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Comparative proteomic analysis of Leishmania parasites isolated from visceral and cutaneous leishmaniasis patients

Published online by Cambridge University Press:  11 November 2021

Melike Dinç Open the ORCID record for Melike Dinç [Opens in a new window] ,
Talat Yalçın ,
İbrahim Çavuş  and
Ahmet Özbilgin
Melike Dinç*
Affiliation:
Izmir Institute of Technology, Integrated Research Centers, National Mass Spectrometry Application and Research Center, Izmir, Turkey
Talat Yalçın
Affiliation:
Faculty of Science, Department of Chemistry, Izmir Institute of Technology, Izmir, Turkey
İbrahim Çavuş
Affiliation:
Faculty of Medicine, Department of Parasitology, Manisa Celal Bayar University, Manisa, Turkey
Ahmet Özbilgin
Affiliation:
Faculty of Medicine, Department of Parasitology, Manisa Celal Bayar University, Manisa, Turkey
*
Author for correspondence: Melike Dinç, E-mail: melikedinc@iyte.edu.tr
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Abstract

Leishmaniasis is an infectious disease in which different clinical manifestations are classified into three primary forms: visceral, cutaneous and mucocutaneous. These disease forms are associated with parasite species of the protozoan genus Leishmania. For instance, Leishmania infantum and Leishmania tropica are typically linked with visceral (VL) and cutaneous (CL) leishmaniasis, respectively; however, these two species can also cause other form to a lesser extent. What is more alarming is this characteristic, which threatens current medical diagnosis and treatment, is started to be acquired by other species. Our purpose was to address this issue; therefore, gel-based and gel-free proteomic analyses were carried out on the species L. infantum to determine the proteins differentiating between the parasites caused VL and CL. In addition, L. tropica parasites representing the typical cases for CL were included. According to our results, electrophoresis gels of parasites caused to VL were distinguishable regarding the repetitive down-regulation on some specific locations. In addition, a distinct spot of an antioxidant enzyme, superoxide dismutase, was shown up only on the gels of CL samples regardless of the species. In the gel-free approach, 37 proteins that were verified with a second database search using a different search engine, were recognized from the comparison between VL and CL samples. Among them, 31 proteins for the CL group and six proteins for the VL group were determined differentially abundant. Two proteins from the gel-based analysis, pyruvate kinase and succinyl-coA:3-ketoacid-coenzyme A transferase analysis were encountered in the protein list of the CL group.

Keywords

2D-PAGEcutaneousleishmaniasisparasiteproteomicsshotgunvisceral
Type
Research Article
Information
Parasitology , Volume 149 , Issue 3 , March 2022 , pp. 298 - 305
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Copyright © The Author(s), 2021. Published by Cambridge University Press

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References

Akhoundi, M, Downing, T, Votýpka, J, Kuhls, K, Lukeš, J, Cannet, A, Ravel, C, Marty, P, Delaunay, P, Kasbari, M, Granouillac, B, Gradoni, L and Sereno, D (2017) Leishmania infections: molecular targets and diagnosis. Molecular Aspects of Medicine 57, 1–29.10.1016/j.mam.2016.11.012CrossRefGoogle ScholarPubMed
