Research Article

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

Natasha M. Archer, Nicole Petersen, Martha A. Clark, Caroline O. Buckee, Lauren M. Childs, and Manoj T. Duraisingh
PNAS July 10, 2018 115 (28) 7350-7355; first published June 26, 2018 https://doi.org/10.1073/pnas.1804388115

  1. Edited by Louis H. Miller, National Institutes of Health, Rockville, MD, and approved June 1, 2018 (received for review March 13, 2018)

Significance

Sickle cell trait has repeatedly been identified as a major human malaria resistance factor. Despite this, the exact mechanism of resistance is unclear. These studies demonstrate how the evolutionarily significant sickle hemoglobin affects Plasmodium falciparum infection success and leads to a better understanding of the molecular basis and pathogenesis of malaria infection.

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.

Nearly 80% of individuals born with sickle cell anemia live in sub-Saharan Africa, where most Plasmodium falciparum malaria cases and deaths occur (1). While the genetic mutation in the beta globin gene producing sickle hemoglobin (HbS) causes severe vascular complications that can lead to early death in individuals who are homozygous (SS) for the mutation, in its heterozygous form (AS), it partially protects against severe malaria caused by P. falciparum infection (2⇓–4). Compared with persons with normal hemoglobin (AA), individuals with AS have a 50–90% reduction in parasite density (5). In addition, the AS genotype is underrepresented among children with severe malaria (6, 7). Other studies have demonstrated faster clearance of asymptomatic infection in children with AS (8). However, despite the strong epidemiologic evidence of selection for AS, the biological mechanism underlying the protection against severe malaria is not completely understood.

Various mechanisms to explain AS malaria resistance have been proposed, including sickling of the infected red blood cells (RBCs) (9, 10), increased splenic phagocytosis (11), premature hemolysis and parasite death (12), impaired hemoglobin digestion (13⇓–15), weakened cytoadherence (16, 17) acquired host immunity (18), translocation of HbS-specific parasite-growth inhibiting microRNAs (19), and induction of heme-oxygenase-1 (20). These proposed mechanisms may play roles in AS malaria resistance; however, the altered parasite growth observed in continuous culture and the cases of severe malaria in children with SS (21⇓–23) suggest an alternative mechanism. The current leading hypothesis for the protective effect in individuals with AS proposes that decreased P. falciparum Erythrocyte Membrane Protein 1 (PfEMP1) (24) expression on infected AS RBCs leads to the reduced binding of infected cells to the endothelium (16), resulting in only approximately one-half the cytoadherence seen in infected AS RBCs. While weakened cytoadherence may contribute, at least partially, to the in vivo phenotype observed, it does not explain the poor growth observed in low O2 in vitro. Building on the work of several others (11, 13⇓–15), we propose that oxygen-dependent HbS polymerization is a major driver of AS malaria resistance.

While the concentration of O2 in arterial blood is approximately 13%, O2 concentrations of <7.5% are found in most organs to which infected RBCs sequester, including the bone marrow, brain, and liver (25⇓⇓⇓⇓–30). We hypothesize that HbS polymerization at low O2 plays a fundamental role in AS malaria resistance. To address this hypothesis, we carefully mapped parasite growth inhibition in AS erythrocytes in relation to the dynamics of intracellular growth and O2 concentration. In vitro, we simulated parasite movement from an environment of high O2 encountered in the peripheral circulation to that of low O2 encountered during sequestration. Strikingly, we observed a direct correlation between O2 concentration and parasite growth in AS RBCs and an overlap in the timing of cytoadherence and the period during which the parasite is most sensitive to low O2.

To examine the in vivo consequences of these observations, we have developed a mathematical model of parasite development within RBCs in sequestered and nonsequestered environments, comparing parasite proliferation dynamics under impaired growth in hypoxic AS RBCs versus reduced cytoadherence. We show that HbS polymerization-induced impaired growth leads to greater reductions in parasite proliferation compared with reduced cytoadherence, and propose that HbS polymerization in infected RBCs sequestered in various human tissues with low O2 concentrations is a major mechanism of protection against severe malaria in individuals with AS.

Results

Parasite Growth in AS RBCs Is Stalled at Low O2 Concentration.

