cAMP-Signalling Regulates Gametocyte-Infected Erythrocyte Deformability Required for Malaria Parasite Transmission

Abstract

Blocking Plasmodium falciparum transmission to mosquitoes has been designated a strategic objective in the global agenda of malaria elimination. Transmission is ensured by gametocyte-infected erythrocytes (GIE) that sequester in the bone marrow and at maturation are released into peripheral blood from where they are taken up during a mosquito blood meal. Release into the blood circulation is accompanied by an increase in GIE deformability that allows them to pass through the spleen. Here, we used a microsphere matrix to mimic splenic filtration and investigated the role of cAMP-signalling in regulating GIE deformability. We demonstrated that mature GIE deformability is dependent on reduced cAMP-signalling and on increased phosphodiesterase expression in stage V gametocytes, and that parasite cAMP-dependent kinase activity contributes to the stiffness of immature gametocytes. Importantly, pharmacological agents that raise cAMP levels in transmissible stage V gametocytes render them less deformable and hence less likely to circulate through the spleen. Therefore, phosphodiesterase inhibitors that raise cAMP levels in P. falciparum infected erythrocytes, such as sildenafil, represent new candidate drugs to block transmission of malaria parasites.

Fig 1. PfPKA-mediated phosphorylation contributes to immature GIE stiffness.

A. Retention in microsphilters of stage III GIEs from the B10 clone pre-incubated 30 min to 1 h at 37°C with 10 μM H89, 10 μM KT5720, 10 μM PKI-m, 10 μM compound 2, 10 μM GGTI-298 or 0,1% DMSO (Control). Error bars denote the standard error of the mean. *** and ** Highly significant differences in retention rates (*** P < 0.001; **P < 0.01); ns: non-significant differences in retention rates compared to control; n: number of experiments. Outliers are shown as open circles. B. Retention in microsphilters of stages III GIEs from the B10 clone and the pHL-pfpka-r clone cultivated with and without pyrimethamine for 15 generations. The pHL-pfpka-r clone was pre-incubated 15 min at 37°C with 100μM 8Br-cAMP. Error bars denote the standard error of the mean. ***Highly significant differences in retention rates (P < 0.001); ns: non-significant differences in retention rates; n: number of experiments. Outliers are shown as open circles. C. Western-blot analysis of PfPKA-R expression in stage III GIE from the B10 clone and the pHL-pfpka-r clone cultivated in presence (+ pyr) or absence (- pyr) of pyrimethamine. Immunoblots were probed with rabbit polyclonal antibodies directed against PfPKA-R and with a mAb directed against PfHSP70 to normalize expression. Black star indicates the expected size for PfPKA-R (50.8 kDa); Red star indicates PfPKA-R with post-translational modifications. The experiment has been performed seven times. Error bars denote the standard error of the mean. *Significant differences in phosphorylation signal (*P < 0.05); ns: non-significant differences in phosphorylation signal. D. Quantitation of signal intensities in panel C using Quantity One software (BioRad). Analysis shows a 1.6-fold increase in PfPKA-R expression in the pHL-pfpka-r clone (+ pyr) compared to B10. Decrease of PfPKA-R expression in absence of pyrimethamine (- pyr) indicates a loss of episomal expression of the PfPKA-R protein. E. Immunofluorescence analysis of stage III GIE from the B10 clone and the pHL-pfpka-r clone cultured for 15 generations in the presence (+ pyr) or absence (- pyr) of pyrimethamine. Infected erythrocytes were stained with anti-PfPKA-R antibodies followed by anti-rabbit Alexa 594-conjugated IgG. Pictures were taken under identical exposure conditions. The bars represent 2 μm.

Fig 2. Phosphorylation events contribute to the switch in deformability.

A. Western-blot analysis of Phospho-PKA site (RRXS*/T*) expression in uninfected red blood cells (uRBCs), MACS-purified stage III (stIII) and stage V (stV) GIE from the B10 clone treated (+Calyc) or not (-Calyc) with 50 nM calyculin during 2h at 37°C. Analysis was performed on membrane extracts recovered by centrifugation after 1% Triton X100 treatment. Immunoblot was probed with rabbit monoclonal antibody directed against phospho-PKA sites (RRX*S/*T) and with mouse mAb directed against Band 3 to normalize expression. Red arrows show bands with more intense phosphorylation signal in stage III GIE than in stage V GIE. Green arrow show band with less intense phosphorylation signal in stage III GIE than in stage V GIE. The experiment has been performed three times. Error bars denote the standard error of the mean. ***Highly significant differences in phosphorylation signal (P < 0.001). B. Quantitation of signal intensities in panel D using Quantity One software (BioRad). The strength of the phosphorylation signal in untreated uninfected red blood cells lysate was set to 1 and all other signals are relative to that. C. Retention in microsphilters of stages V GIEs (light grey), or uninfected red blood cells (uRBCs, dark grey). GIEs were pre-incubated at 37°C for 2 h with 50 nM calyculin, or 0.5% DMSO (control). ***Highly significant differences in retention rates compared to control (P < 0.001); ns: non-significant differences in retention rates compared to control; n: number of experiments. D. Graphical representation for the proportion of GIE showing a regular (light green), or deformed (dark green) shape in a population of paraformaldehyde-fixed GIE, as they flow through the microsphilters after pre-incubation at 37°C 2 h with 50 nM calyculin, or 0.5% DMSO (control). E. Differential interference contrast images of paraformaldehyde-fixed GIE, as they flow through the microsphilters. A majority of DMSO-treated GIE (left panel) are twisted and deformed, whereas inhibitor-treated GIE (right panel) keep a regular shape, unlike uninfected erythrocytes (red star).

