Ability of Plasmodium falciparum to invade Southeast Asian ovalocytes varies between parasite lines

Invasion of host red blood cells (RBCs) by the merozoite is a pivotal step in the life cycle of the malaria parasite Plasmodium falciparum and a clear target for vaccine development. The process of invasion is complex and involves multiple ligand-receptor interactions that are still poorly understood. Additional complexity of this process comes from the heterogeneity among P falciparum lines in the use of particular receptors, as demonstrated by experiments with enzyme-treated and mutant RBCs.^1,2 

Malaria is presumed to be the single infectious disease that has had the biggest impact on the human genome, with some genetic traits maintained at relatively high frequencies in malaria endemic populations because of the selective advantage that they confer against this disease.³  Many of these polymorphisms involve proteins that are expressed in the RBC, including hemoglobin and proteins located at the surface of RBCs. However, it is not completely clear yet whether any of these polymorphisms confers a selective advantage against malaria by preventing invasion by P falciparum merozoites.

Southeast Asian ovalocytosis (SAO) is a peculiar RBC abnormality that results from a 27-bp deletion in the gene encoding band 3(AE1Δ27),⁴  the major transmembrane protein in the RBC. Band 3 has a cytoplasmic N-terminal domain and a C-terminal domain with 12 or 14 membrane-spanning regions.⁵  This protein functions as an anion transporter but also has a role in maintaining the integrity of the RBC by anchoring the membrane to the underlying cytoskeleton. The 9-amino acid deletion that defines the SAO trait is located at the boundary between the cytoplasmic domain and the first transmembrane segment and results in major alterations in the folding and function of the membrane domain.^6-9  The SAO trait is found only in the heterozygous state and is presumed to be lethal when homozygous,¹⁰  but the presence of mutated band 3 has dominant effects. The mutant band 3 induces conformational changes in essentially all of the normal fraction of the protein,⁷  which is explained by the predominance of band 3 heterotetramers, higher order hetero-oligomers, and aggregates in SAO RBCs, in contrast to normal RBCs where band 3 is mainly found as dimers.^7,11,12  SAO RBCs are deficient in anion transport⁹  and their mechanical properties are also dramatically altered. These RBCs are oval in shape and exhibit reduced cell deformability and an extreme increase in membrane rigidity.^13-15  Furthermore, mutant band 3 results in a decrease in the expression of several RBC antigens.^8,16 

SAO also has an effect on susceptibility to malaria. Despite lethal selection against homozygous SAO carriers, this trait reaches high prevalence in some parts of the Western Pacific,¹⁷  and the prevalence of this trait correlates with malaria endemicity in Papua New Guinea (PNG).¹⁰  Two independent epidemiologic studies demonstrated that the SAO trait confers a striking protection against cerebral malaria,^18,19  one of the most devastating complications of the disease. Remarkably, no single SAO individual was found to suffer from cerebral malaria in the 2 studies. On the other hand, SAO individuals are fully susceptible to severe malaria anemia and clinical uncomplicated malaria.¹⁸  Although early studies, conducted before the presence of the SAO trait could be determined unambiguously by polymerase chain reaction (PCR), suggested that SAO individuals exhibit some degree of protection against prevalence of malaria parasites or against high-density P falciparum infections,^20-22  later studies demonstrated that SAO individuals suffer high-density infections by P falciparum and this trait only has minor effects, if any, on the prevalence and density of P falciparum infections.^18,23 

