Leishmania is the unicellular parasite transmitted by phlebotomine sand fly bite. It exists in two different forms; extracellular promastigotes, occurring in the gut of sand flies, and intracellular, round-shaped amastigotes residing mainly in vertebrate macrophages. As amastigotes originating from infected animals are often present in insufficient quality and quantity, two alternative types of amastigotes were introduced for laboratory experiments: axenic amastigotes and amastigotes from macrophages infected in vitro. Nevertheless, there is very little information about the degree of similarity/difference among these three types of amastigotes on proteomic level, whose comparison is crucial for assessing the suitability of using alternative types of amastigotes in experiments. In this study, L. mexicana amastigotes obtained from lesion of infected BALB/c mice were proteomically compared with alternatively cultivated amastigotes (axenic and macrophage-derived ones). Amastigotes of all three types were isolated, individually treated and analysed by LC-MS/MS proteomic analysis with quantification using TMT10-plex isobaric labeling. Significant differences were observed in the abundance of metabolic enzymes, virulence factors and proteins involved in translation and condensation of DNA. The most pronounced differences were observed between axenic amastigotes and lesion-derived amastigotes, macrophage-derived amastigotes were mostly intermediate between axenic and lesion-derived ones.

Biotechnology and Biomedicine Centre of the Academy of Sciences and Charles University Vestec u Prahy Czechia

Department of Parasitology Charles University Prague Czechia

Proteomics Core Facility Faculty of Science Charles University Prague Czechia

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Ali K. S., Rees R. C., Terrell-Nield C., Ali S. A. (2013). Virulence loss and amastigote transformation failure determine host cell responses to l eishmania mexicana. Parasite. Immunol. 35 (12), 441–456. doi: 10.1111/pim.12056 PubMed DOI

Atayde V. D., Aslan H., Townsend S., Hassani K., Kamhawi S., Olivier M. (2015). Exosome secretion by the parasitic protozoan leishmania within the sand fly midgut. Cell Rep. 13 (5), 957–967. doi: 10.1016/j.celrep.2015.09.058 PubMed DOI PMC

Bates P. A. (1994). Complete developmental cycle of leishmania mexicana in axenic culture. Parasitology. 108 (1), 1–9. doi: 10.1017/s0031182000078458 PubMed DOI

Bates P. A., Robertson C. D., Tetley L., Coombs G. H. (1992). Axenic cultivation and characterization of leishmania mexicana amastigote-like forms. Parasitology. 105 (2), 193–202. doi: 10.1017/S0031182000074102 PubMed DOI

Bates P. A., Tetley L. (1993). Leishmania mexicana: induction of metacyclogenesis by cultivation of promastigotes at acidic pH. Exp. Parasitol. 76 (4), 412–423. doi: 10.1006/expr.1993.1050 PubMed DOI

Bellatin J. A., Murray A. S., Zhao M., McMaster W. R. (2002). Leishmania mexicana: identification of genes that are preferentially expressed in amastigotes. Exp. Parasitol. 100 (1), 44–53. doi: 10.1006/expr.2001.4677 PubMed DOI

Biyani N., Madhubala R. (2012). Quantitative proteomic profiling of the promastigotes and the intracellular amastigotes of leishmania donovani isolates identifies novel proteins having a role in leishmania differentiation and intracellular survival. Biochim. Biophys. Acta (BBA)-Proteins. Proteomics. 1824 (12), 1342–1350. doi: 10.1016/j.bbapap.2012.07.010 PubMed DOI

Bouvy C., Wannez A., Laloy J., Chatelain C., Dogné J.-M. (2017). Transfer of multidrug resistance among acute myeloid leukemia cells via extracellular vesicles and their microRNA cargo. Leuk. Res. 62, 70–76. doi: 10.1016/j.leukres.2017.09.014 PubMed DOI

Brittingham A., Chen G., McGwire B. S., Chang K.-P., Mosser D. M. (1999). Interaction of leishmania gp63 with cellular receptors for fibronectin. Infect. Immun. 67 (9), 4477–4484. doi: 10.1128/IAI.67.9.4477-4484.1999 PubMed DOI PMC

Brittingham A., Morrison C. J., McMaster W. R., McGwire B. S., Chang K.-P., Mosser D. M. (1995). Role of the leishmania surface protease gp63 in complement fixation, cell adhesion, and resistance to complement-mediated lysis. J. Immunol. 155 (6), 3102–3111. doi: 10.1016/0169-4758(95)80054-9 PubMed DOI

