Erythrocyte G protein as a novel target for malarial chemotherapy

Abstract

Background: Malaria remains a serious health problem because resistance develops to all currently used drugs when their parasite targets mutate. Novel antimalarial drug targets are urgently needed to reduce global morbidity and mortality. Our prior results suggested that inhibiting erythrocyte Gs signaling blocked invasion by the human malaria parasite Plasmodium falciparum.

Methods and findings: We investigated the erythrocyte guanine nucleotide regulatory protein Gs as a novel antimalarial target. Erythrocyte "ghosts" loaded with a Gs peptide designed to block Gs interaction with its receptors, were blocked in beta-adrenergic agonist-induced signaling. This finding directly demonstrates that erythrocyte Gs is functional and that propranolol, an antagonist of G protein-coupled beta-adrenergic receptors, dampens Gs activity in erythrocytes. We subsequently used the ghost system to directly link inhibition of host Gs to parasite entry. In addition, we discovered that ghosts loaded with the peptide were inhibited in intracellular parasite maturation. Propranolol also inhibited blood-stage parasite growth, as did other beta2-antagonists. beta-blocker growth inhibition appeared to be due to delay in the terminal schizont stage. When used in combination with existing antimalarials in cell culture, propranolol reduced the 50% and 90% inhibitory concentrations for existing drugs against P. falciparum by 5- to 10-fold and was also effective in reducing drug dose in animal models of infection.

Conclusions: Together these data establish that, in addition to invasion, erythrocyte G protein signaling is needed for intracellular parasite proliferation and thus may present a novel antimalarial target. The results provide proof of the concept that erythrocyte Gs antagonism offers a novel strategy to fight infection and that it has potential to be used to develop combination therapies with existing antimalarials.

Figure 1. Schematic for the Production of Ghosts with G_s Signaling and Malarial Infection that Closely Mimic Normal Erythrocytes

Erythrocytes were washed and resuspended to 50% Hct in PBS-glucose containing proteins of interest (“cargo,” yellow spheres) as described in Methods. The cell suspension was dialyzed against ATP-supplemented hypotonic potassium buffer, lysing cells, and allowing exogenous cargoes to enter and endogenous cytoplasmic proteins to exit lysed cells (depicted over time as increasing extracellular hemoglobin, change in cell color from red to pink, and loss of membrane integrity). Lysed cells were removed to cold test tubes and resealed at 37 °C for 1 h and washed three times in RPMI and once in RPMI supplemented with 10% human serum (cRPMI) to eliminate extracellular hemoglobin, cargo, and buffer (see Methods). Ghosts prepared in this manner were subsequently characterized in hematological, signaling, and malarial infection assays, as described in Figure 2.

Figure 2. Hematological and Signaling Characteristics and Cargo Loading of Erythrocyte Ghosts

(A) Ghosts (left) were biconcave but retained less pigment than intact erythrocytes (right); bar represents 10 μm. (B) Ghost MCH (normal range for erythrocytes 27.5–33.5 pg/cell), MCV (normal range for erythrocytes 80–100 fl), and RDW (normal range for erythrocytes 11.0%–15.0%) were determined using a Coulter counter (see Methods). Intracellular ATP levels were determined by luciferase-based ATP assays (see Methods); the 95% confidence interval (CI) for ATP levels is indicated; two experiments. (C) Ghosts loaded with high molecular weight rhodamine-labeled antibody were imaged without fixation by fluorescence microscopy; bar represents 25 μm. (D) Flow cytometry of unloaded (white fill) and FITC-dextran–loaded ghosts (green fill) 2 h post-resealing, showed homogenous loading of cargo. For each cell type, 100,000 gated events were captured. (E) Western blot of GST-loaded ghosts after incubation in culture for 24 h and subsequent treatment in the presence or absence of proteinase K (Prot K) and/or Triton X-100 (TX100); see Methods. GST was detected by GST-specific immunoblotting. (F) cAMP production in normal, intact erythrocytes treated with isopreterenol (iso) and/or propranolol (prop) as measured by enzyme-linked immunosorbent assay (see Methods ). Error bars show 95% CI of triplicate measurements in a representative experiment. The mean baseline cAMP concentration in control erythrocytes was 0.61 pmol cAMP (95% CI 0.20–1.01 pmol; three experiments) per 1 × 10⁸ cells. Induced cAMP levels can vary—the maximal value obtained (once) was 14.58 pmol cAMP (95% CI 13.11–16.03 pmol) per 1 × 10⁸ cells. (G) cAMP production in ghosts measured and depicted as described in (F). Error bars show 95% CI of triplicate measurements in a representative experiment. The mean baseline cAMP concentration in control ghosts was 1.12 pmol cAMP (95% CI 0.59–1.64 pmol; three experiments) per 1 × 10⁸ cells. The maximal isoproterenol-induced value obtained in a single ghost cAMP assay was 9.00 pmol cAMP (95% CI 7.37–10.62 pmol) per 1 × 10⁸ cells.

