Central  Google Scholar 

  11. 11.

    de Souza W, de Carvalho TM, Barrias ES (2010) Review on Trypanosoma cruzi: host cell interaction. Int J Cell Biol. https://doi.org/10.1155/2010/295394

    Article  PubMed  PubMed Central  Google Scholar 

  12. 12.

    Lun ZR, Lai DH, Wen YZ, Zheng LL, Shen JL, Yang TB, Zhou WL, Qu LH, Hide G, Ayala FJ (2015) Cancer in the parasitic protozoans Trypanosoma brucei and Toxoplasma gondii. PNAS 112:8835–8842

    CAS  PubMed  Google Scholar 

  13. 13.

    Brun J (1974) Ultrastructure and life cycle of Herpetomonas muscarum, “Herpetomonas mirabilis” and Crithidia luciliae in Chrysomyia chloropyga. Acta Trop 32:219–290

    CAS  PubMed  Google Scholar 

  14. 14.

    Maslov DA, Yurchenko J, Votýpka V, Lukeš J (2013) Diversity and phylogeny of insect trypanosomatids: all that is hidden shall be revealed. Trends Parasitol 29:43–52

    PubMed  Google Scholar 

  15. 15.

    Borghesan TC, Campaner M, Matsumoto TE, Espinosa OA, Razafindranaivo V, Paiva F, Carranza JC, Añez Neves L, Teixeira MMG, Camargo EP (2018) Genetic diversity and phylogenetic relationships of coevolving symbiont-harboring insect trypanosomatids, and their neotropical dispersal by invader African blowflies (Calliphoridae). Front Microbiol 9:131

    PubMed  PubMed Central  Google Scholar 

  16. 16.

    Ximenes AA, Silva-Cardoso L, De Cicco NN, Pereira MG, Lourenço DC, Fampa P, Folly E, Cunha-e-Silva NL, Silva-Neto MA, Atella GC (2015) Lipophorin drives lipid incorporation and metabolism in insect trypanosomatids. Protist 166:297–309

    CAS  Google Scholar 

  17. 17.

    Kluck G, Régis KC, De Cicco NNT, Silva-Cardoso L, Pereira MG, Fampa P, Chagas-Lima AC, Romeiro A, Cunha-Silva NL, Atella GC (2018) An evaluation of lipid metabolism in the insect trypanosomatid Herpetomonas muscarum uncovers a pathway for the uptake of extracellular insect lipoproteins. Parasitol Int 67:97–106

    CAS  PubMed  Google Scholar 

  18. 18.

    Silva-Neto MAC, Carneiro AB, Vieira DP, Mesquita RD, Lopes AH (2002) Platelet-activating factor (PAF) activates casein kinase 2 in the protozoan parasite Herpetomonas muscarum muscarum. Biochem Biophys Res Commun 293:1358–1363

    CAS  PubMed  Google Scholar 

  19. 19.

    Naula C, Parson M, Mottram JC (2005) Protein kinases as drug targets in trypanosomes and Leishmania. Biochim Biophys Acta 1754:151–159

    CAS  PubMed  PubMed Central  Google Scholar 

  20. 20.

    Huang H (2011) Signal transduction in Trypanosoma cruzi. Adv Parasitol 75:325–344

    PubMed  Google Scholar 

  21. 21.

    McDonald L, Cayla M, Ivens A, Mony BM, MacGregor P, Silvester E, McWilliam K, Matthews KR (2018) Non-linear hierarchy of the quorum sensing signalling pathway in bloodstream form African trypanosomes. PLoS Pathog. https://doi.org/10.1371/journal.ppat.1007145

    Article  PubMed  PubMed Central  Google Scholar 

  22. 22.

    Parsons M, Ruben L (2000) Pathways involved in environmental sensing in trypanosomatids. Parasitol Today 16:56–62

    CAS  PubMed  Google Scholar 

  23. 23.

    Newton AC, Bootman MD, Scott JD (2016) Second Messengers. Cold Spring Harb Perspect Biol 8:1–14 (Article no. a005926)

    Google Scholar 

  24. 24.

    Guan XL, Mäser P (2017) Comparative sphingolipidomics of disease-causing trypanosomatids reveal unique lifecycle- and taxonomy-specific lipid chemistries. Sci Rep 7:13617

    PubMed  PubMed Central  Google Scholar 

  25. 25.