Burza S., Croft S. L., Boelaert M. (2018). Leishmaniasis. Lancet. 392 (10151), 951–970. doi: 10.1016/S0140-6736(18)31204-2 PubMed DOI

Buxbaum L. U., Denise H., Coombs G. H., Alexander J., Mottram J. C., Scott P. (2003). Cysteine protease b of leishmania mexicana inhibits host Th1 responses and protective immunity. J. Immunol. 171 (7), 3711–3717. doi: 10.4049/jimmunol.171.7.3711 PubMed DOI

Cameron P., McGachy A., Anderson M., Paul A., Coombs G. H., Mottram J. C., et al. . (2004). Inhibition of lipopolysaccharide-induced macrophage IL-12 production by leishmania mexicana amastigotes: the role of cysteine peptidases and the NF-κB signaling pathway. J. Immunol. 173 (5), 3297–3304. doi: 10.4049/jimmunol.173.5.3297 PubMed DOI

Casgrain P.-A., Martel C., McMaster W. R., Mottram J. C., Olivier M., Descoteaux A. (2016). Cysteine peptidase b regulates leishmania mexicana virulence through the modulation of GP63 expression. PloS Pathog. 12 (5), e1005658. doi: 10.1371/journal.ppat.1005658 PubMed DOI PMC

Chaudhuri G., Chang K.-P. (1988). Acid protease activity of a major surface membrane glycoprotein (gp63) from leishmania mexicana promastigotes. Mol. Biochem. Parasitol. 27 (1), 43–52. doi: 10.1016/0166-6851(88)90023-0 PubMed DOI

Chaudhuri G., Chaudhuri M., Pan A., Chang K. P. (1989). Surface acid proteinase (gp63) of leishmania mexicana: A metalloenzyme capable of protecting liposome-encapsulated proteins from phagolysosomal degradation by macrophages. J. Biol. Chem. 264 (13), 7483–7489. doi: 10.1016/S0021-9258(18)83260-4 PubMed DOI

Coakley G., McCaskill J. L., Borger J. G., Simbari F., Robertson E., Millar M., et al. . (2017). Extracellular vesicles from a helminth parasite suppress macrophage activation and constitute an effective vaccine for protective immunity. Cell Rep. 19 (8), 1545–1557. doi: 10.1016/j.celrep.2017.05.001 PubMed DOI PMC

Contreras I., Gómez M. A., Nguyen O., Shio M. T., McMaster R. W., Olivier M. (2010). Leishmania-induced inactivation of the macrophage transcription factor AP-1 is mediated by the parasite metalloprotease GP63. PloS Pathog. 6 (10), 1–14. doi: 10.1371/journal.ppat.1001148 PubMed DOI PMC

Coombs G. H., Craft J. A., Hart D. T. (1982). A comparative study of leishmania mexicana amastigotes and promastigotes, enzyme activities and subcellular locations. Mol. Biochem. Parasitol. 5 (3), 199–211. doi: 10.1016/0166-6851(82)90021-4 PubMed DOI

Cox J., Hein M. Y., Luber C. A., Paron I., Nagaraj N., Mann M. (2014). Accurate proteome-wide label-free quantification by delayed normalization and maximal peptide ratio extraction, termed MaxLFQ. Mol. Cell proteomics. 13 (9), 2513–2526. doi: 10.1074/mcp.M113.031591 PubMed DOI PMC

Da-Silva S. A. G., Costa S. S., Rossi-Bergmann B. (1999). The anti-leishmanial effect of kalanchoe is mediated by nitric oxide intermediates. Parasitology. 118 (6), 575–582. doi: 10.1017/S0031182099004357 PubMed DOI

Debrabant A., Joshi M. B., Pimenta P. F. P., Dwyer D. M. (2004). Generation of leishmania donovani axenic amastigotes: Their growth and biological characteristics. Int. J. Parasitol. 34 (2), 205–217. doi: 10.1016/j.ijpara.2003.10.011 PubMed DOI

Denise H., McNeil K., Brooks D. R., Alexander J., Coombs G. H., Mottram J. C. (2003). Expression of multiple CPB genes encoding cysteine proteases is required for leishmania mexicana virulence in vivo. Infect. Immun. 71 (6), 3190–3195. doi: 10.1128/IAI.71.6.3190-3195.2003 PubMed DOI PMC

