Leishmania major is the main causative agent of cutaneous leishmaniasis in the Old World. In Leishmania parasites, the lack of transcriptional control is mostly compensated by post-transcriptional mechanisms. Methylation of arginine is a conserved post-translational modification executed by Protein Arginine Methyltransferase (PRMTs). The genome from L. major encodes five PRMT homologs, including the cytosolic protein associated with several RNA-binding proteins, LmjPRMT7. It has been previously reported that LmjPRMT7 could impact parasite infectivity. In addition, a more recent work has clearly shown the importance of LmjPRMT7 in RNA-binding capacity and protein stability of methylation targets, demonstrating the role of this enzyme as an important epigenetic regulator of mRNA metabolism. In this study, we unveil the impact of PRMT7-mediated methylation on parasite development and virulence. Our data reveals that higher levels of LmjPRMT7 can impair parasite pathogenicity, and that deletion of this enzyme rescues the pathogenic phenotype of an attenuated strain of L. major. Interestingly, lesion formation caused by LmjPRMT7 knockout parasites is associated with an exacerbated inflammatory reaction in the tissue correlated with an excessive neutrophil recruitment. Moreover, the absence of LmjPRMT7 also impairs parasite development within the sand fly vector Phlebotomus duboscqi. Finally, a transcriptome analysis shed light onto possible genes affected by depletion of this enzyme. Taken together, this study highlights how post-transcriptional regulation can affect different aspects of the parasite biology.

Department of Biology University of York York United Kingdom

Department of Cell and Molecular Biology Ribeirão Preto Medical School University of São Paulo Ribeirão Preto São Paulo Brazil

Department of Parasitology Charles University Prague Czech Republic

Laboratory of Parasitic Diseases National Institute of Allergy and Infectious Diseases National Institutes of Health Bethesda Maryland United States

Zobrazit více v PubMed

Georgiadou SP, Makaritsis KP, Dalekos GN. Leishmaniasis revisited: Current aspects on epidemiology, diagnosis and treatment. J Transl Intern Med. 2016. 10.1515/jtim-2015-0002 PubMed DOI PMC

Akhoundi M, Kuhls K, Cannet A, Votýpka J, Marty P, Delaunay P, et al.. A Historical Overview of the Classification, Evolution, and Dispersion of Leishmania Parasites and Sandflies. PLoS Neglected Tropical Diseases. 2016. 10.1371/journal.pntd.0004349 PubMed DOI PMC

Sacks D, Noben-Trauth N. The immunology of susceptibility and resistance to Leishmania major in mice. Nat Rev Immunol. 2002. 10.1038/nri933 PubMed DOI

Ready PD. Biology of Phlebotomine Sand Flies as Vectors of Disease Agents. Annu Rev Entomol. 2013. 10.1146/annurev-ento-120811-153557 PubMed DOI

Dostálová A, Volf P. Leishmania development in sand flies: Parasite-vector interactions overview. Parasites and Vectors. 2012. 10.1186/1756-3305-5-276 PubMed DOI PMC

Martínez-Calvillo S, Yan S, Nguyen D, Fox M, Stuart K, Myler PJ. Transcription of Leishmania major Friedlin chromosome 1 initiates in both directions within a single region. Mol Cell. 2003. 10.1016/s1097-2765(03)00143-6 PubMed DOI

El-Sayed NM, Myler PJ, Blandin G, Berriman M, Crabtree J, Aggarwal G, et al.. Comparative genomics of trypanosomatid parasitic protozoa. Science (80-). 2005. 10.1126/science.1112181 PubMed DOI

Clayton CE. Gene expression in Kinetoplastids. Curr Opin Microbiol. 2016. 10.1016/j.mib.2016.04.018 PubMed DOI

Karamysheva ZN, Guarnizo SAG, Karamyshev AL. Regulation of translation in the protozoan parasite leishmania. Int J Mol Sci. 2020. 10.3390/ijms21082981 PubMed DOI PMC