Our recent finding that SAO infected RBCs (IRBCs) have altered adherence properties compared with normal IRBCs (A.C., M. Mellombo, C.S. Mgone, H.P. Beck, J.C.R., and B.M.C., submitted August 2004), together with the results of the epidemiologic studies, suggests that protection of SAO individuals is achieved by differences in postinvasion processes and not in the process of invasion itself. However, band 3 has been proposed to play a role in invasion,^24,25  perhaps by interacting with the merozoite proteins merozoite surface protein 1 (MSP1) and MSP9.^26,27  Additionally, several early studies found SAO RBCs to be highly resistant to invasion in culture by P falciparum.^14,28  The latter observation is at odds with the in vivo observation of SAO individuals suffering from high-density P falciparum infections.¹⁸  A later study found that SAO RBCs can be invaded in culture to at least 55% the level for normal RBCs¹³  and attributed the previous results to deterioration of SAO RBCs upon cold storage.^13,29  Here, we studied the invasion of SAO RBCs immediately after drawing the blood and found that they were resistant to invasion in culture by several, but not all, parasite lines. We identified 2 presumably isogenic parasite lines, one of which invaded SAO RBCs efficiently whereas the other did not, and found that the capacity to invade SAO RBCs efficiently was paralleled by a dramatic change in the use of different receptors for invasion. Efficient invasion of SAO RBCs was highly dependent on a chymotrypsin-sensitive RBC receptor.

Noninfected blood samples were collected from age- and geography-matched SAO and control (non-SAO) individuals from PNG. Blood was collected in citrate-phosphate-dextrose (CPD) anticoagulant and used for invasion assays immediately after collection, unless otherwise specified. The band 3 and glycophorin C exon 3 status of all the samples was determined by PCR as described previously.^4,30 

Blood from SAO individuals with high-density P falciparum infections was collected from individuals attending the Alexishafen Health Center (Sek, Madang Province, PNG). The SAO status was assessed in the health center using wet preparations and later confirmed by PCR. Approval for these studies was obtained from the PNG Medical Research Advisory Committee. Informed consent was provided according to the Declaration of Helsinki.

Cultures were maintained in Petri dishes in standard RPMI-based parasite culture media supplemented with Albumax II³¹  (Gibco BRL, Auckland, New Zealand) under an atmosphere of 93% N₂:5% CO₂:2% O₂, unless otherwise specified. CS2, E8B, D10, and 3D7 laboratory-adapted parasite lines have been described previously.^32-34  We used 2 different 3D7 parasite lines, which we have termed 3D7-A and 3D7-B. Both 3D7-A and 3D7-B were derived from the same cloned parasite line, 3D7, which was derived from NF54.³⁴  However, we obtained them from different laboratories (Swiss Tropical Institute, Basel, Switzerland; and Monash University, Victoria, Australia) where they had been maintained in culture for several years. To confirm that both lines actually correspond to the 3D7 line and to rule out a labeling error or a contamination, we analyzed the highly polymorphic msp2 restriction fragment length polymorphism (RFLP) pattern and sequenced the ama1 domain I, which contains 31 polymorphic positions.³⁵  The msp2 genotyping by RFLP and PCR amplification and sequencing of the ama1 domain I were performed as previously described.³⁵  The msp2 RFLP pattern and the sequence of the ama1 domain I were identical between both parasite lines (not shown) and were consistent with the published data for these markers in 3D7.

Schizonts were purified from synchronized cultures to purity higher than 95% (typically 99%) using percol-sorbitol gradients,³⁶  then added to washed SAO and normal RBCs to an initial parasitemia typically between 1% and 2%. The assays were performed either in 35-mm diameter Petri dishes at 4% hematocrit (experiments with untreated RBCs) or in 96-well plates at 3% hematocrit (experiments with enzyme-treated RBCs). Giemsastained thin smears were prepared after 18 to 22 hours. Slides were examined by counting the number of infected RBCs in 1000 to 3000 RBCs (depending on the parasitemia). The exceptions were slides that were considered as negative for the presence of parasites in a first examination. For these slides, the number of RBCs in a representative field was determined and similar fields were examined until it was estimated that more than 10 000 RBCs had been screened for the presence of parasites.