Figure 3. Erythrocyte Ghosts Support Malarial Invasion and Growth

(A) LY-D–loaded erythrocyte ghosts (yellow) were infected with P. falciparum (detected by blue DNA stain Hoechst 33342; indicated by arrow). Bar represents 5 μm. (B) Ghosts (white bars) or erythrocytes (grey bars) infected at low (left) or high (right) parasitemias were monitored by Giemsa staining of thin blood smears at the indicated times of infection; mean values are shown. Error bars show standard deviation of triplicate measurements of a representative experiment. (C) An infected culture containing 50% ghosts and 50% erythrocytes was monitored for cell type-specific parasitemias by counting the number of Hoechst 33342–stained parasite nuclei in fluorescent ghosts (white bars) versus non-fluorescent erythrocytes (grey bars) using DIC and fluorescence microscopy of live cells (see Methods). Error bars show standard deviation of triplicate measurements of a representative experiment. (D) Giemsa-stained thin blood smears showing ring-, trophozoite-, and schizont-stage parasites in normal (upper) and ghosted (lower) erythrocytes. Bar represents 5 μm. (E) Growth of P. falciparum in ghosts (Ghs) or in erythrocytes (RBCs) in culture over multiple life cycles. Parasites were cultured in ghosts (indicated by dashed line) or in normal erythrocytes (indicated by solid line) for 60 h to ~25% parasitemia, and then were diluted into normal erythrocytes and grown for an additional 50 h. Parasitemia was assessed by counting Giemsa-stained thin blood smears. Error bars show 95% CIs of duplicate measurements at each time point.

Figure 4. Targeting Erythrocyte G_s Down-Regulates β-Adrenergic Signaling and Inhibits Malarial Infection

(A) Ghosts incubated in 40 μM G_s (indicated by hatched bars) or G_scr control peptide (indicated by solid bars) were treated with agonist (isoproterenol [iso]) and/or antagonist (propranolol [prop]), and cAMP was measured as described in Methods. Error bars show 95% CI of triplicate measurements from a representative experiment of three independent experiments. The mean baseline cAMP concentration in control ghosts was 1.08 pmol cAMP (95% CI 0.57–1.60 pmol; four experiments) per 1 × 10⁸ cells. The maximum isoproterenol-induced cAMP level obtained in control-treated ghosts was 12.45 pmol cAMP (95% CI 10.20–14.70 pmol) per 1 × 10⁸ cells. (B) Ghosts incubated with G_s peptide (≤40 μM) or G_scr control peptide (up to 400 μM) were assayed for malarial invasion. Data are represented as percentage inhibition relative to control cultures treated with equimolar concentrations of control peptide. Three experiments in triplicate for each data point except 10 μM (two experiments in triplicate). Error bars show 95% CI for all experiments; asterisks indicate p < 0.001 compared to 400 μM control G_scr peptide–treated cultures. (C) Parasites in ghosts were incubated with the indicated concentrations of peptide and monitored at 15, 32, and 44 h after invasion by examination of Giemsa-stained thin blood smears. The number of ring (R)–, trophozoite (T)–, and schizont (S)–stage parasites per 1,000 total ghosts at each time point are shown; numbers in parentheses indicate the standard deviation of triplicate measurements from one experiment; a total of two experiments were carried out.

Figure 5. β-Blockers Inhibit Maturation of P. falciparum in In Vitro Cultures

(A) [³H]-hypoxanthine incorporation of P. falciparum 3D7 when treated with racemic propranolol (indicated by black diamonds) or its inactive isomer (indicated by grey squares). Error bars show the standard deviation of triplicate measurements. IC₅₀ values were determined by fitting the data as described in Methods; the IC₅₀ value obtained for racemic propranolol (1.2 μM; 95% CI 1.0–1.6 μM) is shown. Three experiments; hatched sign indicates p < 0.001; asterisks indicate p < 0.03 compared to equimolar inactive propranolol. (B) Effect of 2 μM propranolol on intracellular growth in normal erythrocytes. Mock- and drug-treated cultures were monitored at 15, 32, and 44 h after invasion by examination of Giemsa-stained thin blood smears. The number of ring (R)–, trophozoite (T)–, and schizont (S)–stage parasites per 1,000 total erythrocytes at each time point are shown; numbers in parentheses indicate the standard deviation of triplicate measurements from one experiment; the total number of experiments was three. (C) [³H]-hypoxanthine incorporation of P. falciparum 3D7–infected erythrocytes treated with 1 or 10 μM concentrations of adrenergic-acting drugs (nonspecific β₁/β₂–antagonists: propranolol, alprenolol, and nadolol; β₂-specific antagonists: ICI118,551 and butoxamine; β₁-specific antagonists: acebutalol, atenolol, and metoprolol; and α₂-specific agonist [ag]: clonidine). Chloroquine (50 nM) was used as an inhibitory control. Triplicate samples; asterisks denote a treatment with p < 0.001 compared to control-treated cultures.