    Naula C, Seebeck T (2000) Cyclic AMP signaling in trypanosomatids. Parasitol Today 1:35–38

    Google Scholar 

  26. 26.

    Légrádi A, Chitu V, Szukacsov V, Fajka-Boja R, Szucs SK, Monostori E (2004) Lysophosphatidylcholine is a regulator of tyrosine kinase activity and intracellular Ca2+ level in Jurkat T cell line. Immun Lett 91:17–21

    Google Scholar 

  27. 27.

    Bassa BV, Roh DD, Vaziri ND, Kirschembaum MA, Kamanna VS (1999) Lysophosphatidylcholine activates mesangial cell PKC and MAP kinase by PLC gamma-1 and tyrosine kinase-Ras pathways. Am J Physiol 277:328–337

    Google Scholar 

  28. 28.

    Golodne DM, Monteiro RQ, Graça-Souza AV, Silva-Neto MAC, Atella GC (2003) Lysophosphatidylcholine acts as an anti-hemostatic molecule in the saliva of the blood-sucking bug Rhodnius prolixus. J Biol Chem 278:27766–27771

    CAS  PubMed  Google Scholar 

  29. 29.

    Mesquita RD, Carneiro AB, Bafica A, Gazos-Lopes F, Takiya CM, Souto-Padron T, Vieira DP, Ferreira-Pereira A, Almeida IC, Figueiredo RT, Porto BN, Bozza MT, Graça-Souza AV, Lopes AH, Atella GC, Silva-Neto MA (2008) Trypanosoma cruzi infection is enhanced by vector saliva through immunosuppressant mechanisms mediated by lysophosphatidylcholine. Infect Immun 76:5543–5552

    CAS  PubMed  PubMed Central  Google Scholar 

  30. 30.

    Silva-Neto MAC, Carneiro AB, Silva-Cardoso L, Atella GC (2012) Lysophosphatidylcholine: a novel modulator of Trypanosoma cruzi transmission. J Parasitol Res 2012:625838

    PubMed  Google Scholar 

  31. 31.

    Silva-Neto MAC, Lopes AH, Atella GC (2016) Here, there, and everywhere: the ubiquitous distribution of the immunosignaling molecule lysophosphatidylcholine and its role on Chagas disease. Front Immunol 7:62–65

    PubMed  PubMed Central  Google Scholar 

  32. 32.

    Gazos-Lopes F, Oliveira MM, Hoelz LV, Vieira DP, Marques AF, Nakayasu ES, Gomes MT, Salloum NG, Pascutti PG, Souto-Padrón T, Monteiro RQ, Lopes AH, Almeida IC (2014) Structural and functional analysis of a platelet-activating lysophosphatidylcholine of Trypanosoma cruzi. PLoS Negl Trop Dis 8:e3077

    PubMed  PubMed Central  Google Scholar 

  33. 33.

    Chagas-Lima AC, Pereira MG, Fampa P, Lima MS, Kluck GEG, Atella GC (2019) Bioactive lipids regulate Trypanosoma cruzi development. Parasitol Res 118:2609–2619. https://doi.org/10.1007/s00436-019-06331-9

    Article  PubMed  Google Scholar 

  34. 34.

    Yuan Y, Jackson SP, Newnham HH, Mitchell CA, Salem HH (1995) An essential role for lysophosphatidylcholine in the inhibition of platelet aggregation by secretory phospholipase A2. Am Soc Hematol 11:4166–4174

    Google Scholar 

  35. 35.

    Ridgley EL, Ruben L (2001) Phospholipase from Trypanosoma brucei releases arachidonic acid by sequential sn-1, sn-2 deacylation of phospholipids. Mol Biochem Parasitol 114:29–40

    CAS  PubMed  Google Scholar 

  36. 36.

    Shuaibu MN, Kanbara H, Yanagi T, Ameh DA, Bonire JJ, Nok AJ (2001) Phospholipase A2 from Trypanosoma brucei gambiense and Trypanosoma brucei brucei: inhibition by organotins. J Enzyme Inhib 16:433–441

    CAS  PubMed  Google Scholar 

  37. 37.

    Chao W, Olson M (1993) Platelet-activating factor: receptors and signal transduction. Biochem J 292:617–629

    CAS  PubMed  PubMed Central  Google Scholar 

  38. 38.