To examine the effect of HbS on P. falciparum 3D7 IG06 growth, we first determined the effect of 1% O2 concentration on parasite growth in AA and AS RBCs. Consistent with the morphological assessment reported by Pasvol et al. (14), we found with staging by light microscopy that parasites growing in AS remain stalled at the late ring/early trophozoite stage, despite normal maturation of parasites in equally hypoxic AA RBCs (Fig. 1 A and B). The growth differential was first appreciated at 28 h postinfection (hpi), at which point approximately 75% of the parasites in AA erythrocytes were late trophozoites, while parasites in AS RBCs were either rings or early trophozoites, staging that remained constant over time in AS RBCs. Using flow cytometry to measure DNA content, we demonstrated that, as expected, DNA replication commenced at around 24–28 h (31) in parasites growing in hypoxic AA cells. In contrast, we found an absolute block in DNA replication in parasites growing in hypoxic AS erythrocytes (Fig. 1C). These data confirm, beyond what had been previously described using light microscopy, that parasite growth in AS RBCs is stalled at low O2 concentration.

Fig. 1.
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.

High O2 Concentration Reverses the Stalled Growth Phenotype in AS RBCs.

To examine the relationship between O2 and parasite growth, we incubated D10 parasites (selected to allow visualization of knob formation) at varying concentrations of O2 (1%, 3%, 5%, 7.5%, 10%, and 16%). D10 parasites growing in AA erythrocytes at all concentrations of O2 tested demonstrated DNA replication with equal kinetics from ring to schizont stage (Fig. 2A, Left). D10 parasites grew and replicated similarly in AS erythrocytes at high O2 concentrations (10% and 16%), while no DNA replication was evident at low concentrations (1%, 3%, and 5%) (Fig. 2A, Right). Significantly, an intermediate level of growth progression, 66% of the DNA content reached at 44 hpi in 10% and 16% O2, was evident at an O2 concentration of 7.5% (Fig. 2A, Right), the concentration corresponding to that measured in potential sites of sequestration in remote tissues (25⇓⇓⇓⇓–30). The effect of hypoxia on Pf growth was also observed in two additional falciparum laboratory strains, W2mef and FCR3-var2CSA (SI Appendix).

Fig. 2.
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.

When parasites were staged microscopically, there was a complete arrest in progression at the early trophozoite stage at 1% and 3% O2, a significant slowdown in progression to the late trophozoite stage at 5% O2, an intermediate progression to schizogony at 7.5% O2, and full progression to schizogony at 10% and 16% O2 (Fig. 2B, Right). However, when data on the parasite multiplication rate (PMR) (32) encapsulating an entire cycle of growth from rings to rings were collected, a complete lack of ring formation at 1%, 3%, and 5% O2 and a limited amount of reinvasion was noted at 7.5% O2 (Fig. 2C, Right). Wild-type levels were seen at 10% and 16% O2. In other words, while 83% and 91% of parasites in AS RBCs at 7.5% and 10% O2, respectively, become schizonts, a difference of <10%, the PMR in 7.5% O2 was only 36% that of parasites in AS RBCs cultured in 10% O2. This indicates that the partial growth inhibition to schizogony observed at 5% and 7.5% O2 results in more profound effects overall on proliferation. There was no significant difference in the presence of RBC knobs at either O2 concentration.

HbS Polymerization Is Associated with Impaired Hemoglobin Processing.

Hemoglobin processing was impaired, as demonstrated by the lack of hemozoin, an insoluble crystalline structure of free heme produced by the Pf 3D7 IG06 parasite, at 38 hpi in AS-infected RBCs incubated in 1% O2 (Fig. 3A, Left). We compared growth of parasites in AA and AS RBCs at low O2 concentration in multiple (n = 7) experiments using six different AS blood samples, and found that 97% of parasites in AA contained hemozoin within the food vacuole at 38 hpi (what should be the end of the intraerythrocytic life cycle for Pf 3D7 IG06), while only 4% of parasites in AS RBCs had any microscopic evidence of malaria pigment. We also found that the formation of hemozoin was dependent on O2 concentration. One hundred percent of D10 parasites in AS infected RBCs had hemozoin within the food vacuole at 44 hpi when cultured at 10% and 16% O2, that percentage decreased sharply at 1% and 3% O2. At 5% and 7.5%, we again observed an intermediate phenotype (Fig. 3A, Right).

Fig. 3.
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.)

Knowing that hemoglobin processing is affected and that HbS is known to form polymers at low O2, we confirmed the negative effect of HbS polymerization on DNA replication and growth using CO, a potent antisickling agent. We mapped the growth of Pf 3D7 IG06 in hypoxic AA and AS RBCs, but now in the presence and absence of CO. Parasites in CO-treated hypoxic AS cells replicated their DNA, as well as parasites in CO-exposed AA RBCs, suggesting that the prevention of HbS polymerization allowed for normal hemoglobin processing and albeit reduced, parasite growth (Fig. 3B). We directly measured the presence of HbS polymers in AA and AS erythrocytes exposed to different concentrations of O2 by electron microscopy. In electron micrographs, the deoxygenated sickle polymers were visible as discrete, black wavy lines in AS RBCs in 1% O2 (Fig. 3C, Bottom Left), consistent with the notion that HbS polymerization results in the inability of the parasite to process hemoglobin for growth and replication. The HbS polymers were notably absent in AS RBCs at 10% O2 and in AA RBCs at both 1% and 10% O2.