De Pablos L. M., Ferreira T. R., Dowle A. A., Forrester S., Parry E., Newling K., et al. . (2019). The mRNA-bound proteome of leishmania mexicana: novel genetic insight into an ancient parasite. Mol. Cell Proteomics. 18 (7), 1271–1284. doi: 10.1074/mcp.RA118.001307 PubMed DOI PMC

de Paiva R. M. C., Grazielle-Silva V., Cardoso M. S., Nakagaki B. N., Mendonca-Neto R. P., Canavaci A. M. C., et al. . (2015). Amastin knockdown in leishmania braziliensis affects parasite-macrophage interaction and results in impaired viability of intracellular amastigotes. PloS Pathog. 11 (12), e1005296. doi: 10.1371/journal.ppat.1005296 PubMed DOI PMC

de Rezende E., Kawahara R., Peña M. S., Palmisano G., Stolf B. S. (2017). Quantitative proteomic analysis of amastigotes from leishmania (L.) amazonensis LV79 and PH8 strains reveals molecular traits associated with the virulence phenotype. PloS Negl. Trop. Dis. 11 (11), e0006090. doi: 10.1371/journal.pntd.0006090 PubMed DOI PMC

Dey R., Meneses C., Salotra P., Kamhawi S., Nakhasi H. L., Duncan R. (2010). Characterization of a leishmania stage-specific mitochondrial membrane protein that enhances the activity of cytochrome c oxidase and its role in virulence. Mol. Microbiol. 77 (2), 399–414. doi: 10.1111/j.1365-2958.2010.07214.x PubMed DOI PMC

Eperon S., Mcmahon-Pratt D. I. (1989). Extracellular cultivation and morphological characterization of amastigote-like forms of leishmania panamensis and l. braziliensis. J. Protozool. 36 (5), 502–510. doi: 10.1111/j.1550-7408.1989.tb01086.x PubMed DOI

Feng X., Feistel T., Buffalo C., McCormack A., Kruvand E., Rodriguez-Contreras D., et al. . (2011). Remodeling of protein and mRNA expression in leishmania mexicana induced by deletion of glucose transporter genes. Mol. Biochem. Parasitol. 175 (1), 39–48. doi: 10.1016/j.molbiopara.2010.08.008 PubMed DOI PMC

Fiebig M., Kelly S., Gluenz E. (2015). Comparative life cycle transcriptomics revises leishmania mexicana genome annotation and links a chromosome duplication with parasitism of vertebrates. PloS Pathog. 11 (10), e1005186. doi: 10.1371/journal.ppat.1005186 PubMed DOI PMC

Gannavaram S., Connelly P. S., Daniels M. P., Duncan R., Salotra P., Nakhasi H. L. (2012). Deletion of mitochondrial associated ubiquitin fold modifier protein Ufm1 in leishmania donovani results in loss of β-oxidation of fatty acids and blocks cell division in the amastigote stage. Mol. Microbiol. 86 (1), 187–198. doi: 10.1111/j.1365-2958.2012.08183.x PubMed DOI PMC

Gonen H., Dickman D., Schwartz A. L., Ciechanover A. (1996). “Protein synthesis elongation factor EF-1α is an isopeptidase essential for ubiquitin-dependent degradation of certain proteolytic substrates,” in Intracellular protein catabolism (Boston, MA: Springer; ), 209–219. PubMed

Hallé M., Gomez M. A., Stuible M., Shimizu H., McMaster W. R., Olivier M., et al. . (2009). The leishmania surface protease GP63 cleaves multiple intracellular proteins and actively participates in p38 mitogen-activated protein kinase inactivation. J. Biol. Chem. 284 (11), 6893–6908. doi: 10.1074/jbc.M805861200 PubMed DOI PMC

Hart D. T., Coombs G. H. (1982). Leishmania mexicana: Energy metabolism of amastigotes and promastigotes. Exp. Parasitol. 54 (3), 397–409. doi: 10.1016/0014-4894(82)90049-2 PubMed DOI

Holzer T. R., McMaster W. R., Forney J. D. (2006). Expression profiling by whole-genome interspecies microarray hybridization reveals differential gene expression in procyclic promastigotes, lesion-derived amastigotes, and axenic amastigotes in leishmania mexicana. Mol. Biochem. Parasitol. 146 (2), 198–218. doi: 10.1016/j.molbiopara.2005.12.009 PubMed DOI