    Tsoupras AB, Iatrou C, Frangia C, Demopoulos CA (2009) The implication of platelet activating factor in cancer growth and metastasis: potent beneficial role of PAF-inhibitors and antioxidants. Infect Disord Drug Targets 9:390–399

    CAS  PubMed  Google Scholar 

  39. 39.

    Tsuda M, Tozaki-Saitoh H, Inoue K (2011) Platelet-activating factor and pain. Biol Pharm Bull 34:1159–1162

    CAS  PubMed  Google Scholar 

  40. 40.

    Benveniste J (1974) Platelet-activating factor, a new mediator of anaphylaxis and immune complex deposition from rabbit and human basophils. Nature 249:581–590

    CAS  PubMed  Google Scholar 

  41. 41.

    Hwang SB, Lam MH, Pong SS (1986) Ionic and GTP regulation of binding of platelet-activating factor to receptors and platelet-activating factor induced activation of GTPase in rabbit platelet membranes. J Biol Chem 261:532–537

    CAS  PubMed  Google Scholar 

  42. 42.

    Dutra PML, Rodrigues CO, Jesus JB, Lopes AHCS, Souto-Padrón T, Meyer-Fernandes JR (1998) A novel ecto-phosphatase activity of Herpetomonas muscarum muscarum inhibited by platelet-activating factor. Biochem Biophys Res Commun 253:164–169

    CAS  PubMed  Google Scholar 

  43. 43.

    Lopes AH, Dutra PML, Rodrigues CO, Soares MJ, Angluster J, Cordeiro RS (1997) Effect of platelet-activating factor on the process of cellular differentiation of Herpetomonas muscarum muscarum. J Eukaryot Microbiol 44:321–325

    CAS  PubMed  Google Scholar 

  44. 44.

    Rodrigues CO, Dutra PML, Souto-Padrón T, Cordeiro RSB, Lopes AHCS (1996) Effect of platelet-activating factor on cell differentiation of Trypanosoma cruzi. Biochem Biophys Res Commun 223:735–740

    CAS  PubMed  Google Scholar 

  45. 45.

    Rodrigues CO, Dutra PML, Barros FS, Souto-Padrón T, Meyer-Fernandes JR, Lopes AH (1999) Platelet-activating factor induction of secreted phosphatase activity in Trypanosoma cruzi. Biochem Biophys Res Commun 266:36–42

    CAS  PubMed  Google Scholar 

  46. 46.

    Dutra PML, Rodrigues CO, Romeiro A, Grillo LAM, Dias FA, Attias M, De Souza W, Lopes AHCS, Meyer-Fernandes JR (2000) Characterization of ectophosphatase activities in trypanosomatid parasites of plants. Phytopathology 90:1032–1038

    CAS  PubMed  Google Scholar 

  47. 47.

    Rosa MSS, Vieira RB, Pereira AF, Dutra PML, Lopes AHCS (2001) Platelet-activating factor (PAF) modulates peritoneal mouse macrophage infection by Leishmania amazonensis. Curr Microbiol 43:33

    CAS  PubMed  Google Scholar 

  48. 48.

    Roitman C, Roitman I, De Azevedo HP (1972) Growth of an insect trypanosomatid at 37ºC in a defined medium. J Protozool 19:346–349

    CAS  PubMed  Google Scholar 

  49. 49.

    Santos DO, Oliveira MM (1988) Effect of cAMP on macromolecule synthesis in the pathogenic protozoa Trypanosoma cruzi. Mem Inst Oswaldo Cruz 83:287–292

    CAS  PubMed  Google Scholar 

  50. 50.

    Pawlowic MC, Zhang K (2012) Leishmania parasites possess a platelet-activating factor acetylhydrolase important for virulence. Mol Biochem Parasitol 186:11–20

    CAS  PubMed  PubMed Central  Google Scholar 

  51. 51.

    Passos CL, Ferreira C, Soares DC, Saraiva EM (2015) Leishmanicidal effect of synthetic trans-resveratrol analogs. PLoS One 10:e0141778

    PubMed  PubMed Central  Google Scholar 

  52. 52.

    Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685

    CAS  Google Scholar 

  53. 53.

    Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275

    CAS  Google Scholar 

  54. 54.

    Shitamukai A, Matsuzaki F (2012) Control of asymmetric cell division of mammalian neural progenitors. Dev Growth Differ 54:277–286

    CAS  PubMed  Google Scholar 

  55. 55.

