Malaria that is caused by Plasmodium falciparum is a significant global health problem. Genetic characteristics of the host influence the severity of disease and the ultimate outcome of infection, and there is evidence of coevolution of the plasmodium parasite with its host. In humans, pyruvate kinase deficiency is the second most common erythrocyte enzyme disorder. Here, we show that pyruvate kinase deficiency provides protection against infection and replication of P. falciparum in human erythrocytes, raising the possibility that mutant pyruvate kinase alleles may confer a protective advantage against malaria in human populations in areas where the disease is endemic.
Genetic studies of susceptibility to malaria in a mouse model for the erythroid stage of the disease, with the use of infection with P. chabaudi, have localized a number of major loci affecting the extent of parasite replication at the peak of infection. Recombinant congenic mouse strains AcB55 and AcB61 are very resistant to infection with P. chabaudi; resistance in these strains segregates as a recessive monogenic trait caused by a mutation (Ile90Asn) in the gene for pyruvate kinase (Pklr).8,9 The purpose of this study was to determine whether pyruvate kinase deficiency protects humans against malaria and to elucidate the molecular basis of a putative protective effect.
Pyruvate kinase catalyzes the rate-limiting step of glycolysis, converting phosphoenolpyruvate to pyruvate with the generation of one molecule of ATP. In the absence of mitochondria (which are lacking in mature erythrocytes), the enzyme is critical to energy production. Pyruvate kinase deficiency is the most frequent abnormality of the glycolytic pathway and, together with a deficiency in glucose-6-phosphate dehydrogenase (G6PD), is the most common cause of nonspherocytic hemolytic anemia. Pyruvate kinase deficiency is inherited as an autosomal recessive trait and is caused by loss-of-function mutations in PKLR. The prevalence of homozygous pyruvate kinase deficiency is estimated at 1 case per 20,000 persons; more than 158 mutations have been described.10,11
Methods
Subjects
From January 2006 to June 2007, subjects attending hematology clinics at the Toronto General Hospital and the Hospital for Sick Children who were identified as having pyruvate kinase deficiency on the basis of the clinical presentation and the results of an enzyme assay were eligible for enrollment in this study. Their asymptomatic relatives were also eligible for enrollment. The study was approved by the institutional review board at each center, and all subjects provided written informed consent.
We ruled out the presence of other hemolytic disorders by hemoglobin electrophoresis and assessment of the G6PD level. Subjects with homozygous pyruvate kinase deficiency included a 39-year-old man of Italian ancestry (Subject 1) and two women: 39-year-old Subject 2, also of Italian ancestry, and 19-year-old Subject 3, of French ancestry. All subjects had nonspherocytic anemia. Subject 3 was transfusion-dependent, and Subjects 1 and 2 had undergone splenectomy. A blood sample was drawn from Subject 3 before she underwent transfusion. The majority of humans with pyruvate kinase deficiency are compound heterozygotes with respect to the mutation of PKLR.10,12 The subjects in this study had not been previously genotyped to determine the genetic basis of their enzyme deficiency.
Identification of PKLR Mutation
Genomic DNA was isolated from the buffy coat of blood samples from subjects with pyruvate kinase deficiency (case subjects) and persons without pyruvate kinase deficiency (control subjects) with the use of proteinase K, phenol–chloroform extraction, and isopropanol precipitation. DNA (60 ng) — specifically, the 12 coding exons of PKLR, including intron–exon junctions — was used as a template for amplication by polymerase chain reaction (PCR), with 22 to 25 cycles at annealing temperatures ranging from 56° to 58°C. PCR products were purified and sequenced with the use of cycle sequencing with fluorescent nucleotides. Traces were analyzed with the use of BioEdit (www.mbio.ncsu.edu/BioEdit/bioedit.html), and all mutations were confirmed by sequence analysis.