Ishihama Y., Rappsilber J., Mann M. (2006). Modular stop and go extraction tips with stacked disks for parallel and multidimensional peptide fractionation in proteomics. J. Proteome Res. 5 (4), 988–994. doi: 10.1021/pr050385q PubMed DOI

Ivens A. C., Peacock C. S., Worthey E. A., Murphy L., Aggarwal G., Berriman M., et al. . (2005). The genome of the kinetoplastid parasite, leishmania major. Sci. (80-. ). 309 (5733), 436–442. doi: 10.1126/science.1112680 PubMed DOI PMC

Karamysheva Z. N., Gutierrez Guarnizo S. A., Karamyshev A. L. (2020). Regulation of translation in the protozoan parasite leishmania. Int. J. Mol. Sci. 21 (8), 2981. doi: 10.3390/ijms21082981 PubMed DOI PMC

Kulak N. A., Geyer P. E., Mann M. (2017). Loss-less nano-fractionator for high sensitivity, high coverage proteomics. Mol. Cell Proteomics. 16 (4), 694–705. doi: 10.1074/mcp.O116.065136 PubMed DOI PMC

Kulkarni M. M., McMaster W. R., Kamysz E., Kamysz W., Engman D. M., McGwire B. S. (2006). The major surface-metalloprotease of the parasitic protozoan, leishmania, protects against antimicrobial peptide-induced apoptotic killing. Mol. Microbiol 62 (5), 1484–97. doi: 10.1111/j.1365-2958.2006.05459.x PubMed DOI

Lahav T., Sivam D., Volpin H., Ronen M., Tsigankov P., Green A., et al. . (2011). Multiple levels of gene regulation mediate differentiation of the intracellular pathogen leishmania. FASEB J. 25 (2), 515–525. doi: 10.1096/fj.10-157529 PubMed DOI PMC

Lasse C., Azevedo C. S., de Araújo C. N., Motta F. N., Andrade M. A., Rocha A. P., et al. . (2020). Prolyl oligopeptidase from leishmania infantum: Biochemical characterization and involvement in macrophage infection. Front. Microbiol. 11, 1–12. doi: 10.3389/fmicb.2020.01060 PubMed DOI PMC

Leprohon P., Légaré D., Girard I., Papadopoulou B., Ouellette M. (2006). Modulation of leishmania ABC protein gene expression through life stages and among drug-resistant parasites. Eukaryot. Cell. 5 (10), 1713–1725. doi: 10.1128/EC.00152-06 PubMed DOI PMC

Loeuillet C., Bañuls A.-L., Hide M. (2016). Study of leishmania pathogenesis in mice: experimental considerations. Parasit. Vectors. 9 (1), 1–12. doi: 10.1186/s13071-016-1413-9 PubMed DOI PMC

Lynn M. A., Marr A. K., McMaster W. R. (2013). Differential quantitative proteomic profiling of leishmania infantum and leishmania mexicana density gradient separated membranous fractions. J. Proteomics. 82, 179–192. doi: 10.1016/j.jprot.2013.02.010 PubMed DOI

Maroli M., Feliciangeli M. D., Bichaud L., Charrel R. N., Gradoni L. (2013). Phlebotomine sandflies and the spreading of leishmaniases and other diseases of public health concern. Med. Vet. Entomol. 27 (2), 123–147. doi: 10.1111/j.1365-2915.2012.01034.x PubMed DOI

Masuda T., Tomita M., Ishihama Y. (2008). Phase transfer surfactant-aided trypsin digestion for membrane proteome analysis. J. Proteome Res. 7 (2), 731–740. doi: 10.1021/pr700658q PubMed DOI

Maxfield L., Crane J. S. (2019). Leishmaniasis. treasure island (FL) (Treasure island, FL: Stat Pearls Publishing; ).

