Lysine acetylation in sexual stage malaria parasites is a target for antimalarial small molecules

Abstract

Therapies to prevent transmission of malaria parasites to the mosquito vector are a vital part of the global malaria elimination agenda. Primaquine is currently the only drug with such activity; however, its use is limited by side effects. The development of transmission-blocking strategies requires an understanding of sexual stage malaria parasite (gametocyte) biology and the identification of new drug leads. Lysine acetylation is an important posttranslational modification involved in regulating eukaryotic gene expression and other essential processes. Interfering with this process with histone deacetylase (HDAC) inhibitors is a validated strategy for cancer and other diseases, including asexual stage malaria parasites. Here we confirm the expression of at least one HDAC protein in Plasmodium falciparum gametocytes and show that histone and nonhistone protein acetylation occurs in this life cycle stage. The activity of the canonical HDAC inhibitors trichostatin A (TSA) and suberoylanilide hydroxamic acid (SAHA; Vorinostat) and a panel of novel HDAC inhibitors on early/late-stage gametocytes and on gamete formation was examined. Several compounds displayed early/late-stage gametocytocidal activity, with TSA being the most potent (50% inhibitory concentration, 70 to 90 nM). In contrast, no inhibitory activity was observed in P. falciparum gametocyte exflagellation experiments. Gametocytocidal HDAC inhibitors caused hyperacetylation of gametocyte histones, consistent with a mode of action targeting HDAC activity. Our data identify HDAC inhibitors as being among a limited number of compounds that target both asexual and sexual stage malaria parasites, making them a potential new starting point for gametocytocidal drug leads and valuable tools for dissecting gametocyte biology.

FIG 1

Expression of HDACs in P. falciparum gametocytes. (a) Diagnostic RT-PCR on transcripts from trophozoites (Troph) of gametocyte-less strain F12, purified NF54 stage III and V gametocytes (Gam III and Gam V, respectively), and stage V gametocytes at 30 min postactivation (aGam V) with 100 μM xanthurenic acid at room temperature. RT-PCR was carried out with PfHDAC-specific primers. The expression levels of Pfama1 and Pfccp2 were used to verify blood-stage and gametocyte-specific expression, respectively. Samples lacking reverse transcriptase (−RT) were used as controls for genomic DNA contamination. The fructose-1,6-bisphosphate aldolase-encoding gene (Pfaldo) served as a loading control. All PCR products had nucleotide lengths of 200 to 300 bp. Lane M, 100-bp marker. (b) Western blot analysis of protein lysates from trophozoite-stage 3D7 P. falciparum parasites (asexual) and P. falciparum 3D7c gametocytes (method 1) on days 4 (stages I to III), 6 (stages III and IV), 8 (stages IV and V), and 10 (stages IV and V) relative to the start of induction (M1-d0). Protein lysates were analyzed by SDS-PAGE and two-color Western blot analysis using an Odyssey infrared imaging system (LI-COR Biosciences). The same membrane was probed with anti-PfHDAC1 rabbit antiserum (red) and anti-Pfs16 mouse antiserum (green). (c) Immunofluorescence image of late-stage gametocytes showing a bright-field image (top left), nuclear staining with DAPI (4′,6-diamidino-2-phenylindole; top right), predominantly nuclear localization with anti-PfHDAC1 antiserum (bottom left), and an overlay (bottom right).

FIG 2

Lysine acetylation during gametocyte development. (a) Samples were collected from P. falciparum 3D7c parasites (method 1) on days 4 (stages I to III), 6 (stages III and IV), 8 (stages IV and V), and 10 (stages IV and V) relative to the start of gametocyte induction (M1-d0). (b) Protein lysates were analyzed by SDS-PAGE and two-color Western blot analysis using an Odyssey infrared imaging system (LI-COR Biosciences). Membranes were probed with anti-pan-acetyl lysine (K103) mouse antiserum and anti-PfGAPDH rabbit antiserum or anti-tetra-acetylated (lysines 5, 8, 12, and 16) histone H4 rabbit antiserum and anti-Pfs16 mouse antiserum. Arrow, an ∼60-kDa protein showing altered acetylation during gametocyte development. p.i., postinduction.

FIG 3

Structures of the HDAC inhibitors used in this study.

FIG 4

Activity of TSA, SAHA, and CAY10603 against gametocytes. (a) ATP activity assay with cryopreserved and thawed MACS-purified stage V 3D7c gametocytes following 48 h of exposure to 10 μM methylene blue (MB) or 5 μM TSA, SAHA, or CAY10603. Controls contained vehicle only (0.5% DMSO). The mean percent ATP production ± SD is shown for three independent experiments, each of which was carried out in triplicate wells (**, P < 0.001). (b) MitoTracker Red CM-H₂XRos imaging-based viability assay with stage IV and V NF45^Pfs16-GFP gametocytes. Cells were incubated for 72 h with 4 μM methylene blue, TSA, SAHA, or CAY10603 and then incubated for 16 h with MitoTracker Red CM-H₂XRos (and compound) before imaging. Controls contained vehicle only (0.5% DMSO). The mean percent growth ± SD is shown for two independent experiments, each of which was carried out in duplicate wells (**, P < 0.001). (c and d) Stage III and IV gametocytes (∼0.5% gametocytemia, 5% hematocrit) were cultured with 5 μM TSA, SAHA, CAY10603, or the antimalarial drug chloroquine (CQ) for 2 days (c) or 5 days (d), with medium (without compound) being changed on day 2. Control cultures were incubated in the same way but contained vehicle only (0.05% DMSO). Thin blood films were prepared and stained with Giemsa, and the number of gametocytes per 100 cells (percent gametocytemia) was determined by microscopy (>3,000 cells were counted). The mean percent gametocytemia ± SD compared to that for the vehicle controls (taken as 100%) from two independent experiments is shown (**, P < 0.001).

FIG 5

HDAC inhibitors cause hyperacetylation of gametocyte histones. Stage III and IV P. falciparum 3D7c gametocytes were treated with 5 μM methylene blue (MB), chloroquine (CQ), TSA, SAHA, CAY10603, or vehicle only (0.05% DMSO as a control [C]) for 6 h. Protein lysates were analyzed by SDS-PAGE and Western blotting using antisera recognizing acetylated forms of histone H3 and H4: anti-tetra-acetylated H4 (lysines 5, 8, 12, and 16), anti-acetylated H3 (N terminus [N-term]), and anti-acetylated H3 (lysine 9 [lys 9]). Antisera recognizing Pfs16 and PfGAPDH were used as loading controls.