    Fragel-Madeira L, Meletti T, Mariante RM, Monteiro RQ, Einicker-Lamas M (2011) Platelet activating factor blocks interkinetic nuclear migration in retinal progenitors through an arrest of the cell cycle at the S/G2 transition. PLoS ONE 6:e16058

    PubMed  PubMed Central  Google Scholar 

  56. 56.

    Archer SK, Inchaustegui D, Queiroz R, Clayton C (2011) The cell cycle regulated transcriptome of Trypanosoma brucei. PLoS One 31:e18425

    Google Scholar 

  57. 57.

    Ramanadham S, Ali T, Ashley JW, Bone RN, Hancock WD, Lei X (2015) Calcium-independent phospholipases A2 and their roles in biological processes and diseases. J Lipid Res 56:1643–1668. https://doi.org/10.1194/jlr.R058701

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  58. 58.

    Dutra PML, Vieira DP, Meyer-Fernandes JR, Silva-Neto MAC, Lopes AH (2009) Stimulation of Leishmania tropica protein kinase CK2 activities by platelet-activating factor (PAF). Acta Trop 111:247–254

    CAS  PubMed  Google Scholar 

  59. 59.

    Torkhovskaya TI, Ipatova OM, Zakharova TS, Kochetova MM, Khalilov EM (2007) Lysophospholipid receptors in cell signaling. Biochemistry (Mosc) 72:125–131

    CAS  Google Scholar 

  60. 60.

    Flemming PK, Dedman AM, Xu SZ (2006) Sensing of lysophospholipids by TRPC5 calcium channel. J Biol Chem 281:4977–4982

    CAS  PubMed  Google Scholar 

  61. 61.

    Kabarowski JHS, Zhu K, Le LQ et al (2001) Lysophosphatidylcholine as a ligand or the immunoregulatory receptor G2A. Science 293:702–705

    CAS  PubMed  Google Scholar 

  62. 62.

    Wang L, Radu CG, Yang LV, Bentolila LA, Riedinger M, Witte ON (2005) Lysophosphatidylcholine-induced surface redistribution regulates signaling of the murine G protein-coupled receptor G2A. Mol Biol Cell 16:2234–2247

    CAS  PubMed  PubMed Central  Google Scholar 

  63. 63.

    Gould MK, de Koning HP (2011) Cyclic-nucleotide signalling in protozoa. FEMS Microbiol Rev 35:515–541. https://doi.org/10.1111/j.1574-6976.2010.00262.x

    CAS  Article  PubMed  Google Scholar 

  64. 64.

    Kougias P, Chai H, Lin PH, Lumsden AB, Yao Q, Chen C (2006) Lysophosphatidylcholine and secretory phospholipase A2 in vascular disease: Mediators of endothelial dysfunction and atherosclerosis. Med Sci Monit 12:RA5–RA16

    CAS  PubMed  Google Scholar 

  65. 65.

    Lopes AH, Gomes MT, Dutra FL, Vermelho AB, Meyer-Fernandes JR, Silva-Neto MAC, Souto-Padrón T, Vieira DP (2010) Intracellular signaling pathways involved in cell differentiation in trypanosomatids. Open Parasitol J 4:102–110

    CAS  Google Scholar 

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Acknowledgments

We thank Dr. Antonio F. Pereira and Dr. Rafael Linden for valuable discussions, as well as Paulo C. Miguel and Ricardo Pereira for technical assistance. We are grateful to Unidade de Microscopia Multiusuário Souto-Padrón and Lins (UniMicro) for the support on microscopy. This work was supported by grants from the Brazilian Agencies Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ) and Instituto Nacional de Ciência e Tecnologia em Entomologia Molecular (INCTEM).

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Correspondence to Danielle P. Vieira.

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FLD, performed the analysis, wrote the paper; DPV, performed the analysis, contributed data, wrote the paper; FSC, performed the analysis, contributed data; CMA, performed the analysis, contributed data; GCA, contributed data; MACSN, Conceived and designed the analysis, wrote the paper; AHL, conceived and designed the analysis, wrote the paper. All authors have agreed with the content of the manuscript. The authors declare no conflict of interests.

