Expanded Proteomic Survey of the Human Parasite Leishmania major Focusing on Changes in Null Mutants of the Golgi GDP-Mannose/Fucose/Arabinopyranose Transporter LPG2 and of the Mitochondrial Fucosyltransferase FUT1

Abstract

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, 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 6,500 proteins, nearly doubling the experimentally known Leishmania major proteome. This deep proteomic analysis revealed significant quantitative differences in both Δlpg2^- and Δfut1^s mutants with FUT1-dependent changes linked to marked alterations within mitochondrion-associated proteins, while LPG2-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 within Leishmania glycopeptides isolated using hydrophilic interaction liquid chromatography (HILIC) enrichment. This work provides a critical resource for the community on the observable Leishmania proteome as well as highlighting phenotypic changes associated with LPG2 or FUT1, ablation of which may guide the development of future therapeutics. IMPORTANCE Leishmania is a widespread trypanosomatid protozoan parasite of humans, with ~12 million cases currently, 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 6,500 proteins and thus provides a great resource to investigators seeking to decode how this parasite is transmitted and causes disease and to identify new targets for therapeutic intervention. The ultradeep proteomics approach identified potential proteins underlying the "persistence-without-pathology" phenotype of mutants with deletion of the Golgi nucleotide transporter LPG2, showing many alterations and several candidates. Studies of a rare mutant with deletion 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 that this vital protein's elusive target(s) may be more complex than the methods used could detect or that this target may not be a protein but perhaps another glycoconjugate or glycolipid.

Keywords: N-linked glycans; N-linked glycoconjugates; fucose; glycoproteome; kinetoplast; mitochondria; trypanosomatid protozoan parasite; ultradeep proteomics.

FIG 1

Overview of glycosylation pathways relevant to LPG2 or FUT1 biology and deletion mutants. LPG2 encodes is a Golgi GDP-sugar transporter, essential for LPG synthesis. Mutant Δlpg2^– parasites grow comparably to WT parasites in culture but do not produce pathology. FUT1 encodes a fucosyltransferase located in the parasite mitochondria, whose substrates remain unknown. Null-mutant Δfut1^s parasites exhibit severe defects, including significantly decreased growth rate, abnormal mitochondrial morphology, decreased mitochondrial membrane potential (Ψ), and loss of kDNA compactness or the kDNA network altogether. Arap, arabinopyranose (green star); Man, mannose (green circle); Fuc, fucose (red triangle); LPG, lipophosphoglycan. This figure was created with BioRender.com.

FIG 2

(A) Comparison of proteins identified in this work with previous studies. The combined data set (Table S1) from all lines and replicas was compared to L. major proteins with MS-based evidence annotated in TriTrypDB (https://tritrypdb.org/tritrypdb/app). Of the proteins identified here, 3,484 showed previous MS evidence and 3,260 had no prior MS-based reports; 308 proteins with previous MS-based evidence did not appear in our data sets. (B) Distribution of identified proteins among WT, Δfut1^s, and Δlpg2^– L. major. The total proteome (Table S1) was further parsed by considering only proteins identified in at least two of the four biological replicates, in one or more parasite lines, yielding a total of 5,841 (Table S3). The Venn diagram displays the overlap of proteins among the three lines.

FIG 3

Representation of the predicted L. major mitochondrial proteome. Proteins from WT or mutant parasite lines (Table S3) were compared to proteins in MiNT (predicted nuclearly encoded mitochondrial protein), supplemented with maxicircle (mitochondrion)-encoded proteins as described in Materials and Methods. (A) A total of 1,268 proteins, representing 81% of the mitochondrial proteome, were identified in this study. (B) Distribution of mitochondrial proteins by cell line.

FIG 4

Patterns of variation of significantly differentially expressed proteins among WT and FUT1 and LPG2 knockout L. major lines. ANOVA using an FDR of 0.05 and an S0 of 1 was performed on LFQ values for the high-confidence proteome (Table S3), to identify proteins that are significantly different between WT, Δfut1^s, and Δlpg2^– clones. From these data, a heat map was generated, clustering the parasite lines, each with four replicas (top dendrogram), and the differentially expressed genes (left dendrogram). The cluster properties are summarized on the right, and the specific proteins included in each cluster can be found in Table S5 and S6.

FIG 5

Schematic overview of a Leishmania mitochondrion showing proteins with significantly altered expression in Δfut1^s relative to WT L. major. The specific proteins are listed in Table 1. Only mitochondrial proteins showing differential expression (Table 2) are shown. In the panel depicting the electron transport chain, the proteins decreased in the Δfut1^s parasites are shown in blue, those not detected in our data set are shown in white, and those that were unchanged are shown in gray. It should be noted that trypanosomatids possess a single mitochondrion per cell whose structure differs from the typical metazoan mitochondrion shape depicted here. This figure was created with BioRender.com.