McAlister G. C., Nusinow D. P., Jedrychowski M. P., Ẅhr M., Huttlin E. L., Erickson B. K., et al. . (2014). MultiNotch MS3 enables accurate, sensitive, and multiplexed detection of differential expression across cancer cell line proteomes. Anal. Chem. 86 (14), 7150–7158. doi: 10.1021/ac502040v PubMed DOI PMC

McConville M. J., Saunders E. C., Kloehn J., Dagley M. J. (2015). Leishmania carbon metabolism in the macrophage phagolysosome-feast or famine? F1000Research. 4 (F1000 Faculty Rev), 1–11. doi: 10.12688/f1000research.6724.1 PubMed DOI PMC

McGwire B. S., Chang K. P., Engman D. M. (2003). Migration through the extracellular matrix by the parasitic protozoan leishmania is enhanced by surface metalloprotease gp63. Infect. Immun 71(2), 1008–10. doi: 10.1128/IAI.71.2.1008-1010.2003 PubMed DOI PMC

Medina-Acosta E., Karess R. E., Schwartz H., Russell D. G. (1989). The promastigote surface protease (gp63) of leishmania is expressed but differentially processed and localized in the amastigote stage. Mol. Biochem. Parasitol. 37 (2), 263–273. doi: 10.1016/0166-6851(89)90158-8 PubMed DOI

Mottram J. C., Coombs G. H. (1985). Leishmania mexicana: enzyme activities of amastigotes and promastigotes and their inhibition by antimonials and arsenicals. Exp. Parasitol. 59 (2), 151–160. doi: 10.1016/0014-4894(85)90067-0 PubMed DOI

Mottram J. C., Robertson C. D., Coombs G. H., Barry J. D. (1992). A developmentally regulated cysteine proteinase gene of leishmania mexicana. Mol. Microbiol. 6 (14), 1925–1932. doi: 10.1111/j.1365-2958.1992.tb01365.x PubMed DOI

Murray A. S., Lynn M. A., McMaster W. R. (2010). The leishmania mexicana A600 genes are functionally required for amastigote replication. Mol. Biochem. Parasitol. 172 (2), 80–89. doi: 10.1016/j.molbiopara.2010.03.008 PubMed DOI

Naderer T., Ellis M. A., Sernee M. F., De Souza D. P., Curtis J., Handman E., et al. . (2006). Virulence of leishmania major in macrophages and mice requires the gluconeogenic enzyme fructose-1, 6-bisphosphatase. Proc. Natl. Acad. Sci. 103 (14), 5502–5507. doi: 10.1073/pnas.0509196103 PubMed DOI PMC

Naderer T., McConville M. J. (2008). The leishmania–macrophage interaction: a metabolic perspective. Cell Microbiol. 10 (2), 301–308. doi: 10.1111/j.1462-5822.2007.01096.x PubMed DOI

Nandan D., Yi T., Lopez M., Lai C., Reiner N. E. (2002). Leishmania EF-1$α$ activates the src homology 2 domain containing tyrosine phosphatase SHP-1 leading to macrophage deactivation. J. Biol. Chem. 277 (51), 50190–50197. doi: 10.1074/jbc.M209210200 PubMed DOI

Nugent P. G., Karsani S. A., Wait R., Tempero J., Smith D. F. (2004). Proteomic analysis of leishmania mexicana differentiation. Mol. Biochem. Parasitol. 136 (1), 51–62. doi: 10.1016/j.molbiopara.2004.02.009 PubMed DOI

Opperdoes F. R., Coombs G. H. (2007). Metabolism of leishmania: proven and predicted. Trends Parasitol. 23 (4), 149–158. doi: 10.1016/j.pt.2007.02.004 PubMed DOI

Paape D., Barrios-Llerena M. E., Le Bihan T., Mackay L., Aebischer T. (2010). Erratum to gel free analysis of the proteome of intracellular leishmania mexicana. Mol. Biochem. Parasitol. 169, 108–114. doi: 10.1016/j.molbiopara.2009.10.009 PubMed DOI

Paape D., Lippuner C., Schmid M., Ackermann R., Barrios-Llerena M. E., Zimny-Arndt U., et al. . (2008). Transgenic, fluorescent leishmania mexicana allow direct analysis of the proteome of intracellular amastigotes. Mol. Cell Proteomics. 7 (9), 1688–1701. doi: 10.1074/mcp.M700343-MCP200 PubMed DOI PMC

Pan A. A., Duboise S. M., Eperon S., Rivas L., Hodgkinson V., Traub-Cseko Y., et al. . (1993). Developmental life cycle of leishmania–cultivation and characterization of cultured extracellular amastigotes 1. J. Eukaryot. Microbiol. 40 (2), 213–223. doi: 10.1111/j.1550-7408.1993.tb04906.x PubMed DOI

