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

The 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 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 knownLeishmania majorproteome. This deep proteomic analysis revealed significant quantitative differences in both Δlpg2-and Δfut1smutants 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 peptides in 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.