Time of Exposure to Hypoxia Dictates Parasite Growth Potential in AS RBCs.

At high O2 concentrations, similar to those in the peripheral blood, AS RBCs do not sickle; therefore, there must be a feature specific to the infected RBC environment that allows this mechanism of resistance to occur. During infection in vivo, cytoadherence of parasitized RBCs begins around 16 hpi and appears to be complete by 24 hpi (33). We sought to identify the temporal window within which movement of infected erythrocytes from the peripheral circulation to the low O2 environment found in remote tissues such as the bone marrow results in growth inhibition and its relationship to cytoadherence.

We measured the effect of transfer from high O2 to low O2 on the growth of parasite 3D7 IG06 by shifting parasites from a 10% O2 chamber to a 1% O2 concentration chamber at 16, 20, 24, 28, and 32 hpi (Fig. 4A). When moved at 16 and 20 hpi to a 1% O2 chamber, parasite development did not progress. If moved at 24 and 28 hpi, the parasite progressed, albeit at a reduced rate. However, if moved at 32 hpi, parasites progressed to nearly 90% of normal levels. Representative images of parasites at the time they were moved and at 38 hpi (end of growth) when switched at 16, 20, 24, 28, and 32 hpi confirm this phenotype (Fig. 4B). We also measured PMR (32) (Fig. 4C). PMR was greatly reduced if the shift to the hypoxic environment occurred before 32 hpi, while a normal PMR was observed when the shift occurred at 32 hpi. This suggested that the time during which parasites begin to sequester in the hypoxic microcirculation was also the point at which they are most sensitive to low O2-induced growth inhibition.

Fig. 4.
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.

HbS Polymerization-Induced Growth Inhibition Is Expected to Reduce Parasitemia More than Reduced Cytoadherence.

We developed a mathematical model to examine the effects of both HbS polymerization and reduced cytoadherence on parasite densities. Specifically, we used the staging data in Fig. 2B and PMR data from Fig. 4B to derive PMR and deduce proliferation rates at physiologic O2 concentrations or with a 50% [as estimated from Cholera et al. (16) and fitted from Kriek et al. (34)] reduction in PfEMP1 function (SI Appendix). Fig. 5A shows a schematic of our model structure and the parameters we varied to test the competing hypotheses: cycle length, mortality of both nonsequestered and sequestered parasites, and transition rate of nonsequestered and sequestered (parasite multiplication not shown). We conducted a sensitivity analysis to explore the impact of different low O2 concentrations, as this is variable and difficult to measure in vivo.

Fig. 5.
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 (μNE and μNL) and sequestered (μS) parasites, and transition rate of nonsequestered (λN) and (λS) 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.

The range of PMRs resulting from stalled development led to far greater, and sustained, reductions in parasite proliferative ability compared with reduced PfEMP1 expression for realistic parameter ranges (Fig. 5B). This reduced proliferative capacity in the stalled development was observed at O2 concentrations <10%, and the dynamical implications of stalling (Fig. 5C) showed that even at 5% and 7.5% O2, growth is significantly reduced but not entirely abolished, supporting the observed partial protection against malaria described in the literature. Thus, impaired growth in low O2 conditions, particularly if parasites are sequestered in variable O2 environments, may lead to significantly reduced, but nevertheless dynamic, growth rates. The model also demonstrates that measuring parasitemia at a single time point may be misleading, as growth patterns include both peaks (when parasites are nonsequestered) and troughs (when parasites are sequestered) (SI Appendix).

