Complete quantitative histone PTM landscape of asexual and sexual P falciparum parasites

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Parasite production and isolation

P. falciparum 3D7 (drug sensitive) and P. falciparum NF54-PfS16-GFP-Luc (kindly provided by the Fidock lab, Columbia University, USA) [244] parasite cultures were maintained at 5% haematocrit in O+ human erythrocytes and RPMI-1640 cell culture medium supplemented with 24 mM sodium bicarbonate, 0.024 mg/ml gentamicin, 25 mM HEPES, 0.2% v/v glucose, 0.2 mM hypoxanthine and 0.5% w/v AlbuMAX II Lipid Rich Bovine Serum Albumin. The parasite cultures were kept at 37ºC with moderate shaking at 60 rpm and gassed with a mixture of 5% O2, 5% CO2 and 90% N2 [245]. Synchronous asexual parasites (>90%, Pf3D7) were obtained using 10% w/v D-sorbitol [246] and isolated as ring, trophozoite and schizont stages at 14, 32 and 42 hours post invasion (hpi), respectively. Gametocytes were induced from a >90% synchronised asexual P. falciparum NF54-PfS16-GFP-Luc culture (described previously [247], maintained in culture medium lacking glucose) at 0.5% parasitaemia (6% haematocrit) by enforcing environmental stress to asexual parasites [248]. Gametocyte cultures were maintained for 12 days, treated with 50 mM N-acetyl-D-glucosamine and the stage I, II, III, IV and V gametocytes were isolated on day 3, 5, 7, 9 and 12 of gametocytogenesis, respectively. The asexual and sexual parasites were released from the red blood cells (RBCs) using 0.06% w/v saponin in phosphate buffered saline (PBS), followed by several wash steps with PBS to eliminate the presence of any residual erythrocyte material.

Histone isolation and chemical derivatisation

Histones were isolated using a modified protocol from Trelle et al. (2009) [100]. Briefly, nuclei was released from the pure and isolated P. falciparum parasites using a hypotonic buffer containing 10 mM Tris-HCl, pH 8.0, 3 mM MgCl2, 0.2% v/v Nonidet P40, 0.25 M sucrose in the presence of a protease inhibitor cocktail. The mixture was centrifuged at 500g and 4ºC for 10 min and this hypotonic buffer wash step was repeated twice.
Subsequently, the chromatin pellet was homogenised in the hypotonic buffer lacking Nonidet P40, to which 10 mM Tris-HCl, pH 8.0, 0.8 M NaCl, 1 mM EDTA (including protease inhibitor cocktail) was added, followed by a 10 min incubation on ice. Histones were acid-extracted from the chromatin with 0.25 M HCl and rotation at 4ºC for 1 h. The histone-containing supernatant was mixed with an equal volume of 20% v/v trichloroacetic acid, incubated on ice for 15 min and pelleted. The histone-enriched pellet 35 was washed with acetone, air-dried and reconstituted using dddH2O. All samples were dried using a SpeedVac concentrator (SC100, Savant) and reconstituted with 100 mM ammonium bicarbonate (pH 7-9). Histones were prepared for MS analysis via propionic anhydride chemical derivatisation and in-solution trypsin digestion as previously described [249]. Each of the eight stages was analysed in three independent biological experiments with three technical triplicates each. For the initial semi-quantitative study, the experimental procedures are outlined in Appendix III: Methods.

Complete quantitative histone PTM landscape of asexual and sexual

P. falciparum parasites We performed a quantitative assessment of histone samples using high-resolution nanoLC-MS/MS; data processing/peak area extraction was performed by using EpiProfile [243], and spectra identification was performed by using Proteome Discoverer (Thermo). This enabled confident identification and quantification of histone PTMs, as well as horizontal comparison of the histone PTM landscape during P. falciparum parasite development. For the quantitative chromatin proteomics study, a complete validated dataset of 125 individual peptides were confidently identified (<20% coefficient of variance between biological replicates), of which 17% constituted the unmodified proportion of the peptides (see Appendix IV: Supplementary File 1). Reproducibly high sequence coverage was obtained in all three biological replicates, particularly for the core histones at 61 ± 15% (Figure 2.2D). In total, the variant histones were associated with lower sequence coverage compared to their core partners (H2A.Z vs. H2A, P=0.84; H2Bv vs. H2B, P=0.69). As expected, due to the centromere-restricted localisation of H3Cen [250], this protein was present in low abundance with significantly lower sequence coverage (P<0.01) than any of the other histones.

Dynamic histone PTM profiles between asexual and sexual

development The most significant observation in this study is the dynamics and plasticity of histone PTMs as the parasite differentiates during asexual and sexual development. The quantitative dataset allowed head-to-head comparison of the changes in the histone PTMs across all stages (ring, trophozoite and schizont) of the P. falciparum parasite’s asexual developmental cycle, as well as all five distinct morphological stages (stage I-V) of sexual gametocytogenesis. A close positive correlation (r2=0.44, Figure 2.6A) was observed between early asexual stages (ring and trophozoites). However, the transcriptionally active and differentiating schizont stage was diverged from early asexual development, as expected.
Gametocyte histone PTMs is in totality divergent from that found in asexual parasites, with certain gametocyte stages showing closer total correlation (e.g. stage III & V: r2=0.43, Figure 2.6A). Within these gametocyte stages, a subset of 13 conserved modifications (H3K4ac, H3K9me1, H3.3K9me2, H3K18me1, H3K23me1, H3K27me2&me3, H3K36me2&me3, H3K56me1&me2, H3K79me3 and H3.3K79me3) was highly correlated between stage I & III (r2 = 0.78), I & V (r2 = 0.70) and III & V (r2 = 0.83) gametocytes (Figure 2.6A). Such a pattern of highly correlated and conserved modifications within the subset of 13 modifications was only observed in certain sexual stages but not in asexual parasites or stage II/IV gametocytes (Figure 2.7).

