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

ABSTRACT: 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 (DDA) has been extensively used to understand the biochemical processes within the parasite. However, DDA is problematic for the detection of low abundance proteins, protein 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 (DIA) approach. The spectral library includes proteins expressed in the three morphologically distinct RBC stages (ring, trophozoite, schizont), the RBC compartment of trophozoite-iRBCs, and the cytosolic fractions from uninfected RBCs (uRBC). This spectral library contains 87% of P. falciparum proteins with previously blood-stage protein-level evidence as well as 692 previously unidentified proteins. The P. falciparum spectral library was successfully applied to generate semi-quantitative proteomics datasets that characterise the three distinct asexual parasite stages in RBCs, and compare artemisinin resistant (Cam3.IIR539T) and 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.

INSTRUMENT(S): Q Exactive HF, Q Exactive

ORGANISM(S): Plasmodium Falciparum Homo Sapiens (human)

TISSUE(S): Blood Cell, Blood

DISEASE(S): Malaria

SUBMITTER: Ghizal Siddiqui

LAB HEAD: Darren Creek

PROVIDER: PXD027241 | Pride | 2021-08-06

REPOSITORIES: Pride

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Project description:In eukaryotes, the chromatin architecture has a pivotal role in regulating all DNA-related processes. For P. falciparum, the causative agent of human malaria, the nucleosome landscape of the extremely AT-rich intergenic regulatory regions is largely unexplored. With the aid of a highly controlled MNase-seq procedure we reveal how positioning of nucleosomes provides a structural and regulatory framework to the transcriptional unit. We observe strong positioning of nucleosomes around splice sites that could aid co-transcriptional splicing events. In addition, nucleosome depleted regions are apparent hallmarks of transcription start sites (TSSs) and may support pre-initiation complex assembly. Moreover, we reveal nucleosome occupancy dynamics on strong TSSs during intraerythrocytic development, which correlate with gene expression changes and we observe a characteristic nucleosome architecture of functional - but not inert - TGCATGCA DNA motifs. Collectively, these findings highlight the regulatory capacity of the nucleosome landscape of this deadly human pathogen. Mnase-seq during the intra-erythrocytic asexual cycle of Plasmodium falciparum var2csa-panned 3D7 parasites for 8 time-points, every 5 hours starting from 5 hours post invasion until 40 hours post-invasion (T5-T40). Cycle length of these parasites is ~43 hours, synchronicity window is ~ 8 hours. T40 has 2 technical replicates (independent digestions; T40A, T40B). Additionally, pellet control sample (T15), histone H4-ChIP control (T40A) and sonicated and amplified genomic DNA. Chromatin was digested using a combined MNase + exonuclease III treatment. Libraries were prepared according to a Plasmodium-optimized library preparation procedure including KAPA polymerase-mediated PCR amplification. Strand-specific RNA-seq for expression quantification during the intra-erythrocytic asexual cycle of Plasmodium falciparum var2csa-panned 3D7 parasites for 8 time-points every 5 hours starting from 5 hours post invasion invasion until 40 hours post-invasion (T5-T40). Cycle length of these parasites is ~43 hours, synchronicity window is ~ 8 hours. These samples are originating from the exact same batch of parasites as are the MNase-Seq libraries. Libraries were prepared according to a Plasmodium-optimized library preparation procedure including KAPA polymerase-mediated PCR amplification.

Project description:The human malaria parasite Plasmodium falciparum has a complex and multi-stage life cycle that requires extensive immune escape, invasion of human liver and blood cells, and transmission through the female Anopholes mosquito. To date, the regulatory elements orchestrating these critical parasite processes remain largely unknown. However, there is mounting evidence across a broad range of species that intergenic long non-coding RNA (lncRNA) and antisense RNA can regulate chromatin state and gene expression. To pursue such functional roles for lncRNAs in P. falciparum, we performed deep, strand-specific RNA sequencing of fifteen non-polyA-selected blood stage samples, and assembled and characterized the properties of 660 intergenic lncRNAs, 474 antisense RNAs, and 1381 circular RNAs (circRNAs). We further validated the non-canonical splice junctions of seven P. falciparum circRNAs, an emerging class of non-coding RNA with regulatory potential and unexplored functional significance in P. falciparum. Our comprehensive analysis of P. falciparum lncRNAs indicates a functional role for these transcripts; P. falciparum intergenic lncRNAs and antisense RNAs are developmentally regulated in a similar periodic fashion to annotated transcripts, and sense-antisense pair expression is significantly anti-correlated. Notable outliers include intergenic lncRNAs that strongly peak in expression during parasite invasion, such as the telomere-associated lncRNA-TARE family, antisense transcripts that drop in expression during parasite invasion, and a highly correlated, multi-exonic, antisense counterpart to P. falciparum Gametocyte Developmental Protein 1 (PfGDV1). Taken together, our results present over two thousand P. falciparum intergenic lncRNA, antisense, and circRNA candidates and highlight promising P. falciparum lncRNAs for future investigation. We harvested fifteen blood stage samples from two biological replicate time-courses. The first time-course comprised of eleven samples that finely map temporal changes during P. falciparum blood stage development. We harvested samples over 56 hours, at roughly 4-hour time intervals, from a tightly synchronized P. falciparum 3D7 parasite population. As the asexual blood stage is an approximately 48-hour cycle, this time-course allowed us to profile gene expression during RBC rupture and parasite invasion. The second time-course comprised of four samples harvested in synchronous P. falciparum 3D7 parasites approximately four hours before and after the ring to trophozoite and trophozoite to schizont morphological stage transitions, which occur during the blood stage at 24 hours post invasion (hpi) and 36 hpi, respectively.

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

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