Gametogenesis in malaria parasites is mediated by the cGMP-dependent protein kinase

Abstract

Malaria parasite transmission requires differentiation of male and female gametocytes into gametes within a mosquito following a blood meal. A mosquito-derived molecule, xanthurenic acid (XA), can trigger gametogenesis, but the signalling events controlling this process in the human malaria parasite Plasmodium falciparum remain unknown. A role for cGMP was revealed by our observation that zaprinast (an inhibitor of phosphodiesterases that hydrolyse cGMP) stimulates gametogenesis in the absence of XA. Using cGMP-dependent protein kinase (PKG) inhibitors in conjunction with transgenic parasites expressing an inhibitor-insensitive mutant PKG enzyme, we demonstrate that PKG is essential for XA- and zaprinast-induced gametogenesis. Furthermore, we show that intracellular calcium (Ca2+) is required for differentiation and acts downstream of or in parallel with PKG activation. This work defines a key role for PKG in gametogenesis, elucidates the hierarchy of signalling events governing this process in P. falciparum, and demonstrates the feasibility of selective inhibition of a crucial regulator of the malaria parasite life cycle.

Figure 1. Zaprinast Can Stimulate Rounding Up and Exflagellation of Mature Gametocytes in the Absence of XA

Zaprinast was added to mature gametocytes to assess the effects on gametogenesis compared to XA. The concentration of XA used was 20 μM, and the concentration of zaprinast was 400 μM, unless stated otherwise. (A) Micrographs of Giemsa-stained Stage V gametocytes prior to stimulation of gametogenesis (left panel) and after addition of XA (centre panel) or zaprinast (right panel). The scale bar indicates 10 μm. (B) Increasing concentrations of zaprinast were added to stimulate gametogenesis, and cells were scored as either round or crescent-shaped, and plotted as a percentage rounded-up. Results are based on triplicate counts of a representative experiment from the same flask of gametocytes on a single day (except for 12.5 and 25 μM, which are based on a single count only). Error bars indicate the standard error of the mean (± SEM). The experiment was carried out twice with very similar results. (C) The number of centres of exflagellation per 10,000 gametocytes was scored following addition of increasing concentrations of zaprinast. Results are based on triplicate counts of a representative experiment from the same flask of gametocytes on a single day. Error bars indicate mean ± SEM. The experiment was carried out twice with very similar results. (D) Merged confocal images of Giemsa-stained stage V gametocytes prior to stimulation of gametogenesis (left panel) and after addition of XA (centre panel) or zaprinast (right panel). Blue indicates DAPI-stained nuclei and red the anti-α tubulin antibody (Tat1) staining of cells. The scale bars indicate 5 μm. (E) Transmission electron micrographs of male or female gametocytes after stimulation of gametogenesis with XA (left and centre panels) or zaprinast (right panel). a, axonemes; m, mitochondrion; N, nucleus. Scale bars indicate 0.5 μm.

Figure 2. Intracellular Ca²⁺ Is Not Required for the Initial Step of P. falciparum Gametogenesis

To investigate the role of intracellular Ca²⁺ in rounding up and exflagellation, stage V Pfrh3⁻ control gametocytes were pretreated with the BAPTA-AM (BP) before stimulation of gametogenesis with either XA or zaprinast (ZP). (A) (Upper panel) Gametocytes were scored as either round or crescent-shaped, and plotted as percentage rounded-up. Results show the means of duplicate counts, and error bars indicate mean ± SEM. (Lower panel) The number of centres of exflagellation per 10,000 gametocytes was scored. Results show the means of duplicate counts and error bars indicate mean ± SEM. (B) Emergence from host erythrocytes before and after stimulation of gametogenesis with or without BAPTA-AM was scored using IFA with an anti-erythrocyte Band 3 monoclonal antibody. The proportion of cells not reacting with the antibody (indicative of complete emergence) was counted and plotted as a percentage. Results show the means from quadruplicate counts from two independent experiments, and error bars indicate mean ± SEM.