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Protein sample preparation for mass spectrometry analysis

The MS analysis was performed at the Garcia Lab at the University of Pennsylvania (Philadelphia, USA) by Dr. Katarzyna Kulej and Dr. Simone Sidoli. The candidate (Ms N. Coetzee) was trained on the Q-Exactive mass spectrometer and was able to do her own sample preparation and could observe the MS operating procedure. All data analysis was independently performed by the candidate. All samples were thawed at room temperature and kept on ice for the duration of the sample preparation.
The protein samples were prepared by using the Wessel/Flügge method of chloroform/methanol precipitation and extraction (adapted for larger sample volumes) [277]. This method was optimised to concentrate protein samples of larger volumes and to remove unwanted contaminants such as salts, detergents, lipids and nucleic acids. Briefly, protein samples were mixed with an equal volume of methanol:chloroform (3:1 ratio), followed by a brief vortex step and centrifugation at 9,000g for 5 min (Eppendorf centrifuge 5415C) at 4°C. Each sample separated into an upper methanol layer containing unwanted salts and contaminants; an interphase aqueous precipitated protein layer and a bottom chloroform layer containing lipids. The top layer was removed and discarded without disturbing the interphase protein layer, and the samples were washed twice with methanol followed by centrifugation at 17,000g for 3 min (AccuSpin Micro17, Fischer Scientific) and complete removal of methanol:chloroform. The resulting precipitated proteins were dried at room temperature to allow for evaporation of the resultant methanol:chloroform. All samples were dissolved in a solution containing 6 M urea, 2 M thiourea and 50 mM ammonium bicarbonate, pH 7-8.

Table of Contents :

  • Chapter 1 : Introduction
    • 1.1 Malaria in general
    • 1.2 P. falciparum development
    • 1.2.1 Asexual development
    • 1.2.2 Sexual development
    • 1.3 Regulation of P. falciparum life cycle development
    • 1.4 A primer on epigenetic gene regulation
    • 1.4.1 Histone PTMs
    • 1.5 Epigenetic gene regulation in P. falciparum
    • 1.5.1 The nucleosome & histone PTM landscape of P. falciparum parasites
    • 1.5.2 Epigenetic regulation is involved in specific developmental processes during P. falciparum life cycle development
    • 1.6 Parasite biology as a catalyst for intervention strategies
    • 1.7 Hypothesis and objectives
    • 1.8 Research outputs
  • Chapter
    • 2.1 Introduction
    • 2.2 Materials & Methods
    • 2.2.1 Parasite production and isolation
    • 2.2.2 Histone isolation and chemical derivatisation
    • 2.2.3 Quantitative nanoLC-MS/MS-based histone PTM identification
    • 2.2.4 Data analysis
    • 2.2.5 Western Blot validations
    • 2.3 Results
    • 2.3.1 Histone abundance profile during development
    • 2.3.2 Complete quantitative histone PTM landscape of asexual and sexual P falciparum parasites
    • 2.3.3 Dynamic histone PTM profiles between asexual and sexual development
    • 2.4 Discussion
  • Chapter
    • 3.1 Introduction
    • 3.2 Materials & Methods
    • 3.2.1 Parasite production and isolation
    • 3.2.2 Cell lysis & total proteome isolation
    • 3.2.3 Protein sample preparation for mass spectrometry analysis
    • 3.2.4 Data analysis
    • 3.2.5 Transcriptomics data
    • 3.3 Results
    • 3.3.1 The P. falciparum proteome during gametocytogenesis
    • 3.3.2 Gametocytogenesis is characterised by stage-specific protein abundance profiles
    • 3.3.3 Sex-specific proteins peak early during gametocyte development
    • 3.3.4 A quantitative protein set is not translationally repressed during female gametocyte development
    • 3.3.5 Epigenetically driven regulation of protein expression during gametocytogenesis
    • 3.4 Discussion
  • Chapter
    • 4.1 Introduction
    • 4.2 Materials & Methods
    • 4.2.1 SYBR Green I-based fluorescence assay to determine inhibition against the asexual P. falciparum parasite stages
    • 4.2.2 Parasite lactate dehydrogenase assay to determine inhibition against the early and late stage P. falciparum gametocytes
    • 4.3 Results
    • 4.3.1 Antiplasmodial activity, cross-resistance and selectivity against asexual P falciparum stages
    • 4.3.2 Gametocytocidal activity & dual reactivity against early and late stage P falciparum gametocytes
    • 4.3.3 HDACi and HKMTi are potent multi-stage targeting epi-drugs
    • 4.4 Discussion
  • Chapter 5 : Concluding discussion

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