Parasite Culture
P. falciparum clones ITG and 3D7 (mycoplasma-free) were maintained in continuous culture.13 To assess parasite invasion and maturation, schizonts from synchronized cultures14 were mixed with erythrocytes from case subjects and control subjects, as described previously.15 In all samples, invasion of erythrocytes was assessed at 24 hours, 72 hours, and 120 hours, and maturation was assessed at 48 hours, 96 hours, and 144 hours.
Phagocytosis Assay
Human monocytes were isolated and purified from the peripheral blood of healthy donors, as described previously.16 Thioglycollate-elicited macrophages were harvested from the peritoneal fluid of C57BL/6 mice.17 A total of 1.5x105 cells per well were plated on glass coverslips in 24-well plates and incubated for 5 days. All washed erythrocytes, including those infected with P. falciparum and those uninfected, underwent opsonization with 50% fresh autologous serum for 30 minutes at 37°C. Erythrocytes were then washed twice, resuspended at 10% hematocrit, and incubated with macrophages adhered to glass coverslips at a target-to-effector ratio of approximately 40:1. Phagocytosis assays were performed and assessed as described previously.18 All experiments were performed in duplicate and repeated at least three times.
Erythrocyte Membrane Analysis
Bound hemichromes, IgG, and C3c fragments were measured as described previously.15,18 For ring-stage infected erythrocytes, the values were normalized to 100% parasitemia with the use of the following formula: I=(Tot–Nxn)÷(1–n), as described previously,15 in which I indicates the amount of bound IgG and C3c in 100% rings; Tot, the amount of bound IgG and C3c in the whole culture; N, the amount of bound IgG and C3c in erythrocytes without parasites; and n, the fraction of erythrocytes without parasites. For mature-stage infected erythrocytes, the percentage of parasitemia was 5 to 10%.
Statistical Analysis
We performed comparisons with the use of either Student's t-test (two-tailed) or the Mann–Whitney test.
Results
PKLR Mutations
The characteristics of the three subjects with homozygous pyruvate kinase deficiency who presented with nonspherocytic hemolytic anemia are shown in Table 1. To confirm the diagnosis, we derived genomic DNA from the subjects with pyruvate kinase deficiency and from their asymptomatic relatives and sequenced all exons and intron–exon junctions of PKLR. We identified a homozygous G-to-A mutation at position 1269 at the 3' end of exon 9 in two related case subjects (Subjects 1 and 2), which has been previously described as a loss-of-function mutation. It is predicted to cause missplicing of PKLR, resulting in a shortened half-life of the messenger RNA transcript.10,11 Subject 3 was found to be homozygous for a single-base deletion at nucleotide position 823 in exon 7 of PKLR, leading to a frameshift and premature termination of the open-reading frame. The highly deleterious nature of this latter mutation may be responsible for the severe pyruvate kinase deficiency in Subject 3, who was transfusion-dependent. We also identified asymptomatic relatives of Subjects 1 and 2 who were heterozygous for the G-to-A mutation at position 1269 (Table 1). These relatives are designated as Subjects 4 and 5.
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We examined phagocytic uptake of P. falciparum (ring-stage and mature-stage)–infected erythrocytes from case subjects and control subjects by macrophages derived from human and mouse monocytes (Figure 2A and 2B). Phagocytosis of ring-stage–infected erythrocytes from case subjects with homozygous mutations (Subjects 1, 2, and 3) was markedly higher than phagocytosis of parasitemia-matched infected erythrocytes from control subjects (P<0.001). We also observed significantly enhanced clearance by macrophages of ring-stage–infected erythrocytes derived from asymptomatic relatives who were heterozygous for the PKLR mutation (P=0.003) (Figure 2C).