Guccione E, Richard S. The regulation, functions and clinical relevance of arginine methylation. Nat Rev Mol Cell Biol. 2019. 10.1038/s41580-019-0155-x PubMed DOI

Pahlich S, Zakaryan RP, Gehring H. Protein arginine methylation: Cellular functions and methods of analysis. Biochimica et Biophysica Acta—Proteins and Proteomics. 2006. 10.1016/j.bbapap.2006.08.008 PubMed DOI

Jarrold J, Davies CC. PRMTs and Arginine Methylation: Cancer’s Best-Kept Secret? Trends Mol Med. 2019. 10.1016/j.molmed.2019.05.007 PubMed DOI

Ferreira TR, Alves-Ferreira EVC, Defina TPA, Walrad P, Papadopoulou B, Cruz AK. Altered expression of an RBP-associated arginine methyltransferase 7 in Leishmania major affects parasite infection. Mol Microbiol. 2014. 10.1111/mmi.12819 PubMed DOI

Ferreira TR, Dowle AA, Parry E, Alves-Ferreira EVC, Hogg K, Kolokousi F, et al.. PRMT7 regulates RNA-binding capacity and protein stability in Leishmania parasites. Nucleic Acids Res. 2020. 10.1093/nar/gkaa211 PubMed DOI PMC

Späth GF, Beverley SM. A lipophosphoglycan-independent method for isolation of infective Leishmania metacyclic promastigotes by density gradient centrifugation. Exp Parasitol. 2001. 10.1006/expr.2001.4656 PubMed DOI

Volf P, Volfova V. Establishment and maintenance of sand fly olonies. J Vector Ecol. 2011. 10.1111/j.1948-7134.2011.00106.x PubMed DOI

Myskova J, Votypka J, Volf P. Leishmania in Sand Flies: Comparison of Quantitative Polymerase Chain Reaction with Other Techniques to Determine the Intensity of Infection. J Med Entomol. 2008. 10.1603/0022-2585(2008)45[133:lisfco]2.0.co;2 PubMed DOI

Belkaid Y, Mendez S, Lira R, Kadambi N, Milon G, Sacks D. A Natural Model of Leishmania major Infection Reveals a Prolonged “Silent” Phase of Parasite Amplification in the Skin Before the Onset of Lesion Formation and Immunity. J Immunol. 2000. 10.4049/jimmunol.165.2.969 PubMed DOI

Bolger AM, Lohse M, Usadel B. Trimmomatic: A flexible trimmer for Illumina sequence data. Bioinformatics. 2014;30:2114–20. 10.1093/bioinformatics/btu170 PubMed DOI PMC

Andrews S. FastQC: A Quality Control Tool for High Throughput Sequence Data [Online]. 2010.

Ewels P, Magnusson M, Lundin S, MultiQC KM. Summarize analysis results for multiple tools and samples in a single report. Bioinformatics. 2016;32:3047–8. 10.1093/bioinformatics/btw354 PubMed DOI PMC

Langmead B, Salzberg SL. Fast gapped-read alignment with Bowtie 2. Nat Methods. 2012;9:357–9. 10.1038/nmeth.1923 PubMed DOI PMC

Okonechnikov K, Conesa A, García-Alcalde F. Qualimap 2: Advanced multi-sample quality control for high-throughput sequencing data. Bioinformatics. 2015;32:292–4. 10.1093/bioinformatics/btv566 PubMed DOI PMC

Liao Y, Smyth GK, FeatureCounts SW. An efficient general purpose program for assigning sequence reads to genomic features. Bioinformatics. 2014;30:923–30. 10.1093/bioinformatics/btt656 PubMed DOI

Robinson MD, Oshlack A. A scaling normalization method for differential expression analysis of RNA-seq data. Genome Biol. 2010;11. 10.1186/gb-2010-11-3-r25 PubMed DOI PMC

Law CW, Chen Y, Shi W, Smyth GK. Voom: Precision weights unlock linear model analysis tools for RNA-seq read counts. Genome Biol. 2014;15:1–17. 10.1186/gb-2014-15-2-r29 PubMed DOI PMC