Figure 6. Analysis of the Maturation Defect in Propranolol-Treated Parasites

(A) Effect of propranolol on sorbitol sensitivity of infected erythrocytes. Normal or infected erythrocytes were mock-treated or incubated with 2 or 10 μM propranolol for 30 h and then resuspended in 5% sorbitol as described in Methods. Bars represent percentage lysis as calculated by the hemoglobin absorbance (570 nm) of the supernatant after 0 and 15 min of sorbitol treatment divided by the hemoglobin absorbance of a saponin-treated sample (where 100% of hemoglobin is released) from the same culture. Error bars show the standard deviation from triplicate samples. (B) Effect of propranolol on export of a parasite-encoded protein (PfHRPII) fused to GFP to the host erythrocyte. Fluorescence micrographs of infected erythrocytes that were mock-treated or exposed to 2 or 10 μM propranolol for 30 h (see Methods) are shown, with single optical sections. Bar represents 5 μm. The fraction of GFP exported to the erythrocyte (e) from the parasite (p) was determined (see Methods) and is relatively unchanged by drug treatment. Error bars show the standard deviation from ten samples per treatment. (C) Giemsa-stained thin blood smears showing the morphology of parasites when infected erythrocytes were treated with 2 or 10 μM propranolol for 15, 32, and 44 h of culture. Infected erythrocytes treated with 2 μM propranolol showed significant retardation in schizont formation by 44 h. At 10 μM propranolol, a growth defect was observed by 32 h post-invasion. Bar represents 3 μm; all images are displayed at the same scale.

Figure 7. Propranolol Inhibits Malarial Growth in Combination with Existing Antimalarial Drugs

(A) [³H]-hypoxanthine incorporation (see Methods) of P. falciparum 3D7 treated with chloroquine alone (CQ, indicated by black diamonds) or with ratios of both drugs (CQ:propranolol, 3:1 [black spheres]; 1:1 [grey triangles]; and 1:3 [grey squares]). Error bars show standard deviation of triplicate samples. IC₅₀ values were determined by fitting the data as described in Methods. IC₅₀ CQ alone: 28.9 nM (95% CI 22.3–36.6 nM). The IC₅₀ CQ values when treated with combinations of both drugs in different ratios of CQ:propranolol were as follows: 3:1, 14.7 nM (95% CI 11.9–18.1 nM); 1:1, 5.9 nM (95% CI 4.9–7.0.6 nM); and 1:3, 2.9 nM (95% CI 2.5–3.2 nM). (B) [³H]-hypoxanthine incorporation of P. falciparum chloroquine-resistant FCB strain treated with chloroquine alone (CQ, indicated by black diamonds) or with ratios of both drugs (CQ:propranolol, 3:1 [black spheres]; 1:1 [grey triangles]; and 1:3 [grey squares]). Error bars show standard deviation of triplicate samples. IC₅₀ values were determined by fitting the data as described in Methods. IC₅₀ CQ alone: 63.2 nM (95% CI 52.4–76.4 nM). The IC₅₀ CQ values when treated with combinations of both drugs in different ratios of CQ:propranolol were as follows: 3:1, 26.7 nM (95% CI 24.6–29.0 nM); 1:1, 9.7 nM (95% CI 9.3–10.1 nM); and 1:3, 6.0 nM (95% CI 5.8–6.2 nM). (C) [³H]-hypoxanthine incorporation of P. falciparum 3D7 treated with artemisinin alone (black diamonds) or with ratios of both drugs (artemisinin:propranolol, 3:1 [black spheres]; 1:1 [grey triangles]; and 1:3 [grey squares]). Error bars show standard deviation of triplicate samples. IC₅₀ values were determined by fitting the data as described in Methods. IC₅₀ artemisinin alone: 3.2 nM (95% CI 2.9–3.6 nM). The IC₅₀ artemisinin values when treated with combinations of both drugs in different ratios of artemisinin:propranolol were as follows: 3:1, 2.3 nM (95% CI 2.0–2.6 nM); 1:1, 1.3 nM (95% CI 1.2–1.4 nM); and 1:3, 0.56 nM (95% CI 0.5–0.6 nM). (D) Combinations of propranolol with chloroquine (CQ) in a 4-d Peter's test of P. berghei ANKA–infected Balb/c mice (see Methods). IC₅₀ CQ alone: 1.6 mg/kg/d (95% CI 1.2–2.2 mg/kg/d). The IC₅₀ CQ values when treated with combinations of both drugs in different ratios of CQ:propranolol were as follows: 2:1, 1.2 mg/kg/d (95% CI 1.0–1.4 mg/kg/d); 1:1, 1.0 mg/kg/d (95% CI 0.8–1.3 mg/kg/d); and 1:2, 0.7 mg/kg/d (95% CI 0.4–1.0 mg/kg/d). Error bars show standard deviation of measurements from five mice per treatment group.