Resistance to Plasmodium falciparum in sickle cell trait erythrocytes is driven by oxygen-dependent growth inhibition

Abstract

Sickle cell trait (AS) confers partial protection against lethal Plasmodium falciparum malaria. Multiple mechanisms for this have been proposed, with a recent focus on aberrant cytoadherence of parasite-infected red blood cells (RBCs). Here we investigate the mechanistic basis of AS protection through detailed temporal mapping. We find that parasites in AS RBCs maintained at low oxygen concentrations stall at a specific stage in the middle of intracellular growth before DNA replication. We demonstrate that polymerization of sickle hemoglobin (HbS) is responsible for this growth arrest of intraerythrocytic P. falciparum parasites, with normal hemoglobin digestion and growth restored in the presence of carbon monoxide, a gaseous antisickling agent. Modeling of growth inhibition and sequestration revealed that HbS polymerization-induced growth inhibition following cytoadherence is the critical driver of the reduced parasite densities observed in malaria infections of individuals with AS. We conclude that the protective effect of AS derives largely from effective sequestration of infected RBCs into the hypoxic microcirculation.

Keywords: Plasmodium falciparum; malaria; oxygen; red blood cell; sickle hemoglobin.

Fig. 1.

Low O₂ concentration stalls growth of Pf3D7 IG06 parasites. P. falciparum 3D7 IG06 parasites grown in AA (wild-type) and AS (sickle cell trait) erythrocytes at 15, 28, 32, and 38 hpi in 1% O₂ concentration. (A) Representative Pf3D7 IG06 parasites in thin smears of AA (Top) and AS (Bottom) erythrocytes at 15, 28, 32, and 38 hpi. (Scale bars: 10 μm.) (B) Staging in AA (Top) and AS (Bottom) of 300 Pf3D7 IG06 parasites per time point. (C) Flow cytometry analysis of DNA content in synchronized, RNase-treated, SYBR Green-stained Pf3D7 IG06 parasites in AA and AS RBCs.

Fig. 2.

P. falciparum DNA replication and growth increase with rising O₂ concentration. P. falciparum D10 parasites grown in AA and AS erythrocytes cultured at variable O₂ concentrations using 1%, 3%, 5%, 7.5%, 10%, and 16% O₂ with 5% CO₂ and balanced with N. (A) Flow cytometry analysis of DNA content of synchronized, RNase-treated, SYBR Green-stained PfD10 parasites in AA (Top) and AS (Bottom) RBCs. (B) Staging of parasites in AA (Top) and AS (Bottom) cells at 44 hpi. (C) PMR, an assessment of number of ring-infected RBCs/total number of RBCs in culture at 64 hpi divided by number of ring-infected RBCs/total number of RBCs at 19 hpi of PfD10 parasites in AA (Top) and AS (Bottom) cells. ns, P > 0.05; ****P < 0.001.

Fig. 3.

HbS polymerization is associated with impaired hemoglobin processing by P. falciparum in AS RBCs in low O₂. P. falciparum 3D7 IG06 parasites grown in AA (wild-type) and AS erythrocytes. (A) The average hemozoin formation in AA and AS parasites at 38 hpi in seven different experiments using six AS blood samples. (B) Percentage maximum DNA content (max DNA content = infected AA RBCs at 38 hpi) of infected RBCs in 1% O2 in the absence (AA and AS) and presence (AA CO and AS CO) of CO at 12, 28, 32, and 38 hpi. Error bars represent means ± SD. ns, P > 0.05; ****P < 0.001. (C) Electron micrograph of Pf D10 parasites in AA (Top) and AS (Bottom) RBCs at 44 hpi demonstrating knobs (red arrowheads) on AA and AS RBCs but the presence of HbS polymers (blue arrowheads) and altered growth at low O₂ in AS RBCs. (Scale bars: 0.8 μm.)

Fig. 4.

Timing of movement into hypoxia dictates parasite growth potential in AS RBCs. P. falciparum 3D7 IG06 parasites grown in AS erythrocytes. (A) Percentage maximal DNA content at 38 hpi (max DNA content = infected AA RBCs at 38 hpi) of Pf3D7 IG06 parasites in AS RBCs when switched from 10% to 1% O₂. Switch times are 16 (red), 20 (orange), 24 (green), 28 (blue), and 32 (purple) hpi. (B) Representative images of parasites when switched at 16, 20, 24, 28, and 32 hpi (Top) and at 38 hpi (Bottom) for each experiment. (Scale bars: 10 μm.) (C) PMR of parasites in AS RBCs cultured at 10% O₂ (black) as well as those moved from 10% to 1% O₂ at switch times 16, 20, 24, 28, and 32 hpi. ns, P > 0.05; ****P < 0.001.

Fig. 5.

Mathematical modeling suggests that HbS polymerization-induced growth inhibition reduces parasitemia more than impaired cytoadherence. (A) Schematic of intraerythrocytic parasite life cycle within the high O₂ peripheral blood and low O₂ microcirculation, showing splenic clearance and sequestration as well as the parameters used to fit our model, including cycle length (c), mortality of both nonsequestered (μ_NE and μ_NL) and sequestered (μ_S) parasites, and transition rate of nonsequestered (λ_N) and (λ_S) sequestered parasites (parasite multiplication number not shown). As a ring, the parasite circulates in the peripheral blood. As PfEMP1 function increases, the parasite is more likely to sequester or be cleared by the spleen. The parasite within an AS RBC sequesters but then stalls within the low O₂ compartment. (B) Derived PMR based on model parameters of P. falciparum parasites cultured in AA erythrocytes at 1% O₂; AS RBCs at 5%, 7.5%, 10%, and 16% O₂; and AA RBCs with only 50% functional PfEMP1. (C) Predicted relative parasite load change under the foregoing conditions.