Discussion

Here we have demonstrated that low O2 concentrations impair parasite growth in RBCs containing HbAS. While the Hb polymerization observed does not typically occur in heterozygous carriers of HbS, it can occur in extreme conditions like the prolonged hypoxia occurring during sequestration. In our experiments, both infected and uninfected cells have prolonged exposure to hypoxic conditions and may even sickle at the cellular level. In vivo, we postulate that only infected RBCs that express PfEMP1 will demonstrate significant Hb polymerization, because they are the only cells that sequester into the hypoxic microvasculature. This was particularly significant at 5% and 7.5% O2 concentrations, which most closely simulate the environment in the microcirculation (25⇓⇓⇓⇓–30). At these O2 concentrations, we observed an intermediate phenotype that accounts for the incomplete phenotype of reduced parasite densities observed in AS individuals. It was also notable that in the 1% and 3% O2 environments, sickling was extensive and occurred in every parasitized cell. Parasite growth was stalled, as demonstrated by the lack of DNA replication. Morphologically, the parasites looked similar to those previously described in experiments in which P. falciparum parasites were grown in isoleucine-deprived culture media and stalled at the late/ring early trophozoite stage, but could be rescued with the introduction of isoleucine up to 72 h later (35). If sequestration occurred into a 1% or 3% O2 environment, then likely no infections would be seen in individuals with AS. The O2 concentrations within the postcapillary venules have been found to be closer to levels of O2 (between 5% and 10% O2) that could support an intermediate phenotype (25⇓⇓⇓⇓–30). Previous studies have observed stalling of parasites at low O2 in AS RBCs (11, 13⇓–15, 36).

Here we have extended the foregoing foundational studies by determining the growth effects along the physiologic range of O2 and characterizing the relationship between growth inhibition and hemoglobin polymerization. Our experiments show that the capacity to digest hemoglobin increases as O2 concentration increases to the level of normal DNA replication at 10% and above. We also demonstrate that CO, which prevents O2 from binding to hemoglobin and maintains the hemoglobin protein in the relaxed state as if it were bound to O2, restores DNA replication and growth, establishing the causal relationship between HbS polymerization and growth inhibition. CO is mildly toxic in P. falciparum, as evidenced by the slight delay in parasite growth in both AA and AS CO-treated RBCs and in studies demonstrating protection against malaria in mice (37), however the growth of the parasite in AS and AA RBCs at low O2 is equivalent when CO is present. We also show, via electron microscopy, that HbS polymer (12) formation occurs only at low O2 concentrations.

Some studies have observed that parasites can grow normally in AS RBCs at 3% and 5% O2 (19, 38⇓–40). This may be due to technical differences in culture format and growth assessments. We measured the O2 concentration in our incubator chambers directly using a Drager Pac III gas monitor sensor. We made precise estimates of growth by light microscopy and DNA replication by flow cytometry, and further visualized polymers with electron micrographs. We note that it is difficult to detect the growth defect from rings to early schizonts by light microscopy in both 5% and 7.5% O2 as some parasites mature; nonetheless, the effect of low O2 on proliferation, as evidenced by our PMR studies, is profound (Fig. 2).

We experimentally mapped the time at which low O2 has an effect on parasite growth to between 20 and 32 hpi. Since cytoadherence and hemoglobin digestion occur concomitantly, we postulated that once hypoxia-inducing cytoadherence occurs, growth inhibition results due to the inaccessibility of hemoglobin for protease digestion, a critical step in parasite development during which essential amino acids for protein expression, DNA replication, and parasite proliferation are made available (41). This is consistent with a model in which cytoadherence leads to HbS polymerization-induced impaired parasite growth in hypoxic AS RBCs.

The potential impact of reduced PfEMP1 function versus reduced HbS polymerization-induced impaired parasite growth on proliferation also has not been quantified. AS individuals are able to harbor infection; however, parasite density is greatly depressed (2, 5, 7, 18). We sought to compare the effect of impaired hemoglobin processing within physiologic O2 environments with the current leading hypothesis of AS resistance, i.e., reduced PfEMP1 expression and function. We used published PfEMP1 data (16), as performing the scientifically robust studies needed to properly assess function was beyond the scope of this work. Through modeling, we found that HbS polymerization-induced growth inhibition can produce such low parasite densities. In addition, despite assuming 50% PfEMP1 expression and function, the hypoxia-induced impairment in hemoglobin processing had a significantly greater effect on proliferation. It is interesting to note that the difference is dependent on when parasitemia is measured (SI Appendix), and may be a reason why impaired growth has been less well supported as a standalone hypothesis. Several of the other proposed resistance mechanisms (16⇓⇓⇓–20), including reduced cytoadherence, translocation of HbS-specific parasite growth- inhibiting microRNAs, and induction of heme-oxygenase, may contribute variably to parasite proliferation depending on the level of immunity and physiologic conditions such as temperature, acidosis, etc. However, our modeling shows that the mechanism of growth inhibition due to HbS polymerization in a hypoxic environment can be the major driver of infection reduction in individuals with AS.