Akopyants, NS, Kimblin, N, Secundino, N, Patrick, R, Peters, N, Lawyer, P, Dobson, DE, Beverley, SM and Sacks, DL (2009) Demonstration of genetic exchange during cyclical development of Leishmania in the sand fly vector. Science (New York, N.Y.) 324, 265–268.10.1126/science.1169464CrossRefGoogle ScholarPubMed
Alawieh, A, Musharrafieh, U, Jaber, A, Berry, A, Ghosn, N and Bizri, AR (2014) Revisiting leishmaniasis in the time of war: the Syrian conflict and the Lebanese outbreak. International Journal of Infectious Diseases 29, 115–119.10.1016/j.ijid.2014.04.023CrossRefGoogle ScholarPubMed
Alves Souza, N, Souza Leite, R, de Oliveira Silva, S, Groenner Penna, M, Figueiredo Felicori Vilela, L, Melo, MN and de Andrade, ASR (2019) Detection of mixed Leishmania infections in dogs from an endemic area in southeastern Brazil. Acta Tropica 193, 12–17.10.1016/j.actatropica.2019.02.016CrossRefGoogle ScholarPubMed
Bardou, P, Mariette, J, Escudié, F, Djemiel, C and Klopp, C (2014) jvenn: an interactive Venn diagram viewer. BMC Bioinformatics 15, 293.10.1186/1471-2105-15-293CrossRefGoogle ScholarPubMed
Brosch, M, Yu, L, Hubbard, T and Choudhary, J (2009) Accurate and sensitive peptide identification with Mascot percolator. Journal of Proteome Research 8, 3176–3181.10.1021/pr800982sCrossRefGoogle ScholarPubMed
Calvopina, M, Gomez, EA, Uezato, Hi, Kato, H, Nonaka, S and Hashiguchi, Y (2005) Atypical clinical variants in new world cutaneous leishmaniasis: disseminated erysipeloid, and recidiva cutis due to Leishmania (V.) panamensis. The American Journal of Tropical Medicine and Hygiene 73, 281–284. doi: 10.4269/ajtmh.2005.73.281CrossRefGoogle ScholarPubMed
Celesia, BM, Cacopardo, B, Massimino, D, Gussio, M, Tosto, S, Nunnari, G and Pinzone, MR (2014) Atypical presentation of PKDL due to Leishmania infantum in an HIV-infected patient with relapsing visceral leishmaniasis. Case Reports in Infectious Diseases 2014, 370286.10.1155/2014/370286CrossRefGoogle Scholar
Chappuis, F, Sundar, S, Hailu, A, Ghalib, H, Rijal, S, Peeling, RW, Alvar, J and Boelaert, M (2007) Visceral leishmaniasis: what are the needs for diagnosis, treatment and control? Nature Reviews Microbiology 5, 873–882.10.1038/nrmicro1748CrossRefGoogle Scholar
Craig, R and Beavis, RC (2004) TANDEM: matching proteins with tandem mass spectra. Bioinformatics (Oxford, England) 20, 1466–1467.10.1093/bioinformatics/bth092CrossRefGoogle ScholarPubMed
Da-Cruz, AM, Filgueiras, DV, Coutinho, Z, Mayrink, W, Grimaldi, G Jr., De Luca, PM, Mendonça, SCF and Coutinho, SG (1999) Atypical mucocutaneous leishmaniasis caused by Leishmania braziliensis in an acquired immunodeficiency syndrome patient: T-cell responses and remission of lesions associated with antigen immunotherapy. Memórias do Instituto Oswaldo Cruz 94, 537–542.10.1590/S0074-02761999000400020CrossRefGoogle Scholar
Dai, Y, Whittal, RM and Li, L (1999) Two-layer sample preparation: a method for MALDI-MS analysis of complex peptide and protein mixtures. Analytical Chemistry 71, 1087–1091.10.1021/ac980684hCrossRefGoogle ScholarPubMed
Dereure, J, Vanwambeke, SO, Malé, P, Martinez, S, Pratlong, F, Balard, Y and Dedet, J-P (2009) The potential effects of global warming on changes in canine leishmaniasis in a focus outside the classical area of the disease in southern France. Vector Borne and Zoonotic Diseases (Larchmont, N.Y.) 9, 687–694.10.1089/vbz.2008.0126CrossRefGoogle Scholar
