TY - JOUR A1 - Engstler, Markus A1 - Beneke, Tom T1 - Gene editing and scalable functional genomic screening in Leishmania species using the CRISPR/Cas9 cytosine base editor toolbox LeishBASEedit JF - eLife N2 - CRISPR/Cas9 gene editing has revolutionised loss-of-function experiments in Leishmania, the causative agent of leishmaniasis. As Leishmania lack a functional non-homologous DNA end joining pathway however, obtaining null mutants typically requires additional donor DNA, selection of drug resistance-associated edits or time-consuming isolation of clones. Genome-wide loss-of-function screens across different conditions and across multiple Leishmania species are therefore unfeasible at present. Here, we report a CRISPR/Cas9 cytosine base editor (CBE) toolbox that overcomes these limitations. We employed CBEs in Leishmania to introduce STOP codons by converting cytosine into thymine and created http://www.leishbaseedit.net/ for CBE primer design in kinetoplastids. Through reporter assays and by targeting single- and multi-copy genes in L. mexicana, L. major, L. donovani, and L. infantum, we demonstrate how this tool can efficiently generate functional null mutants by expressing just one single-guide RNA, reaching up to 100% editing rate in non-clonal populations. We then generated a Leishmania-optimised CBE and successfully targeted an essential gene in a plasmid library delivered loss-of-function screen in L. mexicana. Since our method does not require DNA double-strand breaks, homologous recombination, donor DNA, or isolation of clones, we believe that this enables for the first time functional genetic screens in Leishmania via delivery of plasmid libraries. KW - CRISPR/Cas9 KW - Leishmania KW - cytosine base editor (CBE) toolbox KW - gene editing KW - scalable functional genomic screening KW - LeishBASEedit Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-350002 VL - 12 ER - TY - JOUR A1 - Cull, Benjamin A1 - Lima Prado Godinho, Joseane A1 - Fernandes Rodrigues, Juliany Cola A1 - Frank, Benjamin A1 - Schurigt, Uta A1 - Williams, Roderick AM A1 - Coombs, Graham H A1 - Mottram, Jeremy C T1 - Glycosome turnover in Leishmania major is mediated by autophagy JF - Autophagy N2 - Autophagy is a central process behind the cellular remodeling that occurs during differentiation of Leishmania, yet the cargo of the protozoan parasite's autophagosome is unknown. We have identified glycosomes, peroxisome-like organelles that uniquely compartmentalize glycolytic and other metabolic enzymes in Leishmania and other kinetoplastid parasitic protozoa, as autophagosome cargo. It has been proposed that the number of glycosomes and their content change during the Leishmania life cycle as a key adaptation to the different environments encountered. Quantification of RFP-SQL-labeled glycosomes showed that promastigotes of L. major possess ~20 glycosomes per cell, whereas amastigotes contain ~10. Glycosome numbers were significantly greater in promastigotes and amastigotes of autophagy-defective L. major Δatg5 mutants, implicating autophagy in glycosome homeostasis and providing a partial explanation for the previously observed growth and virulence defects of these mutants. Use of GFP-ATG8 to label autophagosomes showed glycosomes to be cargo in ~15% of them; glycosome-containing autophagosomes were trafficked to the lysosome for degradation. The number of autophagosomes increased 10-fold during differentiation, yet the percentage of glycosome-containing autophagosomes remained constant. This indicates that increased turnover of glycosomes was due to an overall increase in autophagy, rather than an upregulation of autophagosomes containing this cargo. Mitophagy of the single mitochondrion was not observed in L. major during normal growth or differentiation; however, mitochondrial remnants resulting from stress-induced fragmentation colocalized with autophagosomes and lysosomes, indicating that autophagy is used to recycle these damaged organelles. These data show that autophagy in Leishmania has a central role not only in maintaining cellular homeostasis and recycling damaged organelles but crucially in the adaptation to environmental change through the turnover of glycosomes. KW - ATG8 KW - Leishmania KW - TEM KW - glycosome KW - protozoan parasite KW - ATG KW - autophagy-related KW - GFP KW - green fluorescent protein KW - MVT KW - multivesicular tubule KW - RFP KW - red fluorescent protein KW - transmission electron microscopy KW - adaptation KW - autophagy KW - mC KW - mCherry KW - fluorescent protein Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-150277 VL - 10 IS - 12 ER - TY - JOUR A1 - Moll, Heidrun A1 - Röllinghoff, Martin T1 - T-cell reactivity to purified lipophosphoglycan from Leishmania major: A model for analysis of the cellular immune response to microbial carbohydrates. N2 - The major macromolecule on the surface o/Leishmania majorpromastigotes is a lipophosphoglycan (LPG). This glycoconjugate plays a key role in determining infectivity and survival of para-sites in the mammalian host cell. In addition, L. major LPG is able to induce a host-protective immune response. In this article, we summarise the evidence for recognition of highly purified LPG by T cells and we discuss the potential mechanisms of T-cell Stimulation by this non-protein antigen. KW - Leishmania KW - T lymphocytes KW - glycosyl phosphatidyl-inostitols. Y1 - 1991 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-33022 ER -