Our studies demonstrate the importance of considering the physiologic environment encountered by parasitized AS cells in understanding the role of AS in malaria protection. This may also be relevant to other RBC disorders, including other hemoglobinopathies and, importantly, sickle cell disease (SS). Growth of P. falciparum is SS RBCs is also likely depressed, but this is likely complicated by the heterogeneity of the RBCs in patients with the disease. In the context of AS, while P. falciparum has evolved to cytoadhere and prevent splenic clearance, the host has evolved to continue to produce a mutation in hemoglobin, despite its lethality in the homozygous state, which protects against parasite infection by inducing growth inhibition in sequestered parasites, representing a clear case of coevolution of the host and parasite.

Materials and Methods

Plasmodium Parasite Culture.

The following in vitro culture-adapted P. falciparum laboratory strains were used: 3D7, D10, W2mef, and FCR3-var2CSA parasites (gift from Joe Smith, Center for Infectious Disease Research, Seattle). The D10 laboratory strain was enriched for knobs using gelatin flotation (42). Most experiments were performed using 3D7 IG06, with a shortened life cycle of approximately 40 h (gift from Daniel Goldberg, Washington University School of Medicine, St. Louis). P. falciparum lines were cultured (43) in human O+ erythrocytes from Research Blood Components in complete RPMI 1640 supplemented with 0.5% AlbuMAX (Invitrogen) and 0.25% sodium bicarbonate at 37 °C in 1% O2, 5% CO2, and balance N. The FCR3-var2CSA line was cultured at 4% hematocrit in RPMI 1640 supplemented with 10% human serum (Interstate Blood) and 0.25% sodium bicarbonate. AS blood was generously donated by parents of SS patients at Boston Children’s Hospital. Complete blood counts (Sysmex XN analyzer) and HPLC (Tosoh G7 HPLC analyzer) were performed to assess RBC size and confirm AS status.

Growth Assays.

For all growth assays, synchronized schizonts were obtained by magnet purification with the MACS system. Heparin (Sigma-Aldrich) was added at 4–6 h after magnet purification to maintain a tightly synchronized culture. After synchronization, parasites were allowed to mature to the schizont stage before being MACS-purified and introduced into 1% hematocrit wild-type (AA) or AS blood. RBC concentration was measured with a hemocytometer. Each experiment began with a starting parasitemia of 0.25–1% schizonts. Parasite growth was quantified for one to two life cycles by microscopy using May–Grunwald–Giemsa stained cytospins taken throughout the life cycle. Hemozoin quantification and parasite staging methods are described in SI Appendix. The O2 concentration in experimental modular incubator chambers (Billups-Rothenberg) was confirmed using a Dräger Pac III gas monitor detector sensor. For CO studies, CO was introduced into the enclosed chamber system at the start of the experiment and again at every 21 h of the assay to prevent sickling in low O2 environments. The CO-Hb (ABL800 FLEX blood gas analyzer) of blood was >20% at 21 h after gassing.

Flow Cytometry-Based Detection of DNA Content.

DNA content was analyzed by measuring mean fluorescence intensity (MFI) via flow cytometry as described previously (31). Parasite culture samples were fixed in 4% paraformaldehyde and 0.0075% glutaraldehyde (Electron Microscopy Sciences) in PBS, permeabilized by 0.1% Triton X-100 (Sigma-Aldrich), treated with RNase A from bovine pancreas (Sigma-Aldrich), and stained with SYBR Green, at a 1:10,000 dilution (Invitrogen).

Scanning Electron Microscopy.

A pellet of RBCs was fixed, washed, embedded, and sectioned as detailed by Ganter et al. (31) Images of the most advanced-stage parasites in AA and AS blood at 1% and 10% O2 were acquired.

Model.

We adapted an ordinary differential equation model of parasite development in RBCs (44) to track the population of P. falciparum-infected RBCs in the peripheral blood and in sequestered areas with low O2 concentrations. We assume that splenic clearance of infected RBCs (i.e., the death rate of nonsequestered infected cells) is related to PfEMP1 function, and that the function of PfEMP1 increases over time such that few trophozoites or schizonts are seen in the peripheral bloodstream, consistent with clinical observations. We also assume that sequestration (transition to a sustained low O2 environment) is facilitated by PfEMP1 expression via cytoadherence to host blood vessels. We compare proliferation rates that result from reducing PfEMP1 function by modulating the fraction of cells that become sequestered and have impaired growth in low O2 environments, “stalling” development in sequestered compartments, and using experimental data for parameterization.

In brief, the lifecycle was modeled as a series of 10-min transitions through various stages of parasite development, timed to reflect P. falciparum in AA cells. Mortality of nonsequestered cells via splenic clearance began at the early trophozoite stage, as in Gravenor et al. (44), consistent with the timing of PfEMP1 function. At the same time, sequestration of cells was modeled as a sigmoidal function of the developmental stage (hpi) fitted from Kriek et al. (34) (SI Appendix), corresponding to approximately 50% of cells sequestered by 18.58 hpi.