Dorfer, V, Pichler, P, Stranzl, T, Stadlmann, J, Taus, T, Winkler, S and Mechtler, K (2014) MS Amanda, a universal identification algorithm optimized for high accuracy tandem mass spectra. Journal of Proteome Research 13, 3679–3684.10.1021/pr500202eCrossRefGoogle ScholarPubMed
Elamin, EM, Guizani, I, Guerbouj, S, Gramiccia, M, El Hassan, AM, Di Muccio, T, Taha, MA and Mukhtar, MM (2008) Identification of Leishmania donovani as a cause of cutaneous leishmaniasis in Sudan. Transactions of the Royal Society of Tropical Medicine and Hygiene 102, 54–57.10.1016/j.trstmh.2007.10.005CrossRefGoogle ScholarPubMed
Emami, S, Tavangar, P and Keighobadi, M (2017) An overview of azoles targeting sterol 14α-demethylase for antileishmanial therapy. European Journal of Medicinal Chemistry 135, 241–259.10.1016/j.ejmech.2017.04.044CrossRefGoogle ScholarPubMed
Eng, JK, Jahan, TA and Hoopmann, MR (2013) Comet: an open-source MS/MS sequence database search tool. Proteomics 13, 22–2410.1002/pmic.201200439CrossRefGoogle ScholarPubMed
Espinoza-Morales, D, Lucchetti Rodríguez, A, Silva-Caso, W, Suarez-Ognio, L, Pons, MJ and del Valle Mendoza, J (2017) An atypical case of disseminated cutaneous leishmaniasis due to Leishmania peruviana in the valleys of Ancash-Peru. Asian Pacific Journal of Tropical Medicine 10, 1101–1103.10.1016/j.apjtm.2017.10.001CrossRefGoogle ScholarPubMed
Ferreira, E, Cruz, I, Cañavate, C, de Melo, LA, Pereira, AAS, Madeira, FAM, Valério, SAN, Cunha, HM, Paglia, AP and Gontijo, CMF (2015) Mixed infection of Leishmania infantum and Leishmania braziliensis in rodents from endemic urban area of the new world. BMC Veterinary Research 11, 71.10.1186/s12917-015-0392-yCrossRefGoogle Scholar
Garrido-Jareño, M, Sahuquillo-Torralba, A, Chouman-Arcas, R, Castro-Hernández, I, Molina-Moreno, JM, Llavador-Ros, M, Gómez-Ruiz, MD, López-Hontangas, JL, Botella-Estrada, R, Salavert-Lleti, M and Pemán-García, J (2020) Cutaneous and mucocutaneous leishmaniasis: experience of a Mediterranean hospital. Parasites and Vectors 13, 24.10.1186/s13071-020-3901-1CrossRefGoogle ScholarPubMed
González, C, Wang, O, Strutz, SE, González-Salazar, C, Sánchez-Cordero, V and Sarkar, S (2010) Climate change and risk of leishmaniasis in North America: predictions from ecological niche models of vector and reservoir species. PLoS Neglected Tropical Diseases 4, e585.10.1371/journal.pntd.0000585CrossRefGoogle ScholarPubMed
Gromova, I and Celis, JE (2006) Chapter 27 – Protein detection in gels by silver staining: a procedure compatible with mass spectrometry, 3rd Edn. In Celis, JEBT-CB (ed.), Burlington: Academic Press, pp. 219–223.Google Scholar
Guimarães, LH, Queiroz, A, Silva, JA, Silva, SC, Magalhães, V, Lago, EL, Machado, PRL, Bacellar, O, Wilson, ME, Beverley, SM, Carvalho, EM and Schriefer, A (2016) Atypical manifestations of cutaneous leishmaniasis in a region endemic for Leishmania braziliensis: clinical, immunological and parasitological aspects. PLoS Neglected Tropical Diseases 10, e0005100.10.1371/journal.pntd.0005100CrossRefGoogle Scholar
Gupta, N and Pevzner, PA (2009) False discovery rates of protein identifications: a strike against the two-peptide rule. Journal of Proteome Research 8, 4173–4181.10.1021/pr9004794CrossRefGoogle ScholarPubMed
Hajjaran, H, Mousavi, P, Burchmore, R, Mohebali, M, Mohammadi Bazargani, M, Salekdeh, GH, Kazemi-Rad, E and Khoramizadeh, MR (2015) Comparative proteomic profiling of Leishmania tropica: investigation of a case infected with simultaneous cutaneous and viscerotropic leishmaniasis by 2-dimensional electrophoresis and mass spectrometry. Iranian Journal of Parasitology 10, 366–380.Google Scholar