Washed RBCs were treated with either neuraminidase (Calbiochem, Nottingham, United Kingdom; catalog no. 480700, 32 U/μL in RPMI-HEPES [RPMI-N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid] buffer, pH 6.8, 1.8% hematocrit); TPCK (N-alpha-tosyl-l-phenylalanine chloromethyl ketone)-treated trypsin (Sigma, St Louis, MO; catalog no. T-8802, 1.2 mg/mL in RPMI-HEPES, pH 7.4, 0.6% hematocrit); or TLCK (N-tosyl-l-lysine chloromethyl ketone)-treated chymotrypsin (Sigma; catalog no. C-3142, 1.2 mg/mL in RPMI-HEPES, pH 7.4, 0.6% hematocrit) for 1.5 hours at 37°C in a rotating wheel. Treatment with trypsin or chymotrypsin was always followed by one wash in RPMI-HEPES and a 10-minute treatment at room temperature with soybean trypsin inhibitor (Sigma; catalog no. T-6522, 0.5 mg/mL). For double treatment with neuraminidase plus trypsin, RBCs were first digested with neuraminidase, then washed twice, then digested with trypsin. RBCs treated with either enzyme were washed twice before resuspension in complete culture media and used immediately in invasion assays.

SAO RBCs assayed immediately after venopuncture were highly resistant to invasion by several laboratory-adapted parasite lines. The level of invasion was about 20% of that for normal (non-SAO) RBCs from matched donors for the parasite lines CS2, E8B, D10, and 3D7-B (Table 1). Parasites of the FCR3 line and 3 field isolates obtained from non-SAO PNG donors also failed to invade SAO RBCs efficiently (data not shown). In contrast to these results, parasites of the 3D7-A line invaded SAO RBCs almost as efficiently as normal RBCs. Invasion of SAO RBCs by 3D7-A was 80% of that for normal cells (Table 1). This parasite line grew in culture in SAO RBCs for several cycles almost as well as in normal RBCs and reached parasitemias as high as 12% in SAO RBCs.

Even in the parasite lines that failed to invade SAO RBCs efficiently, the few rings that had invaded these cells developed into morphologically normal pigmented trophozoites and schizonts, and these schizonts produced fully viable merozoites capable to invade normal RBCs efficiently. Thus, SAO RBCs were resistant to invasion by several parasite lines, but the parasites from these lines developed normally in these cells after invasion.

We tested several different culture conditions, including different concentrations of oxygen (2%, 5%, and 20%), different pH of the culture media, and different supplementation of the culture media (human serum instead of Albumax II). We also studied the effect of collecting blood in different anticoagulants (CPD, CPDA [citrate phosphate dextrose adenine], and EDTA [ethylenediaminetetraacetic acid]). None of these conditions increased the invasion of SAO RBCs by any of the lines relative to invasion in standard culture conditions. Instead, increasing the pH of the culture media resulted in even lower levels of invasion of SAO RBCs.

The dramatic difference in invasion of SAO RBCs between 3D7-A and 3D7-B parasites was consistent for RBCs from 8 age- and geography-matched pairs of SAO-control donors, and there were only minor differences in invasion by either line among SAO or among normal RBCs from different donors. Invasion by other parasite lines was also tested with RBCs from at least 2 pairs of donors. The 3D7-A parasite line also invaded much more efficiently than 3D7-B RBCs from 4 different SAO donors that were not used immediately after collection. The resistance of SAO RBCs to invasion in culture by several parasite lines cannot be explained by co-occurrence of SAO with the Gerbich-negative phenotype because none of the SAO donors in these experiments was either homozygous or heterozygous for the glycophorin C exon 3 deletion (GYPCΔex3), which determines Gerbich negativity in Melanesians.³⁰ 

Storage of SAO RBCs for up to 4 days, either at 4°C or at 25°C, had only a limited effect on invasion by all parasite lines tested. Invasion of SAO RBCs that had been stored for 4 days decreased by less than 50% compared with invasion of fresh SAO RBCs. In contrast, storage at 25°C for periods of time of 8 days or longer resulted in abundant hemolysis and a drastic loss of invasion of SAO RBCs compared with storage for the same period of time at 4°C, which had only a limited effect on their invadability.