Fig 3. GIE filterability is dependent on cAMP concentration.

A. cAMP concentration drops in mature GIE. The total intracellular cAMP concentration was measured in stage III and V GIE using a competitive immunoassay for the quantitative determination of cAMP. GIE were harvested by magnetic isolation and aliquots of 6.10⁶ cells were assayed in duplicate wells. The assay was carried out three times. Error bars denote the standard error of the mean. **Highly significant difference compared to stage III GIE (P < 0.01). B. Western-blot analysis of PKA-R expression in MACS-purified stage III and stage V GIE (5.10e6 parasites/lane). Immunoblots were probed with rabbit polyclonal antibodies directed against PfPKA-R and with a mAb directed against PfHSP70 to normalize expression. Quantity One (BioRad) analysis shows that PfPKA-R levels were not significantly different between stage III and stage V. C. Retention rates in microsphilters of stage III GIE (dark grey), stage V GIE (light grey) and uninfected red blood cells (uRBC, black) pre-incubated 15 min at 37°C with different concentrations of 8Br-cAMP. Error bars denote the standard error of the mean. Outliers are shown as open circles. **Highly significant differences in retention rates compared to control without 8Br-cAMP (P < 0.01); ns: non-significant differences in retention rates compared to control; n: number of experiments.

Fig 4. cAMP degradation by phosphodiesterases regulates GIE mechanical properties.

A. mRNA levels of four PDEs were determined by real time RT-PCR in asexual blood-stages (asex), stage III GIE (st.III) and stage V GIE (st.V). Relative amounts of transcript were normalized to mRNA levels of the PfHK (PF08_0085), Pfs48/45 (PF13_0247) and Pfs25 (PF10_0303) were used as markers of stage III and stage V GIE, respectively. Triplicate PCR reactions were analysed for each sample. B. The total intracellular cAMP concentration was measured in stage III and V GIE from the 3D7 clone and the PfPDEδ-mutant clone 4 using a competitive immunoassay for the quantitative determination of cAMP. GIE were harvested by magnetic isolation and aliquots of 6.10⁶ cells were assayed in triplicate wells. The assay was carried out at least three times for each clone. C. Retention in microsphilters of stages V GIEs from the 3D7 clone and the PfPDEδ-mutant clone 4. Error bars denote the standard error of the mean. Outliers are shown as open circles. n: number of experiments.

Fig 5. Interfering with cAMP levels impairs mature GIE filterability.

A. Stage V GIE were harvested by magnetic isolation and incubated at 37°C for 30 min with 150 μM forskolin, 65 μM zaprinast, or 0.1% DMSO (Control). The total intracellular cAMP concentration was measured on aliquots of 6.10⁶ cells in duplicate wells. The assay was carried out three times. Error bars denote the standard error of the mean. *Significant differences in retention rates compared to control (P < 0.05). B. Retention in microsphilters of stages V GIEs (light grey), or uninfected red blood cells (uRBCs, dark grey). GIEs were pre-incubated at 37°C for 30 min with 150 μM forskolin, 65 μM zaprinast or 0,1% DMSO (control). ***Highly significant differences in retention rates compared to control (DMSO) (P < 0.001); ns: non-significant differences in retention rates compared to control. n: number of experiments. C. Graphical representation for the proportion of GIE showing a regular (light green), or deformed (dark green) shape in a population of paraformaldehyde-fixed GIE, as they flow through the microsphilters after pre-incubation at 37°C for 30 min with 150 μM forskolin, 65 μM zaprinast or 0.1% DMSO (control).

Fig 6. Sildenafil impairs mature GIE filterability.

A. Stage V GIE were harvested by magnetic isolation and incubated at 37°C for 30 min with 100 μM sildenafil, or 0.1% DMSO (Control). The total intracellular cAMP concentration was measured on aliquots of 6.10⁶ cells in duplicate wells. The assay was carried out five times. Error bars denote the standard error of the mean. **Highly significant differences in retention rates compared to control (DMSO) (P < 0.01). B. Graphical representation for the proportion of GIE showing a regular (light green), or deformed (dark green) shape in a population of paraformaldehyde-fixed GIE, as they flow through the microsphilters after pre-incubation at 37°C for 30 min with 100μM sildenafil, or 0.1% DMSO (Control). C. Differential interference contrast images of paraformaldehyde-fixed GIE, as they flow through the microsphilters. D. Retention rates in microsphilters of stage V GIE (light grey) and uninfected red blood cells (uRBC, dark grey) pre-incubated 30 min at 37°C with different concentrations of sildenafil. Error bars denote the standard error of the mean. Outliers are shown as open circles. *** and **Highly significant differences in retention rates compared to control (**P < 0.01; ***P < 0.001); ns: non-significant differences in retention rates compared to control. n: number of experiments.