Pang Z., Zhou G., Ewald J., Chang L., Hacariz O., Basu N., et al. . (2022). Using MetaboAnalyst 5.0 for LC–HRMS spectra processing, multi-omics integration and covariate adjustment of global metabolomics data. Nat. Protoc. 17 (8), 1735–1761. doi: 10.1038/s41596-022-00710-w PubMed DOI

Pan A. A., Pan S. C. (1986). Leishmania mexicana: comparative fine structure of amastigotes and promastigotes in vitro and in vivo. Exp. Parasitol. 62 (2), 254–265. doi: 10.1016/0014-4894(86)90030-5 PubMed DOI

Peacock C. S., Seeger K., Harris D., Murphy L., Ruiz J. C., Quail M. A., et al. . (2007). Comparative genomic analysis of three leishmania species that cause diverse human disease. Nat. Genet. 39 (7), 839–847. doi: 10.1038/ng2053 PubMed DOI PMC

Perez-Riverol Y., Csordas A., Bai J., Bernal-Llinares M., Hewapathirana S., Kundu D. J., et al. . (2019). The PRIDE database and related tools and resources in 2019: Improving support for quantification data. Nucleic Acids Res. 47 (D1), D442–D450. doi: 10.1093/nar/gky1106 PubMed DOI PMC

Pescher P., Blisnick T., Bastin P., Späth G. F. (2011). Quantitative proteome profiling informs on phenotypic traits that adapt leishmania donovani for axenic and intracellular proliferation. Cell Microbiol. 13 (7), 978–991. doi: 10.1111/j.1462-5822.2011.01593.x PubMed DOI

Piel L., Rajan K. S., Bussotti G., Varet H., Legendre R., Proux C., et al. . (2022). Experimental evolution links posttranscriptional regulation to leishmania fitness gain. PloS Pathog. 18 (3), 1–33. doi: 10.1371/journal.ppat.1010375 PubMed DOI PMC

Pral E. M. F., BIJOvSKY A. T., Balanco J. M. F., Alfieri S. C. (1993). Leishmania mexicana: Proteinase activities and megasomes in axenically cultivated amastigote-like forms. Exp. Parasitol. 77 (1), 62–73. doi: 10.1006/expr.1993.1061 PubMed DOI

Pruzinova K., Sadlova J., Myskova J., Lestinova T., Janda J., Volf P. (2018). Leishmania mortality in sand fly blood meal is not species-specific and does not result from direct effect of proteinases. Parasit. Vectors. 11 (1), 1–9. doi: 10.1186/s13071-018-2613-2 PubMed DOI PMC

Rodriguez-Contreras D., Hamilton N. (2014). Gluconeogenesis in leishmania mexicana: contribution of glycerol kinase, phosphoenolpyruvate carboxykinase, and pyruvate phosphate dikinase. J. Biol. Chem. 289 (47), 32989–33000. doi: 10.1074/jbc.M114.569434 PubMed DOI PMC

Rodríguez-Contreras D., Landfear S. M. (2006). Metabolic changes in glucose transporter-deficient leishmania mexicana and parasite virulence. J. Biol. Chem. 281 (29), 20068–20076. doi: 10.1074/jbc.M603265200 PubMed DOI

Rogers M. B., Hilley J. D., Dickens N. J., Wilkes J., Bates P. A., Depledge D. P., et al. . (2011). Chromosome and gene copy number variation allow major structural change between species and strains of leishmania. Genome Res. 21 (12), 2129–2142. doi: 10.1101/gr.122945.111 PubMed DOI PMC

Rosenzweig D., Smith D., Opperdoes F., Stern S., Olafson R. W., Zilberstein D. (2008). Retooling leishmania metabolism: from sand fly gut to human macrophage. FASEB J. 22 (2), 590–602. doi: 10.1096/fj.07-9254com PubMed DOI

Ruiz-Postigo J. A., Grout L. J. S. (2020). Global leishmaniasis surveillance 2017 – 2018, and first report on 5 additional indicators (Geneva: WHO; ), 265–280. Available at: https://www.who.int/wer/2020/wer9525/en/.