To determine whether the different capacity of 3D7-A and 3D7-B to invade SAO RBCs is associated with the use of different receptors for invasion, we studied invasion of enzyme-treated normal RBCs (European origin) by the 2 parasite lines. The 3D7-A parasite line invaded RBCs that had been treated with either neuraminidase, trypsin, or chymotrypsin much more efficiently than 3D7-B (Table 2). The difference was especially large for chymotrypsin-treated RBCs, which were invaded to almost 50% of the extent of untreated RBCs by 3D7-A but were almost completely resistant to invasion by 3D7-B. Noteworthy, 3D7-A invaded RBCs double-digested with neuraminidase plus trypsin, whereas these RBCs were completely resistant to invasion by 3D7-B (no single ring was observed in many experiments; Table 2). The 3D7-A parasites that invaded these double-treated RBCs developed into morphologically normal pigmented trophozoites and fully invasive schizonts. The 3D7-A parasite line could be maintained in culture in these RBCs for several generations, and the parasitemia remained approximately stationary. Further digestion of these double-treated RBCs with chymotrypsin prevented invasion by 3D7-A almost completely (more than 99% inhibition), but a few healthy rings could still be observed in these triple-treated RBCs, in contrast to the complete absence of rings in double-treated RBCs with 3D7-B parasites.

Invasion of SAO RBCs by 3D7-A parasites was highly dependent on the presence of a chymotrypsin-sensitive RBC receptor(s). Comparison of the effect of enzyme treatment of SAO and normal RBCs on invasion by 3D7-A revealed that treatment with either neuraminidase or trypsin had a similar effect on invasion of both types of cells (Figure 1). Double treatment with neuraminidase plus trypsin had a bigger effect on invasion of SAO RBCs, but the difference was not significant. On the other hand, chymotrypsin treatment had a much bigger effect on invasion of SAO RBCs (P < .05). This result indicates that efficient invasion of SAO RBCs depends on a chymotrypsin-sensitive receptor(s) more than invasion into normal RBCs. The difference could not be explained by deletion of the glycophorin C exon 3. One of the control donors was homozygous for the glycophorin C exon 3 deletion, but these RBCs were invaded normally and also showed a normal pattern of susceptibility to enzyme treatment, indicating that Gerbich negativity does not have an effect on invasion of RBCs, even when the repertoire of other receptors available is limited by enzymatic treatment.

On the other hand, the residual invasion of SAO RBCs by 3D7-B was highly dependent on neuraminidase-sensitive, trypsin-sensitive, and chymotrypsin-sensitive receptors (Figure 1), similar to invasion of normal RBCs by this parasite line.

Parasites that produced high-density infections in SAO individuals failed to invade SAO RBCs efficiently in culture. Blood was collected from 3 SAO individuals with high-density P falciparum infections (> 1%) and the parasites cultured in vitro to the late trophozoite stage. The culture was then diluted 1:5 with freshly collected uninfected RBCs from either a different SAO donor or from a control donor. Invasion was determined by counting the number of ring-stage parasites after 24 hours (Figure 2A). Using this approach, the same parasites were tested to invade both types of RBCs, and any differences observed were attributable to the presence of mutated band 3. In the 3 independent experiments, parasites invaded normal RBCs efficiently but failed almost completely to invade SAO RBCs (Figure 2B).

In good agreement, none of 8 isolates obtained from SAO individuals reinvaded efficiently in culture RBCs from the original donor (the invasion rate was always less than one), whereas the majority of isolates from control individuals reinvaded efficiently.