Figure 3. Compound 1 Inhibits Gametogenesis at Its Onset

To investigate the effect of compound 1 on gametogenesis, stage V Pfrh3⁻ gametocytes were induced with and without compound 1. (A) Light micrographs of XA-stimulated gametocytes in the presence (left panel) or absence (right panel) of 2 μM compound 1. The size bar indicates 5 μm. (B) Increasing concentrations of compound 1 were added with XA (or without XA, Con), and cells were scored as either round or crescent-shaped, and plotted as percentage rounded up. Results are based on duplicate counts from a representative experiment, and error bars indicate mean ± SEM. The results of this experiment have been reproduced on more than ten occasions. (C) The number of centres of exflagellation per 10,000 gametocytes was scored. Results are based on duplicate counts from a representative experiment, and error bars indicate mean ± SEM.

Figure 4. Substitution of a Key Active Site Threonine Residue Confers Compound 1 Insensitivity to Recombinant PfPKG

(A) An alignment of regions of PfPKG in the ATP-binding pocket, thought to interact with compound 1, with those of other apicomplexans. Conserved active site residues highlighted in grey were predicted to approach compound 1 in a model of the Eimeria tenella PKG [29] (EtPKG). The threonine residue (T618), substituted in PfPKG in this study, is highlighted in grey and marked with an asterisk. Amino acid sequences are numbered to the right of the blocks of sequence. (B) Dose-response curves showing the effect of compound 1 on recombinant PKG enzyme activity assayed in the presence of 10 μM cGMP. Red diamonds indicate full-length WT PfPKG; black triangles, mutated PfPKG with T₆₁₈ replaced by a glutamine residue; and blue squares, mutated PfPKG with T₆₁₈ replaced by a methionine residue. IC₅₀ values (± SEM): WT 5.8 ± 0.91 nM; T618Q mutant 17,800 ± 4,700 nM; and T618M mutant 1.5 ± 0.61 nM.

Figure 5. Transgenic P. falciparum Expressing an Inhibitor-Insensitive PfPKG Demonstrate an Essential Role for the Enzyme in the Initiation of Gametogenesis

(A) Native cGMP-dependent kinase activity in independent clones of PfPKG_T618Q mutants (A and B) and the Pfrh3⁻ (Psu) control cells were measured in both ring stage parasites and activated gametocytes in the presence of 10 μM cGMP. At least two independent experiments were carried out in triplicate, and the data displayed are from a representative experiment (error bars indicate mean ± SEM). A western blot of each sample is shown beneath, incubated with an anti-human PKGIα antibody. Lanes contain approximately 2 × 10⁸ ring stage parasites or 2 × 10⁶ activated gametocytes. One quarter of these amounts was used in native PKG assays. A recombinant, N-terminally truncated PfPKG (87 kDa) is included as a positive control (Con). Equivalent loading was assessed by reprobing the blot with the anti–α-tubulin antibody Tat-1. (B) Compound 1 (1 μM, 2 μM, and 5 μM) was added along with XA to WT (grey bars) and PfPKG_T618Q (clone A) gametocytes (white bars); cells were scored as either round or crescent-shaped, and plotted as percentage rounded-up. Results show data from duplicate counts from a representative experiment, and error bars indicate mean ± SEM. (C) Quantification of rounding up upon stimulation of WT (grey bars) and PfPKG_T618Q (clone A) gametocytes (white bars) with either XA or zaprinast (ZP) in the presence of either 2 μM compound 1 (C1) or 2 μM compound 2 (C2). Results show data from duplicate counts from a representative experiment, and error bars indicate mean ± SEM.

Figure 6. A Schematic Representing the Progression of Gametogenesis and the Proposed Hierarchy of Signalling Events That Occur

P. falciparum gametogenesis can be stimulated in vitro by addition of XA or zaprinast combined with a decrease in temperature. Within 2 min, a profound morphological change occurs whereby both male and female gametocytes transform from crescent-shaped to spherical. This process can still take place upon chelation of intracellular Ca²⁺ with BAPTA-AM, but gamete emergence is reduced and exflagellation is completely inhibited. By contrast, the primary rounding-up step (and subsequent progression) is completely halted by specific inhibition of the parasite PKG. This protein kinase therefore plays an essential role in mediating the initiation of gametogenesis, and it may act upstream of or in parallel with Ca²⁺ signalling. The involvement of CDPK4 in this process has been demonstrated for P. berghei.