Ritchie ME, Phipson B, Wu D, Hu Y, Law CW, Shi W, et al.. limma powers differential expression analyses for RNA-sequencing and microarray studies. Nucleic Acids Res. 2015;43:e47. 10.1093/nar/gkv007 PubMed DOI PMC

Sádlová J, Price HP, Smith BA, Votỳpka J, Volf P, Smith DF. The stage-regulated HASPB and SHERP proteins are essential for differentiation of the protozoan parasite Leishmania major in its sand fly vector. Phlebotomus papatasi Cell Microbiol. 2010. 10.1111/j.1462-5822.2010.01507.x PubMed DOI PMC

Svárovská A, Ant TH, Seblová V, Jecná L, Beverley SM, Volf P. Leishmania major glycosylation mutants require phosphoglycans (lpg2 -) but not lipophosphoglycan (lpg1-) for survival in permissive sand fly vectors. PLoS Negl Trop Dis. 2010. 10.1371/journal.pntd.0000580 PubMed DOI PMC

Kraeva N, Leštinová T, Ishemgulova A, Majerová K, Butenko A, Vaselek S, et al.. LmxM.22.0250-Encoded Dual Specificity Protein/Lipid Phosphatase Impairs Leishmania mexicana Virulence In Vitro. Pathogens. 2019;8:241. 10.3390/pathogens8040241 PubMed DOI PMC

Sunter JD, Yanase R, Wang Z, Catta-Preta CMC, Moreira-Leite F, Myskova J, et al.. Leishmania flagellum attachment zone is critical for flagellar pocket shape, development in the sand fly, and pathogenicity in the host. Proc Natl Acad Sci U S A. 2019. 10.1073/pnas.1812462116 PubMed DOI PMC

Sádlová J, Svobodová M, Volf P. Leishmania major: Effect of repeated passages through sandfly vectors or murine hosts. Ann Trop Med Parasitol. 1999. PubMed

Lima-Junior DS, Costa DL, Carregaro V, Cunha LD, Silva ALN, Mineo TWP, et al.. Inflammasome-derived IL-1β production induces nitric oxide-mediated resistance to Leishmania. Nat Med. 2013. 10.1038/nm.3221 PubMed DOI

Chaves MM, Sinflorio DA, Thorstenberg ML, Martins MDA, Moreira-Souza ACA, Rangel TP, et al.. Non-canonical NLRP3 inflammasome activation and il-1β signaling are necessary to L. Amazonensis control mediated by P2x7 receptor and leukotriene B4. PLoS Pathog. 2019. 10.1371/journal.ppat.1007887 PubMed DOI PMC

Toledo M. dos S, Cronemberger-Andrade A, Barbosa FMC, Reis NF de C, Dupin TV, Soares RP, et al.. Effects of extracellular vesicles released by peritoneal B-1 cells on experimental Leishmania (Leishmania) amazonensis infection. J Leukoc Biol n/a. 10.1002/JLB.3MA0220-464RR PubMed DOI

Zhang WW, Karmakar S, Gannavaram S, Dey R, Lypaczewski P, Ismail N, et al.. A second generation leishmanization vaccine with a markerless attenuated Leishmania major strain using CRISPR gene editing. Nat Commun. 2020. 10.1038/s41467-020-17154-z PubMed DOI PMC

Mehravaran A, Nasab MR, Mirahmadi H, Sharifi I, Alijani E, Nikpoor AR, et al.. Protection induced by Leishmania Major antigens and the imiquimod adjuvant encapsulated on liposomes in experimental cutaneous leishmaniasis. Infect Genet Evol. 2019. 10.1016/j.meegid.2019.01.005 PubMed DOI

van Zandbergen G, Klinger M, Mueller A, Dannenberg S, Gebert A, Solbach W, et al.. Cutting Edge: Neutrophil Granulocyte Serves as a Vector for Leishmania Entry into Macrophages. J Immunol. 2004. 10.4049/jimmunol.173.11.6521 PubMed DOI

