The association between inherited erythrocyte disorders in humans and resistance to malaria is well documented and includes enzyme deficiencies, hemoglobinopathies and membrane protein abnormalities. The association between pyruvate kinase (PK) deficiency and resistance to malaria has been demonstrated in vivo using a murine model1 and it has been suggested that this phenomenon may extend to humans.
The human PK-LR gene codes for erythrocyte PK, which catalyzes the conversion of phosphoenolpyruvate to pyruvate. It is an essential step in anerobic glycolysis and is critical for ATP production in erythrocytes. There are more than 180 PK mutations, and clinical symptoms appear in patients who are homozygous or compound heterozygotes for an abnormal allele. The frequency of PK deficiency is highly variable, and prevalence studies in North America, Europe and parts of Asia found that the heterozygote allele frequencies ranged between 1% and 3.6%2,3 in these regions. A study in African Americans suggested that the frequency of the heterozygote allele was 2.4 times more common in Africans than in Caucasians.4
Blood from a PK deficient patient and from the heterozygous parent was used for parasite culture experiments. The study was approved by the relevant ethics authority and both subjects gave their informed consent. Clinical and laboratory data are shown in Table 1. A G6PD screening test, hemoglobin electrophoresis and membrane protein studies were performed to exclude co-existing inherited erythrocyte disorders.
P. falciparum strain 3D7 was maintained in culture and synchronized according to standard methods.5 Duplicate erythrocyte cultures of the homozygote (PK deficient patient), heterozygote (parent) and a control (healthy volunteer) were seeded on day one with parasite ring stages to give a hematocrit of 5% and a parasitemia of ~1.5%. Unseeded erythrocyte cultures for each subject were used as a blank to control for reticulocytes and autohemolysis. Parasitemia was monitored daily with Giemsa-stained blood smears and quantitated in duplicate by counting 5 × 10 cells by flow cytometry6,7 for at least two complete cycles (six days). Briefly, a 2 μL aliquot of cells was added to freshly prepared thiazole orange (0.05 μg/mL phosphate buffered saline), incubated at 4°C for 30 mins. and analyzed on a FACSCalibur flow cytometer (BD Biosciences). Thiazole orange is a fluorescent dye that intercalates into nucleic acids and differentiates between non-nucleated erythrocytes and different parasite stages. A typical flow cytometry data set is shown in Figure 1A. Reticulocytes contain RNA and falsely elevate the parasitemia, which was corrected by subtracting blank readings from parasitized culture readings. The complete experiment was performed twice on different occasions.
Erythrocytes from the homozygous PK deficient patient demonstrated a dramatic resistance to P. falciparum infection (Figure 1B). Numerous prickle cells, typical of PK deficiency, were observed and these did not contain any parasites. Although the parasitemia in the heterozygote was slightly lower than the control, there was no statistically significant difference between them on day five (p=0.44) and six (p=0.55). Similar results were obtained in a second set of experiments.
In vitro autohemolysis is a feature of inherited erythrocyte disorders and to asses its effect on cultures, the hemoglobin concentration in the supernatant of each culture was measured daily at 420nm. Over six days the hematocrit decreased from 5% to 4.9% (control), 4.8% (heterozygote) and 4.6% (homozygote), indicating that autohemolysis had minimal impact. Hemolysis due to parasitemia increased with increasing parasitemia (Figure 1C) but was, however, significantly less in the heterozygote compared with the control (p=0.0001 on day five; p=0.008 on day six). The reason for this is not clear, but indicates that a mild protective effect in the heterozygote cannot be excluded. The failure of the parasite-induced hemolysis to increase in the homozygote reflected the resistance to malaria in this subject.