Hashemi, SA, Badirzadeh, A, Sabzevari, S, Nouri, A and Seyyedin, M (2018) First case report of atypical disseminated cutaneous leishmaniasis in an opium abuser in Iran. Revista do Instituto de Medicina Tropical de Säo Paulo 60, e5. https://doi.org/10.1590/S1678-9946201860005Google Scholar
Hide, M and Bañuls, A-L (2006) Species-specific PCR assay for L. infantum/L. donovani discrimination. Acta Tropica 100, 241–245.10.1016/j.actatropica.2006.10.012CrossRefGoogle ScholarPubMed
Ishihama, Y, Oda, Y, Tabata, T, Sato, T, Nagasu, T, Rappsilber, J and Mann, M (2005) Exponentially modified protein abundance Index (emPAI) for estimation of absolute protein amount in proteomics by the number of sequenced peptides per protein*S. Molecular & Cellular Proteomics 4, 1265–1272.10.1074/mcp.M500061-MCP200CrossRefGoogle Scholar
Karakuş, M, Çizmeci, Z, Karabela, ŞN, Erdoğan, B and Güleç, N (2019) The impact of refugees on leishmaniasis in Turkey: a new Syrian/Turkish Leishmania tropica population structure described by multilocus microsatellite typing (MLMT). Parasitology Research 118, 2679–2687.10.1007/s00436-019-06392-wCrossRefGoogle Scholar
Kim, S and Pevzner, PA (2014) MS-GF+ makes progress towards a universal database search tool for proteomics. Nature Communications 5, 5277.10.1038/ncomms6277CrossRefGoogle ScholarPubMed
King, KC, Stelkens, RB, Webster, JP, Smith, DF and Brockhurst, MA (2015) Hybridization in parasites: consequences for adaptive evolution, pathogenesis, and public health in a changing world. PLoS Pathogens 11, e1005098.10.1371/journal.ppat.1005098CrossRefGoogle Scholar
Kouyialis, S, Archontakis, S, Bilinis, C, Nikolaou, S, Stavropoulou, E, Samaras, C, Sarafoglou, C, Nicolaou, I, Parasi, A and Minadaki, M (2005) Report of an atypical case of leishmaniasis presented as acute tonsillitis in an immunocompetent patient. Scandinavian Journal of Infectious Diseases 37, 916–918.10.1080/00365540500262609CrossRefGoogle Scholar
Lambert, J-P, Ethier, M, Smith, JC and Figeys, D (2005) Proteomics: from gel based to gel free. Analytical Chemistry 77, 3771–3787.10.1021/ac050586dCrossRefGoogle ScholarPubMed
Lopes, L, Vasconcelos, P, Borges-Costa, J, Soares-Almeida, L, Campino, L and Filipe, P (2013) An atypical case of cutaneous leishmaniasis caused by Leishmania infantum in Portugal. Dermatology Online Journal 19, 20407.CrossRefGoogle ScholarPubMed
Lypaczewski, P and Matlashewski, G (2021) Leishmania donovani hybridisation and introgression in nature: a comparative genomic investigation. The Lancet Microbe 2, E250–E258. doi: 10.1016/S2666-5247(21)00028-8CrossRefGoogle Scholar
Martínez, DY, Verdonck, K, Kaye, PM, Adaui, V, Polman, K, Llanos-Cuentas, A, Dujardin, J-C and Boelaert, M (2018) Tegumentary leishmaniasis and coinfections other than HIV. PLoS Neglected Tropical Diseases 12, e0006125.CrossRefGoogle ScholarPubMed
Mebrahtu, YB, Van Eys, G, Guizani, I, Lawyer, PG, Pamba, H, Koech, D, Roberts, C, Perkins, PV, Were, JB and Hendricks, LD (1993) Human cutaneous leishmaniasis caused by Leishmania donovani s.l. in Kenya. Transactions of the Royal Society of Tropical Medicine and Hygiene 87, 598–601.10.1016/0035-9203(93)90101-UCrossRefGoogle ScholarPubMed
Meireles, CB, Maia, LC, Soares, GC, Teodoro, IPP, Gadelha, M, da Silva, CGL and de Lima, MAP (2017) Atypical presentations of cutaneous leishmaniasis: a systematic review. Acta Tropica 172, 240–254.CrossRefGoogle ScholarPubMed