For clarity, we assume that merozoite formation, egress, and invasion processes are unchanged in AS RBCs, focusing on the stalling phenotype shown in Fig. 2B to parameterize the impact of growth impairment. This allows us to fit our model to observed data and examine the impact on in vivo dynamics in the absence of all the unmeasured complexities associated with egress and invasion. We model the in vivo trajectory of the acute stage of infection assuming no immune system effects, and calculate PMR as the number of ring stage infected RBCs at 52 hpi divided by the number of ring stage infected RBCs at 16 hpi. Parameter estimation and model fitting details are provided in SI Appendix.

Acknowledgments

We thank all the individuals with sickle cell trait who generously volunteered to donate blood for this study and we thank Drs. Carlo Brugnara, H. Franklin Bunn, Daniel Goldberg, David G. Nathan, and David A. Williams for their feedback on this project. We thank Christof Gruring for his guidance in many of our experiments, and Maria Ericsson, Elizabeth Benecchi, and Margaret Coughlin for preparing the EM images. This work was supported by a Harvard Catalyst Program for Faculty Development and Diversity Inclusion Faculty Fellowship, an American Society of Hematology/Robert Wood Johnson Foundation Harold Amos Medical Faculty Development Award, an Eleanor and Miles Shore Fellowship Program Dana-Farber Cancer Institute Fellowship, the Burroughs Wellcome Fund, and the National Heart, Lung, and Blood Institute (Grant 1R01 HL139337).

Footnotes

  • ↵1To whom correspondence should be addressed. Email: mduraisi{at}hsph.harvard.edu.

  • Author contributions: N.M.A., M.A.C., C.O.B., L.M.C., and M.T.D. designed research; N.M.A., N.P., C.O.B., and L.M.C. performed research; N.M.A., C.O.B., and L.M.C. analyzed data; and N.M.A., N.P., M.A.C., C.O.B., L.M.C., and M.T.D. wrote the paper.

  • The authors declare no conflict of interest.

  • This article is a PNAS Direct Submission.

  • This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1804388115/-/DCSupplemental.

Published under the PNAS license.