Ribeiro-Gomes FL, Peters NC, Debrabant A, Sacks DL. Efficient capture of infected neutrophils by dendritic cells in the skin inhibits the early anti-leishmania response. PLoS Pathog. 2012. 10.1371/journal.ppat.1002536 PubMed DOI PMC

Peters NC, Egen JG, Secundino N, Debrabant A, Kimblin N, Kamhawi S, et al.. In vivo imaging reveals an essential role for neutrophils in leishmaniasis transmitted by sand flies. Science (80-). 2008. PubMed PMC

Thalhofer CJ, Chen Y, Sudan B, Love-Homan L, Wilson ME. Leukocytes infiltrate the skin and draining lymph nodes in response to the protozoan leishmania infantum chagasi. Infect Immun. 2011. 10.1128/IAI.00338-10 PubMed DOI PMC

Peniche AG, Bonilla DL, Palma GI, Melby PC, Travi BL, Osorio EY. A secondary wave of neutrophil infiltration causes necrosis and ulceration in lesions of experimental American cutaneous leishmaniasis. PLoS One. 2017. 10.1371/journal.pone.0179084 PubMed DOI PMC

Lopez Kostka S, Dinges S, Griewank K, Iwakura Y, Udey MC, von Stebut E. IL-17 Promotes Progression of Cutaneous Leishmaniasis in Susceptible Mice. J Immunol 2009. 10.4049/jimmunol.0713598 PubMed DOI PMC

Carlsen ED, Liang Y, Shelite TR, Walker DH, Melby PC, Soong L. Permissive and protective roles for neutrophils in leishmaniasis. Clin Exp Immunol. 2015. 10.1111/cei.12674 PubMed DOI PMC

Huang S, Litt M, Felsenfeld G. Methylation of histone H4 by arginine methyltransferase PRMT1 is essential in vivo for many subsequent histone modifications. Genes Dev. 2005. 10.1101/gad.1333905 PubMed DOI PMC

Bisset R. Lipoic acid metabolism in Leishmania major. University of Glasgow 2009. http://theses.gla.ac.uk/id/eprint/1264.

O’Riordan M, Moors MA, Portnoy DA. Listeria Intracellular Growth and Virulence Require Host-Derived Lipoic Acid. Science (80-). 2003. 10.1126/science.1088170 PubMed DOI

Zhao X, Miller JR, Cronan JE. The reaction of LipB, the octanoyl-[Acyl carrier protein]:Protein N-octanoyltransferase of lipoic acid synthesis, proceeds through an acyl-enzyme intermediate. Biochemistry. 2005. PubMed

Ma Q, Zhao X, Eddine AN, Geerlof A, Li X, Cronan JE, et al.. The Mycobacterium tuberculosis LipB enzyme functions as a cysteine/lysine dyad acyltransferase. Proc Natl Acad Sci U S A. 2006. 10.1073/pnas.0510436103 PubMed DOI PMC

Zhang G, Dai J, Lu Z, Dunaway-Mariano D. The Phosphonopyruvate Decarboxylase from Bacteroides fragilis. J Biol Chem. 2003. 10.1074/jbc.M305976200 PubMed DOI

Pallitsch K, Rogers MP, Andrews FH, Hammerschmidt F, McLeish MJ. Phosphonodifluoropyruvate is a mechanism-based inhibitor of phosphonopyruvate decarboxylase from Bacteroides fragilis. Bioorganic Med Chem. 2017. 10.1016/j.bmc.2017.06.013 PubMed DOI

Berriman M, Ghedin E, Hertz-Fowler C, Blandin G, Renauld H, Bartholomeu DC, et al.. The genome of the African trypanosome Trypanosoma brucei. Science (80-). 2005. 10.1126/science.1112642 PubMed DOI

Rastrojo A, Corvo L, Lombraña R, Solana JC, Aguado B, Requena JM. Analysis by RNA-seq of transcriptomic changes elicited by heat shock in Leishmania major. Sci Rep. 2019. 10.1038/s41598-019-43354-9 PubMed DOI PMC