An expanded proteomic survey of the human parasiteLeishmania majorfocusing on changes in null mutants of the Golgi GDP-Mannose/Fucose/ArabinopyranosetransporterLPG2or the mitochondrial fucosyltransferaseFUT1

Abstract

ABSTRACTThe trypanosomatid protozoan parasiteLeishmaniahas 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 inLeishmania, encoding the Golgi Mannose/Arabinopyranose/Fucose nucleotide-sugar transporterLPG2, and the mitochondrial fucosyltransferaseFUT1. 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 knownLeishmania majorproteome. This deep proteomic analysis revealed significant quantitative differences in bothΔlpg2-andΔfut1smutants withFUT1-dependent changes linked to marked alterations within mitochondrial associated proteins whileLPG2-dependent changes impacted many pathways including the secretory pathway. While the FUT1 enzyme has been shown to fucosylate peptidesin vitro, no evidence for protein fucosylation was identified within our ultradeep analysis nor did we observe fucosylated glycans withinLeishmaniaglycopeptides isolated using HILIC enrichment. Combined this work provides a critical resource for the community on the observableLeishmaniaproteome as well as highlights phenotypic changes associated withLPG2orFUT1ablation which may guide the development of future therapeutics.ImportanceLeishmaniais a widespread trypanosomatid protozoan parasite of humans with ∼12 million cases ranging from mild to fatal, and hundreds of millions asymptomatically infected. This work advances knowledge of the experimental proteome by nearly 2 fold, to more than 6500 proteins a great resource to investigators seeking to decode how this parasite is transmitted and causes disease, and new targets for therapeutic intervention. The ultradeep proteomics approach identified potential proteins underlying the ‘persistence without pathology’ phenotype of deletion mutants of the Golgi nucleotide transporter LPG2, showing many alterations and several candidates. Studies of a rare deletion mutant of the mitochondrial fucosyltransferase FUT1 revealed changes underlying its strong mitochondrial dysfunction, but did not reveal examples of fucosylation of either peptides or N-glycans. This suggests this vital protein’s elusive target(s) may be more complex than the methods used could detect, or may not be a protein, perhaps another glycoconjugate or glycolipid.