The SAO trait confers a marked resistance to invasion in culture by many P falciparum lines that is unmatched by any other known human genetic trait. Contrary to observations with other RBC polymorphisms, such as the sickle cell trait, this resistance occurred under all conditions tested, including different oxygen concentrations. Resistance was observed with SAO RBCs assayed immediately after collection, but we found that storage of these RBCs at 4°C had only a limited effect on their invadability. Thus, we could not support previous suggestions that resistance to invasion of SAO RBCs was the consequence of a structural change induced by cold storage resulting in cation leakage and adenosine triphosphate (ATP) depletion.^13,29  For the first time, we studied invasion of freshly collected SAO RBCs by multiple parasite lines, and our results here clearly indicate that the discrepancy between previous studies relates to the different parasite lines used.^13,14,28 

The presence of mutated band 3 results in major alterations to the mechanical and antigenic properties of the RBC membrane. Consequently, the resistance of SAO RBCs to invasion by many parasite lines cannot be directly attributed to a defective interaction of merozoite ligands with band 3 because it could also be explained by the increased rigidity of SAO RBCs^13-15  or by the decreased mobility of band 3 in SAO that may prevent the formation of a smooth protein-depleted membrane patch that may be necessary for invasion.^37,38  In support of the latter possibility, antibodies against the cytoplasmic domain of band 3 that limit its mobility also inhibited invasion.³⁹  Still another possible explanation for the resistance to invasion of SAO RBCs is a defective interaction of the merozoite with RBC receptor(s) whose distribution is altered by the presence of mutated band 3.^8,16  It is important to note that band 3 might be part of a large membrane complex including glycophorin A and glycophorin B, which are used by P falciparum as receptors for invasion, and the Rh proteins.^8,40 

To characterize the molecular interactions involved in efficient invasion of SAO RBCs, we studied the invasion phenotype of 3D7-A by enzymatic treatment of RBCs. The dramatically increased capacity of 3D7-A compared with 3D7-B (derived from the same cloned parasite) to invade SAO RBCs was paralleled by a strong increase in its ability to invade RBCs treated with either neuraminidase, trypsin, or chymotrypsin. Furthermore, 3D7-A invaded RBCs sequentially treated with neuraminidase plus trypsin. To our knowledge, this is the first report of relatively efficient invasion of such double-treated RBCs. All the RBC receptors that mediate the different invasion pathways so far described are sensitive to either neuraminidase or trypsin. Binding to glycophorin A, B, and C and to the receptors of unknown molecular identity termed receptors E and Y depends on sialic acid residues that are removed by digestion with neuraminidase, and trypsin cleaves glycophorin A and C and receptor X.^1,41-44  The receptor Z, which is the putative receptor for the parasite ligand normocyte binding protein 2b (PfNBP2b),⁴⁵  is resistant to treatment with neuraminidase or low concentration of trypsin (0.067 mg/mL) but sensitive to treatment with high concentration of trypsin (1 mg/mL). Thus, invasion by 3D7-A occurs after disruption of all known RBC receptors for invasion, indicating that it can use an RBC receptor that has a different molecular entity from all the other known receptors for invasion, and henceforth we will refer to it as receptor A. Receptor A is resistant to treatment with neuraminidase and to treatment with a high concentration of trypsin but is probably at least partially sensitive to chymotrypsin, as indicated by experiments with triple-digested RBCs.

The only parasite line that we identified able to invade SAO RBCs efficiently is also the only line described so far that can invade neuraminidase-plus-trypsin-treated cells. We predict that the molecular change(s) in 3D7-A relative to 3D7-B, either a different level of expression of particular gene(s) or a mutation/genetic rearrangement, must determine the more efficient invasion of both enzyme-treated RBCs and SAO RBCs by 3D7-A, probably by conferring to the merozoite the capacity to interact with additional receptor(s) such as the receptor A. Invasion of SAO RBCs by 3D7-A was similarly efficient as invasion of normal RBCs after treatment with neuraminidase or trypsin but was almost completely abolished by digestion with chymotrypsin, indicating that efficient invasion of SAO RBCs mainly depends on a receptor(s) that is relatively neuraminidase and trypsin resistant but sensitive to chymotrypsin. Thus, the only well-characterized receptors for invasion by P falciparum, glycophorin A⁴⁴  and glycophorin C,^42,46  are not involved in efficient invasion of SAO RBCs. The enzyme susceptibility of the receptor essential for invasion of SAO RBCs clearly resembles that described for receptor A. Interestingly, band 3 itself is chymotrypsin sensitive but neuraminidase and trypsin resistant and is paradoxically a strong candidate to be the receptor used by 3D7-A for the efficient invasion of SAO RBCs, despite being the protein that is mutated in these RBCs. The topology of some of the extracellular loops of band 3 is not altered by the 9-amino acid deletion.⁶ 