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To determine whether macrophage uptake of ring-stage–infected erythrocytes from case subjects was mediated by IgG or complement, we carried out phagocytosis assays with complement-inactivated serum and in the presence of Fc-receptor blockade. We found that uptake was predominantly mediated by complement, since inactivation of complement (in autologous serum) caused a significant decrease in uptake, whereas Fc-receptor blockade had no significant effect (Figure 1 of the Supplementary Appendix). As compared with erythrocytes from control subjects, uninfected erythrocytes from case subjects also had enhanced phagocytic uptake associated with increased deposition of hemichrome, IgG, and complement C3c, although at markedly lower levels than those in infected erythrocytes from case subjects. Together, these results showed that erythrocytes from case subjects that were infected with P. falciparum underwent more extensive phagocytosis than did infected erythrocytes from control subjects, a process that occurs through a C3c-mediated pathway.
We examined single-nucleotide polymorphisms (SNPs) in PKLR in populations of varying ancestry (www.HapMap.org), including the Yoruba of Nigeria, where malaria is endemic. We did not observe differences in the prevalence of these SNPs in the various HapMap populations, although our analysis was inconclusive because of the relative paucity of informative PKLR SNPs in the HapMap database.
Discussion
We have shown that pyruvate kinase deficiency has a protective effect against replication of the malarial parasite in human erythrocytes. We have described a dual mechanism for protection against P. falciparum in pyruvate kinase deficiency that included an invasion defect of erythrocytes from case subjects (observed in those with a homozygous mutation) and preferential macrophage clearance of ring-stage–infected erythrocytes from case subjects (observed in both homozygotes and heterozygotes). The pleiotropic effect of pyruvate kinase deficiency on parasite invasion (reduced) and phagocytosis of ring-stage–infected erythrocytes (enhanced) may provide protection against clinical malaria either by causing an overall reduction in the parasite burden or by reducing the number of erythrocytes infected with parasites in the trophozoite and schizont stages that are available to bind within microvascular beds of vital organs.19
In light of the poor overall health status and relative rarity of patients with pyruvate kinase deficiency who have homozygous mutations at PKLR (severe anemia with dependence on transfusion), it is unlikely that full-fledged pyruvate kinase deficiency is relevant to protection against malaria in the field. However, heterozygosity for partial or complete loss-of-function alleles or even compound heterozygosity for mild alleles with appropriate erythropoietic compensation may have little negative effect on overall fitness (including transmission of mutant alleles), while providing a modest but significant protective effect against malaria. Although speculative, this situation would be similar to that proposed for hemoglobinopathies (sickle cell and both 
-thalassemia and β-thalassemia) and G6PD deficiency, in which similar mechanisms of protection that are associated with increased clearance of ring-stage–infected erythrocytes have been reported previously.6,15 Such a mechanism would be manifested as an increase in retention or prevalence of mutant PKLR alleles in regions where malaria is endemic, a hypothesis that can now be formally tested.
Supported by a Team Grant in Malaria (to Drs. Gros and Kain) from the Canadian Institute of Health Research (CIHR), an operating grant (MT-37121, to Dr. Kain) from the CIHR, and a grant from Genome Canada through the Ontario Genomics Institute and the CIHR Canada Research Chair (both to Dr. Kain).
No potential conflict of interest relevant to this article was reported.
Source Information
From the McLaughlin–Rotman Centre for Global Health (K.A., L.S., M.C., K.C.K.) and the Department of Medicine (I.Q., K.C.K.), University Health Network–Toronto General Hospital; Hematological Unit, Hospital for Sick Children (M.K.-A.); and the McLaughlin Centre for Molecular Medicine, University of Toronto (K.C.K.) — all in Toronto; and the Department of Biochemistry and Centre for the Study of Host Resistance, McGill University, Montreal (G.M.-O., P.G.).
This article (10.1056/NEJMoa072464) was published at www.nejm.org on April 16, 2008.
Address reprint requests to Dr. Gros at the Department of Biochemistry, McGill University, 3655 Promenade Sir William Osler, Rm. 907, Montreal, QC H3G 1Y6, Canada, or at philippe.gros{at}mcgill.ca.
References
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