Mule, SN, Saad, JS, Fernandes, LR, Stolf, BS, Cortez, M and Palmisano, G (2020) Protein glycosylation in Leishmania spp. Molecular Omics 16, 407–424.10.1039/D0MO00043DCrossRefGoogle ScholarPubMed
Mwenechanya, R, Kovářová, J, Dickens, NJ, Mudaliar, M, Herzyk, P, Vincent, IM, Weidt, SK, Burgess, KE, Burchmore, RJS, Pountain, AW, Smith, TK, Creek, DJ, Kim, D-H, Lepesheva, GI and Barrett, MP (2017) Sterol 14α-demethylase mutation leads to amphotericin B resistance in Leishmania mexicana. PLOS Neglected Tropical Diseases 11, e0005649.10.1371/journal.pntd.0005649CrossRefGoogle ScholarPubMed
NCBI Resource Coordinators (2017) Database resources of the National Center for Biotechnology Information. Nucleic Acids Research 45, D12–D17.10.1093/nar/gkw1071CrossRefGoogle Scholar
Negrão, F, Abánades, DR, Jaeeger, CF, Rocha, DFO, Belaz, KRA, Giorgio, S, Eberlin, MN and Angolini, CFF (2017) Lipidomic alterations of in vitro macrophage infection by L. infantum and L. amazonensis. Molecular BioSystems 13, 2401–2406.CrossRefGoogle ScholarPubMed
O'Farrell, PH (1975) High resolution two-dimensional electrophoresis of proteins. The Journal of Biological Chemistry 250, 4007–4021.10.1016/S0021-9258(19)41496-8CrossRefGoogle ScholarPubMed
Özbilgin, A, Çulha, G, Uzun, S, Harman, M, Topal, SG, Okudan, F, Zeyrek, F, Gündüz, C, Östan, İ, Karakuş, M, Töz, S, Kurt, Ö, Akyar, I, Erat, A, Güngör, D, Kayabaşı, Ç, Çavuş, İ, Bastien, P, Pratlong, F, Kocagöz, T and Özbel, Y (2016) Leishmaniasis in Turkey: first clinical isolation of Leishmania major from 18 autochthonous cases of cutaneous leishmaniasis in four geographical regions. Tropical Medicine & International Health 21, 783–791.10.1111/tmi.12698CrossRefGoogle ScholarPubMed
Özbilgin, A, Harman, M, Karakuş, M, Bart, A, Töz, S, Kurt, Ö, Çavuş, İ, Polat, E, Gündüz, C, Van Gool, T and Özbel, Y (2017) Leishmaniasis in Turkey: visceral and cutaneous leishmaniasis caused by Leishmania donovani in Turkey. Acta Tropica 173, 90–96.CrossRefGoogle ScholarPubMed
Perkins, DN, Pappin, DJ, Creasy, DM and Cottrell, JS (1999) Probability-based protein identification by searching sequence databases using mass spectrometry data. Electrophoresis 20, 3551–3567.10.1002/(SICI)1522-2683(19991201)20:18<3551::AID-ELPS3551>3.0.CO;2-23.0.CO;2-2>CrossRefGoogle ScholarPubMed
Powell, DW, Weaver, CM, Jennings, JL, McAfee, KJ, He, Y, Weil, PA and Link, AJ (2004) Cluster analysis of mass spectrometry data reveals a novel component of SAGA. Molecular and Cellular Biology 24, 7249–7259.CrossRefGoogle ScholarPubMed
Ready, PD (2010) Leishmaniasis emergence in Europe. Euro Surveillance: Bulletin Europeen sur les maladies transmissibles = European Communicable Disease Bulletin 15, 19505.Google ScholarPubMed
Romano, A, Inbar, E, Debrabant, A, Charmoy, M, Lawyer, P, Ribeiro-Gomes, F, Barhoumi, M, Grigg, M, Shaik, J, Dobson, D, Beverley, SM and Sacks, DL (2014) Cross-species genetic exchange between visceral and cutaneous strains of Leishmania in the sand fly vector. Proceedings of the National Academy of Sciences of the United States of America 111, 16808–16813.CrossRefGoogle ScholarPubMed
Shafi, MT, Bamra, T, Das, S, Kumar, A, Abhishek, K, Kumar, M, Kumar, V, Kumar, A, Mukherjee, R, Sen, A and Das, P (2021) Mevalonate kinase of Leishmania donovani protects parasite against oxidative stress by modulating ergosterol biosynthesis. Microbiological Research 251, 126837.CrossRefGoogle ScholarPubMed