References

  1. ↵
    1. Piel FB,
    2. Hay SI,
    3. Gupta S,
    4. Weatherall DJ,
    5. Williams TN
    (2013) Global burden of sickle cell anaemia in children under five, 2010-2050: Modelling based on demographics, excess mortality, and interventions. PLoS Med 10:e1001484.
    OpenUrlCrossRefPubMed
  2. ↵
    1. Allison AC
    (1954) Protection afforded by sickle-cell trait against subtertian malareal infection. BMJ 1:290–294.
    OpenUrlFREE Full Text
  3. ↵
    1. Haldane JBS
    (1949) The rate of mutation of human genes. Hereditas 35:267–273.
    OpenUrl
  4. ↵
    1. Malaria Genomic Epidemiology Network,
    2. Kirk RA, et al.
    (2014) Reappraisal of known malaria resistance loci in a large multi-centre study. Nat Genet 46:1197–1204.
    OpenUrlCrossRefPubMed
  5. ↵
    1. Williams TN, et al.
    (2005) Sickle cell trait and the risk of Plasmodium falciparum malaria and other childhood diseases. J Infect Dis 192:178–186.
    OpenUrlCrossRefPubMed
  6. ↵
    1. Gonçalves BP, et al.
    (2014) Parasite burden and severity of malaria in Tanzanian children. N Engl J Med 370:1799–1808.
    OpenUrlCrossRefPubMed
  7. ↵
    1. Taylor SM,
    2. Parobek CM,
    3. Fairhurst RM
    (2012) Haemoglobinopathies and the clinical epidemiology of malaria: A systematic review and meta-analysis. Lancet Infect Dis 12:457–468.
    OpenUrlCrossRefPubMed
  8. ↵
    1. Billo MA, et al.
    (2012) Sickle cell trait protects against Plasmodium falciparum infection. Am J Epidemiol 176(Suppl 7):S175–S185.
    OpenUrlCrossRefPubMed
  9. ↵
    1. Miller MJ,
    2. Neel JV,
    3. Livingstone FB
    (1956) Distribution of parasites in the red cells of sickle-cell trait carriers infected with Plasmodium falciparum. Trans R Soc Trop Med Hyg 50:294–296.
    OpenUrlCrossRefPubMed
  10. ↵
    1. Mackey JP,
    2. Vivarelli F
    (1954) Sickle-cell anaemia (letter). Br Med J 1:276.
    OpenUrlFREE Full Text
  11. ↵
    1. Luzzatto L,
    2. Nwachuku-Jarrett ES,
    3. Reddy S
    (1970) Increased sickling of parasitised erythrocytes as mechanism of resistance against malaria in the sickle-cell trait. Lancet 1:319–321.
    OpenUrlCrossRefPubMed
  12. ↵
    1. Roth EF Jr, et al.
    (1978) Sickling rates of human AS red cells infected in vitro with Plasmodium falciparum malaria. Science 202:650–652.
    OpenUrlAbstract/FREE Full Text
  13. ↵
    1. Pasvol G
    (1980) The interaction between sickle haemoglobin and the malarial parasite Plasmodium falciparum. Trans R Soc Trop Med Hyg 74:701–705.
    OpenUrlCrossRefPubMed
  14. ↵
    1. Pasvol G,
    2. Weatherall DJ,
    3. Wilson RJ
    (1978) Cellular mechanism for the protective effect of haemoglobin S against P. falciparum malaria. Nature 274:701–703.
    OpenUrlCrossRefPubMed
  15. ↵
    1. Friedman MJ
    (1978) Erythrocytic mechanism of sickle cell resistance to malaria. Proc Natl Acad Sci USA 75:1994–1997.
    OpenUrlAbstract/FREE Full Text
  16. ↵
    1. Cholera R, et al.
    (2008) Impaired cytoadherence of Plasmodium falciparum-infected erythrocytes containing sickle hemoglobin. Proc Natl Acad Sci USA 105:991–996.
    OpenUrlAbstract/FREE Full Text
  17. ↵
    1. Cyrklaff M, et al.
    (2011) Hemoglobins S and C interfere with actin remodeling in Plasmodium falciparum-infected erythrocytes. Science 334:1283–1286.
    OpenUrlAbstract/FREE Full Text
  18. ↵
    1. Williams TN, et al.
    (2005) An immune basis for malaria protection by the sickle cell trait. PLoS Med 2:e128.
    OpenUrlCrossRefPubMed
  19. ↵
    1. LaMonte G, et al.
    (2012) Translocation of sickle cell erythrocyte microRNAs into Plasmodium falciparum inhibits parasite translation and contributes to malaria resistance. Cell Host Microbe 12:187–199.
    OpenUrlCrossRefPubMed
  20. ↵
    1. Ferreira A, et al.
    (2011) Sickle hemoglobin confers tolerance to Plasmodium infection. Cell 145:398–409.
    OpenUrlCrossRefPubMed
  21. ↵
    1. McAuley CF, et al.
    (2010) High mortality from Plasmodium falciparum malaria in children living with sickle cell anemia on the coast of Kenya. Blood 116:1663–1668.
    OpenUrlAbstract/FREE Full Text
  22. ↵
    1. Komba AN, et al.
    (2009) Malaria as a cause of morbidity and mortality in children with homozygous sickle cell disease on the coast of Kenya. Clin Infect Dis 49:216–222.
    OpenUrlCrossRefPubMed
  23. ↵
    1. Makani J, et al.
    (2010) Malaria in patients with sickle cell anemia: Burden, risk factors, and outcome at the outpatient clinic and during hospitalization. Blood 115:215–220.
    OpenUrlAbstract/FREE Full Text
  24. ↵
    1. Baruch DI, et al.
    (1997) Identification of a region of PfEMP1 that mediates adherence of Plasmodium falciparum-infected erythrocytes to CD36: Conserved function with variant sequence. Blood 90:3766–3775.
