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.
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.
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.
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 , CK2 protein kinase CK2 .
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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).
Conflict of interest
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
- Herpetomonas samuelpessoai
- Phospholipase A2
- Protein kinase CK2
- Platelet-activating factor