Russell D. G., Wilhelm H. (1986). The involvement of the major surface glycoprotein (gp63) of leishmania promastigotes in attachment to macrophages. J. Immunol. 136 (7), 2613–2620. PubMed

Sadlova J., Myskova J., Lestinova T., Votypka J., Yeo M., Volf P. (2017). Leishmania donovani development in phlebotomus argentipes: comparison of promastigote-and amastigote-initiated infections. Parasitology. 144 (4), 403–410. doi: 10.1017/S0031182016002067 PubMed DOI PMC

Saunders E. C., Ng W. W., Chambers J. M., Ng M., Naderer T., Krömer J. O., et al. . (2011). Isotopomer profiling of leishmania mexicana promastigotes reveals important roles for succinate fermentation and aspartate uptake in tricarboxylic acid cycle (TCA) anaplerosis, glutamate synthesis, and growth. J. Biol. Chem. 286 (31), 27706–27717. doi: 10.1074/jbc.M110.213553 PubMed DOI PMC

Saunders E. C., Ng W. W., Kloehn J., Chambers J. M., Ng M., McConville M. J. (2014). Induction of a stringent metabolic response in intracellular stages of leishmania mexicana leads to increased dependence on mitochondrial metabolism. PloS Pathog. 10 (1), e1003888. doi: 10.1371/journal.ppat.1003888 PubMed DOI PMC

Seay M. B., Heard P. L., Chaudhuri G. (1996). Surface zn-proteinase as a molecule for defense of leishmania mexicana amazonensis promastigotes against cytolysis inside macrophage phagolysosomes. Infect. Immun. 64 (12), 5129–5137. doi: 10.1128/iai.64.12.5129-5137.1996 PubMed DOI PMC

Silverman J. M., Clos J., de’Oliveira C. C., Shirvani O., Fang Y., Wang C., et al. . (2010). An exosome-based secretion pathway is responsible for protein export from leishmania and communication with macrophages. J. Cell Sci. 123 (6), 842–852. doi: 10.1242/jcs.056465 PubMed DOI

Siqueira-Neto J. L., Debnath A., McCall L.-I., Bernatchez J. A., Ndao M., Reed S. L., et al. . (2018). Cysteine proteases in protozoan parasites. PloS Negl. Trop. Dis. 12 (8), e0006512. doi: 10.1371/journal.pntd.0006512 PubMed DOI PMC

Souza A. E., Waugh S., Coombs G. H., Mottram J. C. (1992). Characterization of a multi-copy gene for a major stage-specific cysteine proteinase of leishmania mexicana. FEBS Lett. 311 (2), 124–127. doi: 10.1016/0014-5793(92)81382-V PubMed DOI

Steverding D. (2017). The history of leishmaniasis. Parasit Vectors 10 (82), 1–10. doi: 10.1186/s13071-017-2028-5 PubMed DOI PMC

Sundar S., Singh B. (2018). Emerging therapeutic targets for treatment of leishmaniasis. Expert Opin. Ther. Targets 22 (6), 467–486. doi: 10.1080/14728222.2018.1472241 PubMed DOI PMC

Thompson A., Schäfer J., Kuhn K., Kienle S., Schwarz J., Schmidt G., et al. . (2003). Tandem mass tags: a novel quantification strategy for comparative analysis of complex protein mixtures by MS/MS. Anal. Chem. 75 (8), 1895–1904. doi: 10.1021/ac0262560 PubMed DOI

Ueda-Nakamura T., Attias M., de Souza W. (2007). Comparative analysis of megasomes in members of the leishmania mexicana complex. Res. Microbiol. 158 (5), 456–462. doi: 10.1016/j.resmic.2007.03.003 PubMed DOI

Walker J., Vasquez J.-J., Gomez M. A., Drummelsmith J., Burchmore R., Girard I., et al. . (2006). Identification of developmentally-regulated proteins in leishmania panamensis by proteome profiling of promastigotes and axenic amastigotes. Mol. Biochem. Parasitol. 147 (1), 64–73. doi: 10.1016/j.molbiopara.2006.01.008 PubMed DOI

Williams R. A., Tetley L., Mottram J. C., Coombs G. H. (2006). Cysteine peptidases CPA and CPB are vital for autophagy and differentiation in leishmania mexicana. Mol. Microbiol. 61 (3), 655–674. doi: 10.1111/j.1365-2958.2006.05274.x PubMed DOI

Lipopolysaccharide pretreatment increases the sensitivity of the TRPV1 channel and promotes an anti-inflammatory phenotype of capsaicin-activated macrophages

Hide-and-Seek: A Game Played between Parasitic Protists and Their Hosts