    OpenUrlAbstract/FREE Full Text
  25. ↵
    1. Carreau A,
    2. El Hafny-Rahbi B,
    3. Matejuk A,
    4. Grillon C,
    5. Kieda C
    (2011) Why is the partial oxygen pressure of human tissues a crucial parameter? Small molecules and hypoxia. J Cell Mol Med 15:1239–1253.
    OpenUrlCrossRefPubMed
  26. ↵
    1. Harrison JS,
    2. Rameshwar P,
    3. Chang V,
    4. Bandari P
    (2002) Oxygen saturation in the bone marrow of healthy volunteers. Blood 99:394.
    OpenUrlFREE Full Text
  27. ↵
    1. Meixensberger J,
    2. Dings J,
    3. Kuhnigk H,
    4. Roosen K
    (1993) Studies of tissue PO2 in normal and pathological human brain cortex. Acta Neurochir Suppl (Wien) 59:58–63.
    OpenUrlPubMed
  28. ↵
    1. Dings J,
    2. Meixensberger J,
    3. Jäger A,
    4. Roosen K
    (1998) Clinical experience with 118 brain tissue oxygen partial pressure catheter probes. Neurosurgery 43:1082–1095.
    OpenUrlCrossRefPubMed
  29. ↵
    1. Brezis M,
    2. Rosen S
    (1995) Hypoxia of the renal medulla: Its implications for disease. N Engl J Med 332:647–655.
    OpenUrlCrossRefPubMed
  30. ↵
    1. Spencer JA, et al.
    (2014) Direct measurement of local oxygen concentration in the bone marrow of live animals. Nature 508:269–273.
    OpenUrlCrossRefPubMed
  31. ↵
    1. Ganter M, et al.
    (2017) Plasmodium falciparum CRK4 directs continuous rounds of DNA replication during schizogony. Nat Microbiol 2:17017.
    OpenUrl
  32. ↵
    1. Chotivanich K, et al.
    (2000) Parasite multiplication potential and the severity of falciparum malaria. J Infect Dis 181:1206–1209.
    OpenUrlCrossRefPubMed
  33. ↵
    1. Silamut K,
    2. White NJ
    (1993) Relation of the stage of parasite development in the peripheral blood to prognosis in severe falciparum malaria. Trans R Soc Trop Med Hyg 87:436–443.
    OpenUrlCrossRefPubMed
  34. ↵
    1. Kriek N, et al.
    (2003) Characterization of the pathway for transport of the cytoadherence-mediating protein, PfEMP1, to the host cell surface in malaria parasite-infected erythrocytes. Mol Microbiol 50:1215–1227.
    OpenUrlCrossRefPubMed
  35. ↵
    1. Babbitt SE, et al.
    (2012) Plasmodium falciparum responds to amino acid starvation by entering into a hibernatory state. Proc Natl Acad Sci USA 109:E3278–E3287.
    OpenUrlAbstract/FREE Full Text
  36. ↵
    1. Friedman MJ,
    2. Roth EF,
    3. Nagel RL,
    4. Trager W
    (1979) The role of hemoglobins C, S, and Nbalt in the inhibition of malaria parasite development in vitro. Am J Trop Med Hyg 28:777–780.
    OpenUrlAbstract/FREE Full Text
  37. ↵
    1. Weinberg JB,
    2. Lopansri BK,
    3. Mwaikambo E,
    4. Granger DL
    (2008) Arginine, nitric oxide, carbon monoxide, and endothelial function in severe malaria. Curr Opin Infect Dis 21:468–475.
    OpenUrlCrossRefPubMed
  38. ↵
    1. Kilian N, et al.
    (2013) Haemoglobin S and C affect the motion of Maurer’s clefts in Plasmodium falciparum-infected erythrocytes. Cell Microbiol 15:1111–1126.
    OpenUrlCrossRefPubMed
  39. ↵
    1. Orjih AU
    (2005) Comparison of Plasmodium falciparum growth in sickle cells in low oxygen environment and candle-jar. Acta Trop 94:25–34.
    OpenUrlCrossRefPubMed
  40. ↵
    1. Cyrklaff M, et al.
    (2016) Oxidative insult can induce malaria-protective trait of sickle and fetal erythrocytes. Nat Commun 7:13401.
    OpenUrlCrossRef
  41. ↵
    1. Liu J,
    2. Istvan ES,
    3. Gluzman IY,
    4. Gross J,
    5. Goldberg DE
    (2006) Plasmodium falciparum ensures its amino acid supply with multiple acquisition pathways and redundant proteolytic enzyme systems. Proc Natl Acad Sci USA 103:8840–8845.
    OpenUrlAbstract/FREE Full Text
  42. ↵
    1. Goodyer ID,
    2. Johnson J,
    3. Eisenthal R,
    4. Hayes DJ
    (1994) Purification of mature-stage Plasmodium falciparum by gelatine flotation. Ann Trop Med Parasitol 88:209–211.
    OpenUrlPubMed
  43. ↵
    1. Trager W,
    2. Jensen JB
    (2005) Human malaria parasites in continuous culture. 1976. J Parasitol 91:484–486.
    OpenUrlCrossRefPubMed
  44. ↵
    1. Gravenor MB, et al.
    (2002) A model for estimating total parasite load in falciparum malaria patients. J Theor Biol 217:137–148.
    OpenUrlCrossRefPubMed
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Resistance to Plasmodium falciparum in sickle cell trait erythrocytes is driven by oxygen-dependent growth inhibition
Natasha M. Archer, Nicole Petersen, Martha A. Clark, Caroline O. Buckee, Lauren M. Childs, Manoj T. Duraisingh
Proceedings of the National Academy of Sciences Jul 2018, 115 (28) 7350-7355; DOI: 10.1073/pnas.1804388115
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