The data presented in this paper demonstrate for the first time that PK deficient human erythrocytes are resistant to malaria infection in vitro, and support the in vivo findings in the murine model.1 Two interesting questions arise from this study. Firstly, has malaria selected for the relatively high frequency of the abnormal PK allele? And secondly, how does PK deficiency exert its protective effect? The detrimental effect of PK deficiency on the host suggests that, like other inherited erythrocyte disorders, there was a historical driving force that maintained the polymorphic frequency of the abnormal allele. That malaria is the most likely driving force is proposed for several reasons: (i) this study has demonstrated that human PK deficient erythrocytes are resistant to malaria in vitro; (ii) in vivo protection has been shown in the mouse model;1 (iii) until 1900 malaria was encountered in all regions where PK deficiency8 has been reported; and (iv) malaria has been well-documented as a driving force behind other human erythrocyte disorders (Haldane’s malaria hypothesis).
The mechanisms responsible for the resistance of PK deficient erythrocytes to malaria are unknown but may be due to ATP depletion, which impairs parasite invasion in vitro.9 Decreased ATP leads to cross-linking of membrane proteins,10 which may affect invasion, growth or exit of the parasite. Low ATP concentration also increases 2,3-diphosphoglycerate, which i) disrupts spectrin, actin and protein 4.1 interactions causing membrane instability11 and impacting on intracellular parasite survival, and ii) inhibits G6PD leading to increased oxidative stress.12 The scenario in vivo may include other factors such as splenic clearance, early phagocytosis and decreased cyto-adherence of parasitized erythrocytes. Our investigations have also demonstrated that the mutant PK activity decreased at elevated temperature (data not shown), suggesting that fever may exacerbate the low ATP concentration.
The in vitro data in humans presented here confirm the in vivo protection of PK deficiency demonstrated in the mouse model and lay the foundation for future clinical case control studies.
the authors wish to thank the PK-deficient patient and parents for their co-operation in this study and Drs. I. Thomson and P. Keene for the patient referral.
- Min-Oo G, Fortin A, Tam M, Nantel A, Stevenson MM, Gros P. Pyruvate kinase deficiency in mice protects against malaria. Nature Genetics. 2003; 35:357-62. Google Scholar
- Zanella A, Fermo E, Bianchi P, Chiarelli LR, Valenti G. Pyruvate kinase deficiency: the genotype-phenotype association. Blood Rev. 2007; 21:217-31. Google Scholar
- Beutler E, Gelbart T. Estimating the prevalence of pyruvate kinase deficiency from the gene frequency in the general white population. Blood. 2000; 95:3585-8. Google Scholar
- Mohrenweiser HW. Functional hemizygosity in the human genome: direct estimate from twelve erythrocyte enzyme loci. Human Genet. 1987; 77:241-5. Google Scholar
- Methods in Malaria Research.Google Scholar
- Makler MT, Recktenwald D. Thiazole orange: a new dye for Plasmodium species analysis. Cytometry. 1987; 8:568- 70. Google Scholar
- Schulze DLC, Makgatho EM, Coetzer TL, Louw AI, van Rensburg CEJ, Visser L. Development and application of a modified flow cytometric procedure for rapid in vitro quantitation of malaria parasitaemia. S Afr J Science. 1997; 93:156-8. Google Scholar
- Hay SI, Guerra CA, Tatern AJ, Noor AM, Snow RW. The global distribution and population at risk of malaria: past, present and future. Lancet Infect Dis. 2004; 4:327-36. Google Scholar
- Olson JA, Kilejian A. Involvement of spectrin and ATP in infection of resealed erythrocyte ghosts by the human malarial parasite, Plasmodium falciparum. J Cell Biol. 1982;757-62. Google Scholar
- Palek J, Lui SC, Snyder LM. Analysis of spectrin rich complexes in ATP-depleted cells. Blood. 1978; 51:385-95. Google Scholar
- Sheetz MP, Casaly J. 2,3-Diphosphoglycerate and ATP dissociate erythrocyte membrane skeletons. J Biol Chem. 1980; 255:9955-60. Google Scholar
- Min-Oo G, Gros P. Erythrocyte variants and the nature of their malaria protective effect. Cell Microbiol. 2005; 7:753-63. Google Scholar