Project description:The trypanosomatid protozoan parasite Leishmania has a significant impact on human health globally. Understanding the pathways associated with virulence within this significant pathogen is critical for identifying novel vaccination and chemotherapy targets. Within this study we leverage an ultradeep proteomic approach to improve our understanding of two virulence associated genes in Leishmania; the Golgi Mannose/Arabinopyranose/Fucose nucleotide-sugar transporter LPG2, and the mitochondrial fucosyltransferase FUT1. Using deep peptide fractionation followed by complementary fragmentation approaches with higher energy collisional dissociation (HCD) and Electron-transfer dissociation (ETD) allowed the identification of over 6500 proteins, nearly doubling the experimentally observed Leishmania major proteome. This deep proteomic analysis revealed significant quantitative differences in both lpg2- and fut1s mutants with FUT1-dependent changes linked to marked alterations within mitochondrial associated proteins while LPG2-dependent changes impacted multiple aspects of the secretory pathway. While FUT1 has been shown to fucosylate peptides in vitro, no evidence for protein fucosylation was identified within our ultradeep analysis nor did we observe fucosylated glycans within Leishmania glycopeptides isolated using HILIC enrichment. Combined this work provides a critical proteomic resource for the community on the observable Leishmania proteome as well as highlights phenotypic changes associated with LPG2/FUT1 which may guide the development of future therapeutics.
Project description:The genomic DNAs of strains JPCM5 and 263 of L. infantum, strains LV39 and Friedlin of L. major and strains Parrot-TarII and S125 of L. tarentolae were used in comparative genomic hybridizations to reveal the intra-species and inter-species gene content, and to validate L. tarentolae Parrot-TarII genome sequencing results. Leishmania (Sauroleishmania) tarentolae was first isolated in the lizard Tarentola mauritanica. This species is not known to be pathogenic to humans but is often used as a model organism for molecular analyses or protein overproduction. The Leishmania tarentolae Parrot-TarII strain genome sequence was resolved by high-throughput sequencing technologies. The L. tarentolae genome was first assembled de novo and then aligned against the reference L. major Friedlin genome to facilitate contig positioning and annotation, providing a 23-fold coverage of the genome. This is the first non-pathogenic to humans kinetoplastid protozoan genome to be described, and it provides an opportunity for comparison with the completed genomes of the pathogenic Leishmania species. A high synteny was observed in de novo assembled contigs between all sequenced Leishmania species. A number of limited chromosomal regions diverged between L. tarentolae and L. infantum, while remaining syntenic with L. major. Globally, over 90% of the L. tarentolae gene content was shared with the other Leishmania species. There were 250 L. major genes absent from L. tarentolae, and interestingly these missing genes were primarily expressed in the intracellular amastigote stage of the pathogenic parasites. This implies that L. tarentolae may have impaired ability to survive as an intracellular parasite. In contrast to other Leishmania genomes, two gene families were expanded in L. tarentolae, namely the leishmanolysin (GP63) and a gene related to the promastigote surface antigen (PSA31C). Overall, L. tarentolae appears to have a gene content more adapted to the insect stage rather than the mammalian one. This may partly explain its inability to replicate within mammalian macrophages and its suspected preferred life style as promastigote in the lizards.