A new mass spectral library for high-coverage and reproducible analysis of the Plasmodium falciparum-infected red blood cell proteome.

BACKGROUND: Plasmodium falciparum causes the majority of malaria mortality worldwide, and the disease occurs during the asexual red blood cell (RBC) stage of infection. In the absence of an effective and available vaccine, and with increasing drug resistance, asexual RBC stage parasites are an important research focus. In recent years, mass spectrometry-based proteomics using data-dependent acquisition has been extensively used to understand the biochemical processes within the parasite. However, data-dependent acquisition is problematic for the detection of low-abundance proteins and proteome coverage and has poor run-to-run reproducibility. RESULTS: Here, we present a comprehensive P. falciparum-infected RBC (iRBC) spectral library to measure the abundance of 44,449 peptides from 3,113 P. falciparum and 1,617 RBC proteins using a data-independent acquisition mass spectrometric approach. The spectral library includes proteins expressed in the 3 morphologically distinct RBC stages (ring, trophozoite, schizont), the RBC compartment of trophozoite-iRBCs, and the cytosolic fraction from uninfected RBCs. This spectral library contains 87% of all P. falciparum proteins that have previously been reported with protein-level evidence in blood stages, as well as 692 previously unidentified proteins. The P. falciparum spectral library was successfully applied to generate semi-quantitative proteomics datasets that characterize the 3 distinct asexual parasite stages in RBCs, and compared artemisinin-resistant (Cam3.IIR539T) and artemisinin-sensitive (Cam3.IIrev) parasites. CONCLUSION: A reproducible, high-coverage proteomics spectral library and analysis method has been generated for investigating sets of proteins expressed in the iRBC stage of P. falciparum malaria. This will provide a foundation for an improved understanding of parasite biology, pathogenesis, drug mechanisms, and vaccine candidate discovery for malaria.

Post-Genomic Approaches to Understanding Malaria Parasite Biology: Linking Genes to Biological Functions.

Plasmodium species are evolutionarily distant from model eukaryotes, and as a consequence they exhibit many non-canonical cellular processes. In the post-genomic era, functional "omics" disciplines (transcriptomics, proteomics, and metabolomics) have accelerated our understanding of unique aspects of the biology of malaria parasites. Functional "omics" tools, in combination with genetic manipulations, have offered new opportunities to investigate the function of previously uncharacterized genes. Knowledge of basic parasite biology is fundamental to understanding drug modes of action, mechanisms of drug resistance, and relevance of vaccine candidates. This Perspective highlights recent "omics"-based discoveries in basic biology and gene function of the most virulent human malaria parasite, Plasmodium falciparum.

Mutations in the pantothenate kinase of Plasmodium falciparum confer diverse sensitivity profiles to antiplasmodial pantothenate analogues.

The malaria-causing blood stage of Plasmodium falciparum requires extracellular pantothenate for proliferation. The parasite converts pantothenate into coenzyme A (CoA) via five enzymes, the first being a pantothenate kinase (PfPanK). Multiple antiplasmodial pantothenate analogues, including pantothenol and CJ-15,801, kill the parasite by targeting CoA biosynthesis/utilisation. Their mechanism of action, however, remains unknown. Here, we show that parasites pressured with pantothenol or CJ-15,801 become resistant to these analogues. Whole-genome sequencing revealed mutations in one of two putative PanK genes (Pfpank1) in each resistant line. These mutations significantly alter PfPanK activity, with two conferring a fitness cost, consistent with Pfpank1 coding for a functional PanK that is essential for normal growth. The mutants exhibit a different sensitivity profile to recently-described, potent, antiplasmodial pantothenate analogues, with one line being hypersensitive. We provide evidence consistent with different pantothenate analogue classes having different mechanisms of action: some inhibit CoA biosynthesis while others inhibit CoA-utilising enzymes.

A high parasite density environment induces transcriptional changes and cell death in Plasmodium falciparum blood stages.

Transient regulation of Plasmodium numbers below the density that induces fever has been observed in chronic malaria infections in humans. This species transcending control cannot be explained by immunity alone. Using an in vitro system we have observed density dependent regulation of malaria population size as a mechanism to possibly explain these in vivo observations. Specifically, Plasmodium falciparum blood stages from a high but not low-density environment exhibited unique phenotypic changes during the late trophozoite (LT) and schizont stages of the intraerythrocytic cycle. These included in order of appearance: failure of schizonts to mature and merozoites to replicate, apoptotic-like morphological changes including shrinking, loss of mitochondrial membrane potential, and blebbing with eventual release of aberrant parasites from infected erythrocytes. This unique death phenotype was triggered in a stage-specific manner by sensing of a high-density culture environment. Conditions of glucose starvation, nutrient depletion, and high lactate could not induce the phenotype. A high-density culture environment induced rapid global changes in the parasite transcriptome including differential expression of genes involved in cell remodeling, clonal antigenic variation, metabolism, and cell death pathways including an apoptosis-associated metacaspase gene. This transcriptional profile was also characterized by concomitant expression of asexual and sexual stage-specific genes. The data show strong evidence to support our hypothesis that density sensing exists in P. falciparum. They indicate that an apoptotic-like mechanism may play a role in P. falciparum density regulation, which, as in yeast, has features quite distinguishable from mammalian apoptosis. DATABASE: Gene expression data are available in the GEO databases under the accession number GSE91188.

Metabolomics-Based Elucidation of Active Metabolic Pathways in Erythrocytes and HSC-Derived Reticulocytes.

A detailed analysis of the metabolic state of human-stem-cell-derived erythrocytes allowed us to characterize the existence of active metabolic pathways in younger reticulocytes and compare them to mature erythrocytes. Using high-resolution LC-MS-based untargeted metabolomics, we found that reticulocytes had a comparatively much richer repertoire of metabolites, which spanned a range of metabolite classes. An untargeted metabolomics analysis using stable-isotope-labeled glucose showed that only glycolysis and the pentose phosphate pathway actively contributed to the biosynthesis of metabolites in erythrocytes, and these pathways were upregulated in reticulocytes. Most metabolite species found to be enriched in reticulocytes were residual pools of metabolites produced by earlier erythropoietic processes, and their systematic depletion in mature erythrocytes aligns with the simplification process, which is also seen at the cellular and the structural level. Our work shows that high-resolution LC-MS-based untargeted metabolomics provides a global coverage of the biochemical species that are present in erythrocytes. However, the incorporation of stable isotope labeling provides a more accurate description of the active metabolic processes that occur in each developmental stage. To our knowledge, this is the first detailed characterization of the active metabolic pathways of the erythroid lineage, and it provides a rich database for understanding the physiology of the maturation of reticulocytes into mature erythrocytes.