The observation that only one parasite line invaded SAO RBCs efficiently in culture suggested that a similar situation might occur in the field, with some parasites able to infect SAO individuals and others failing to do so. This hypothesis is consistent with the observation that SAO cells can support high parasitemias in vivo.¹⁸  However, we could not prove this hypothesis because parasites obtained from SAO individuals with high-density P falciparum infections, which presumably invaded SAO RBCs efficiently in vivo, failed to invade SAO RBCs in culture. Two possible explanations can account for this observation: (i) SAO RBCs undergo some modification when they are taken out of the circulation that makes them resistant to invasion by several but not all parasite lines; or (ii) parasites undergo some modification when they are removed from the physiologic environment of the circulation that makes them unable to invade SAO RBCs. This hypothetical modification might involve lack of expression of some merozoite protein(s) essential for invasion of these RBCs, presumably including the ligand for receptor A. This expression would have to be induced in each cycle by some signal that occurs in vivo but not in culture conditions. It is well known that the expression of many merozoite genes is tightly regulated and occurs only late in the asexual cycle. Some parasite lines, like 3D7-A and the parasites used by Dluzewski et al¹³  that invaded SAO RBCs efficiently, might have adapted to express this hypothetical protein in the absence of a signal. This is an important observation that clearly deserves further investigation to determine how closely studies of invasion performed in culture reflect the situation in vivo. If the expression of some proteins involved in invasion is lost under the conditions of in vitro culture, the process of invasion in vivo might have an unsuspectedly higher level of complexity. In this regard, it has been shown that under pressure it is possible for P falciparum-cloned populations to be induced to alter their invasion phenotypes in vitro and exhibit invasion through a new receptor pathway.⁴⁷ 

The specific protection against cerebral malaria conferred by the SAO trait^18,19  cannot be satisfactorily explained by differences in the process of invasion alone. Partial resistance to invasion by most or all of the parasite lines circulating in the field would presumably be reflected in protection against a broader spectrum of presentations of the disease, including severe malaria anemia and uncomplicated malaria, and resistance against only a small subset of the parasite lines would probably be without effect. Instead, altered adhesive interactions of SAO-infected RBCs with endothelial cells provide a more plausible explanation for the specific protection against cerebral malaria observed, and we recently showed that SAO-infected RBCs have a different adhesive behavior that possibly determines a different distribution of sequestered parasites, such that fewer would be sequestered in the brain (A.C., M. Mellombo, C.S. Mgone, H.P. Beck, J.C.R., and B.M.C., submitted August 2004). It is noteworthy that a single genetic trait, SAO, has such a dramatic effect on invasion and cytoadherence. It can be speculated that this trait originally expanded among Melanesian populations because of the protection that it conferred against invasion by malaria parasites, but later on invasion pathways that permitted invasion of these mutant RBCs arose in parasite populations. Then the trait would have been maintained because of a second layer of protection, the altered adhesiveness of infected erythrocytes that possibly results in specific protection against cerebral malaria.

The authors are thankful to all the blood donors in the Madang North coast and among Papua New Guinea Institute of Medical Research (PNGIMR) staff for donating blood and to staff at the Alexishafen Health Center for their contribution to this study. We are also thankful to Mata Mellombo and Alice Ura for technical assistance in the lab and in the field and to Livingstone Tavul and Rebecca Samen for technical assistance in the field.