Shevchenko, A, Tomas, H, Havlis, J, Olsen, JV and Mann, M (2006) In-gel digestion for mass spectrometric characterization of proteins and proteomes. Nature Protocols 1, 2856–2860.10.1038/nprot.2006.468CrossRefGoogle ScholarPubMed
Shinoda, K, Tomita, M and Ishihama, Y (2010) emPAI Calc – for the estimation of protein abundance from large-scale identification data by liquid chromatography-tandem mass spectrometry. Bioinformatics (Oxford, England) 26, 576–577.CrossRefGoogle ScholarPubMed
Shoulders, MD and Raines, RT (2009) Collagen structure and stability. Annual Review of Biochemistry 78, 929–958.CrossRefGoogle ScholarPubMed
Siriwardana, HVYD, Noyes, HA, Beeching, NJ, Chance, ML, Karunaweera, ND and Bates, PA (2007) Leishmania donovani and cutaneous leishmaniasis, Sri Lanka. Emerging Infectious Diseases 13, 476–478.CrossRefGoogle ScholarPubMed
Steverding, D (2017) The history of leishmaniasis. Parasites & Vectors 10, 82.CrossRefGoogle ScholarPubMed
Thakur, L, Singh, KK, Shanker, V, Negi, A, Jain, A, Matlashewski, G and Jain, M (2018) Atypical leishmaniasis: a global perspective with emphasis on the Indian subcontinent. PLoS Neglected Tropical Diseases 12, e0006659.CrossRefGoogle ScholarPubMed
The UniProt Consortium (2016) UniProt: the universal protein knowledgebase. Nucleic Acids Research 45, D158–D169.Google Scholar
Toz, SO, Culha, G, Zeyrek, FY, Ertabaklar, H, Alkan, MZ, Vardarlı, AT, Gunduz, C and Ozbel, Y (2013) A real-time ITS1-PCR based method in the diagnosis and species identification of Leishmania parasite from human and dog clinical samples in Turkey. PLoS Neglected Tropical Diseases 7, e2205.CrossRefGoogle ScholarPubMed
Tyanova, S, Temu, T, Sinitcyn, P, Carlson, A, Hein, MY, Geiger, T, Mann, M and Cox, J (2016) The Perseus computational platform for comprehensive analysis of (prote)omics data. Nature Methods 13, 731–740.CrossRefGoogle ScholarPubMed
Vaudel, M, Barsnes, H, Berven, FS, Sickmann, A and Martens, L (2011) SearchGUI: an open-source graphical user interface for simultaneous OMSSA and X!Tandem searches. Proteomics 11, 996–999.CrossRefGoogle ScholarPubMed
Vaudel, M, Burkhart, JM, Zahedi, RP, Oveland, E, Berven, FS, Sickmann, A, Martens, L and Barsnes, H (2015) PeptideShaker enables reanalysis of MS-derived proteomics data sets. Nature Biotechnology 33, 22–24.10.1038/nbt.3109CrossRefGoogle ScholarPubMed
VIB/UGent (2021) http://bioinformatics.psb.ugent.be/webtools/Venn/.Google Scholar
Vincent, IM, Racine, G, Légaré, D and Ouellette, M (2015) Mitochondrial proteomics of antimony and Miltefosine resistant Leishmania infantum. Proteomes 3, 328–346.CrossRefGoogle ScholarPubMed
Wang, Y, Yang, F, Gritsenko, MA, Wang, Y, Clauss, T, Liu, T, Shen, Y, Monroe, ME, Lopez-Ferrer, D, Reno, T, Moore, RJ, Klemke, RL, Camp, DG 2nd and Smith, RD (2011) Reversed-phase chromatography with multiple fraction concatenation strategy for proteome profiling of human MCF10A cells. Proteomics 11, 2019–2026.CrossRefGoogle ScholarPubMed
Wiśniewski, JR, Zougman, A, Nagaraj, N and Mann, M (2009) Universal sample preparation method for proteome analysis. Nature Methods 6, 359–362.CrossRefGoogle ScholarPubMed
Zhang, Y, Fonslow, BR, Shan, B, Baek, M-C and Yates, JR (2013) Protein analysis by shotgun/bottom-up proteomics. Chemical Reviews 113, 2343–2394.CrossRefGoogle ScholarPubMed
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