Malarial control is largely slowed by plasmodium parasite drug resistance and no effective vaccine. Understanding malarial pathogenesis (e.g. identifying innate or adaptive host defense mechanisms against aforementioned parasite) may open up opportunities for disease intervention. Such mechanisms include genetic determinants of endemic malarial disease susceptibility during epidemics and as variations. This was studied in mouse models via P.chabaudi infection in the erythroid stage of disease and major loci influencing parasite replication at the peak of infection were recombinant congenic mouse strains AcB55 and AcB61 which confers resistance caused by a recessive monogenic trait – mutation (Ile90Asn) in the gene for pyruvate kinase (Pklr). This study is to determine whether pyruvate kinase deficiency (PKD) protects humans against malaria and the molecular basis of a putative protective effect.
Methods:
• Subjects
PKD subjects (identified via clinical presentation and enzyme assay) and their asymptotic relatives were garnered for this study. Genetic basis of their enzyme deficiency were not determined. Hemoglobin electrophoresis and G6PD assessment ruled out the possibility of other hemolytic disorders. Subjects 1, 2 and 3 had homozygous PKD and nonspherocytic anemia. 1 and 2 had undergone splenectomy while 3 was transfusion dependent. A blood sample was drawn from 3 before transfusion.
• Identification of PKLR Mutation
Genomic DNA was extracted from blood of PKD (case) subjects and non-PKD (control) subjects. Amplification by PCR was carried out on the 12 coding exons of PKLR (including intron-exon junctions). Purified products were sequenced with cycle sequencing and fluorescent nucleotides and analyzed via BioEdit (sequence analysis).
• Parasite Culture
Schizonts from cloned, synchronized P.falciparum ITG and 3D7 cultures were mixed with erythrocytes from case and control subjects followed by invasion and maturation assessment.
• Phagocytosis Assay
Thioglycollate-elicited macrophages harvested from mice were incubated with washed, opsonized erythrocytes (both infected and non-infected) with target-to-effector ratio of 40:1. Monocytes were obtained from blood of subjects. Experiments were repeated at least thrice.
• Erythrocyte Membrane Analysis
Ring-stage infected erythrocyte values were normalized to 100% parasitemia using I=(Tot–N×n)÷(1−n). I represents amount of bound IgG and C3c in 100% rings, Tot is amount of bound IgG and C3c in the whole culture, N is amount of bound IgG and C3c in erythrocytes without parasites and n is fraction of erythrocytes without parasites. Percentage of parasitemia was 5-10% for mature-stage infected erythrocytes.
• Statistical Analysis
Comparisons done via t-test (two-tailed) or the Mann-Whitney test.
Multiple invasion and maturation assays with erythrocytes from case subjects with homozygous mutation in PKLR showed reduced invasion of P.falciparum during three consecutive growth cycles (compared with control subjects) due to unique property of erythrocytes. There was no significant defect in invasion of heterozygous PKLR mutants. Difference of intracellular maturation of erythrocytes and parasitic growth due to potential biochemical differences in intracellular milieu (including the accumulation of glycolytic metabolic intermediates) of homozygotes and heterozygotes were insignificant.
Enhanced clearance and phagocytosis of ring-stage-infected erythrocytes from homozygous mutant subjects was higher (due to increased levels of membrane-bound hemichromes, IgG and C3c) than heterozygotes and control subjects respectively. Uninfected erythrocytes from case subjects also experienced enhanced phagocytosis compared to control subjects. Macrophage uptake of mature-stage-infected erythrocytes from case and wild-type erythrocytes had insignificant difference. Uptake was mainly mediated by complement. In contrast, Fc-receptor blockade had insignificant effect.
Therefore, PKD protects human erythrocytes against malarial parasite replication. However, protection might not be extended to severe PKD subjects with poor health. Heterozygotic individuals with appropriate erythropoietic compensation may find respite from malarial infection as well, albeit not as significant as that of homozygotes. As a result of correlation between malaria and PKD, retention or prevalence of mutant PKLR alleles in regions where malaria is endemic occurs.
Reference:
>> The New England Journal of Medicine, 2008. Pyruvate Kinase Deficiency and Malaria. [online] Available at: <http://www.nejm.org/doi/full/10.1056/NEJMoa072464#t=article> [Accessed 4 January 2013].