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S1 Figure. Gallery of the three H. samuelpessoai different forms after Panotic staining observed in bright field light microscopy. For light microscopy observation, H. samuelpessoai cultures smears were fixed and stained using Panotic staining kit (Laborclin, PR, Brazil), and analyzed using a Zeiss Axioplan II light microscope equipped with a Color View XS digital video camera (Zeiss, Jena, Germany). The position of the kinetoplast (arrowheads) in relation to the nucleus (thin arrows) and the flagella (thick arrows), is what determines promastigotes (A), paramastigotes (B) and opisthomastigotes (C) morphotypes. Scale bar: 5 m. (TIFF 885 kb)

S2 Figure. Evaluation of cell cycle through analysis of DNA content of H. samuelpessoai. The cells cultures were treated or not with PAF, PC 1, PC2, LPC1 and LPC2, for 24 h, labeled with propidium iodide in cell cycle solution, and analyzed by flow cytometry. Untreated parasites control (A), PAF (B), 10 mM PC (C), 100 mM PC (D), 10 mM LPC (E), 100 mM LPC (F). Representative plots of at least three independent experiments with similar results. Methodology described in 2.4 Materials and Methods. (PNG 153 kb)

Table S1. Evaluation of cell cycle through analysis of DNA content of H. samuelpessoai. The cells cultures were treated or not with PAF, PC 1, PC2, LPC1 and LPC2, for 24 h, labeled with propidium iodide in cell cycle solution, and analyzed by flow cytometry. Our results confirmed that PAF, PC and LPC did not affect the division pattern of the H. samuelpessoai parasites, compared to the untreated controls (P ≤ 0.05). Methodology described in 2.4 Materials and Methods. (PNG 55 kb)

S3 Figure. Evaluation of H. samuelpessoai protein phosphorylation profile in the absence or in the presence of different kinases inhibitors. The parasites were treated in the absence or presence of following drugs: LPC (10−7 M) and LPC (10−7 M) plus different kinases inhibitors: H 89 (50 nM) (PKA inhibitor), BIS I (10 nM) (PKC inhibitor), BIS T (0.4 mM) (PKC inhibitor), KN 93 (0.37 mM) (CAMK II inhibitor) PD 98 (2 mM) (MAPK inhibitor), TBB (0.9 mM) (CK2 inhibitor), ML-7 (0.3 mM) (myosin light chain kinase inhibitor). These parasites were then centrifuged, washed twice with saline and once with buffer A (sucrose 250 mM, 100 mM Tris–HCl, 50 mM MgCl 2, pH 7.4). The sediment was resuspended in lysis buffer (20 mM Tris–HCl, 15 mM NaCl, 1 mM sodium orthovanadate, EDTA 1 mM, 1 mM EGTA, 10 mM NaF, 0.02% sodium azide; pH 8.0). The parasites were, then frozen in liquid nitrogen, thawed, sonicated for 12 min and centrifuged for 15 min at room temperature. The supernatant was used in the gel. For an endogenous phosphorylation analysis, the reaction medium consisting of (50 mM Tris–HCl, 10 mM MgCl 2, 1 mM EDTA, 1 mM EGTA, pH 8.0), H. samuelpessoai cell extract ATP sample and mixture (cold ATP and 32P-ATP - 4.0 x 106 cpm/ml); this reaction medium was incubated for 45 min at 37 °C. The proteins were then separated by SDS-PAGE, 10%. The electrophoresis was performed at 150 volts and 60 mA. The gel was stained (with Comassie Blue dye), dried and exposed to a X-rays film at -70° C for 20 days. Before each application in gel, the amounts of proteins were quantified by the Lowry method to ensure the same sample concentration in all systems. For protein measurement, the spectrophotometer of the Ultrospec 2000 brand, Pharmacia Biotech was used. C: control; L LPC; BIS-T (BT); BIS I (BI); PD 98,059 (PD); KN 93 (KN); H 89 (H); TBB (T); ML-7 (M). (PNG 351 kb)

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Dutra, F.L., Vieira, D.P., Coelho, F.S. et al. Lysophosphatidylcholine triggers cell differentiation in the protozoan parasite Herpetomonas samuelpessoai through the CK2 pathway. Acta Parasit. 65, 108–117 (2020). https://doi.org/10.2478/s11686-019-00135-8

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Keywords

  • Herpetomonas samuelpessoai
  • Lysophosphatidylcholine
  • Phosphatidylcholine
  • Phospholipase A2
  • Protein kinase CK2
  • Platelet-activating factor