ALBERT

Description: Platelets are best known for their roles in hemostasis, but there is accumulating evidence for their importance in immunity and host defence against infection. We have shown that platelets are required for host protection during malarial infection and are a major determinant for survival. Platelets control the growth of intraerythrocytic Plasmodium parasites by directly binding to infected erythrocytes and killing the parasite inside 1. We 2 and others 3 have also described the parasite killing mechanism exerted by platelets. Upon binding to parasite-infected red cells, platelets release a protein with dual chemokine and antimicrobial functions called platelet factor 4 (PF4). PF4 binds to the red cell Duffy antigen chemokine receptor, and is then absorbed into the parasite where it lyses the parasite food vacuole and kills the parasite. This work aimed to further investigate the role of PF4 in vivo using mice a PF4 knockout mouse strain (Pf4tm1Mako/tm1Mako; 4) and blood stage infection with a murine malaria model parasite, P. chabaudi. We confirmed that the homozygous mutant mice did not produce any PF4 protein. Following challenge with P. chabaudi (1 x 104 parasites administered intravenously), rates of survival amongst cohorts of age and sex matched wild type and homozygous mice (both on a C57BL/6 background) were not significantly different (approximately 30% for females and 10% for males). However, a deficiency of PF4 resulted in significant differences in parasite growth during the pre-peak parasitemia phase of the infection. Between days 5 and 8 following infection, proportions of parasite-infected red cells in the circulation of PF4-null mice were double those in wild-type mice. This coincided with a 50-60% reduction in the frequency of growth arrested or dead parasites (detected using terminal deoxynucleotidyl transferase dUTP nick end labelling, also known as TUNEL labelling). Together these data indicate that platelet-derived PF4 has an important in vivo malaria-protective role. The protein is necessary for the platelet to control parasite growth during the early stage of erythrocytic malarial infection. However, the highly virulent nature of the P. chabaudi strain used in these experiments precluded detection of any predicted survival disadvantage in the PF4-null mice. McMorran BJ, Marshall VM, de Graaf C, et al. Platelets kill intraerythrocytic malarial parasites and mediate survival to infection. Science. 2009;323(5915):797-800.McMorran BJ, Wieczorski L, Drysdale KE, et al. Platelet factor 4 and Duffy antigen required for platelet killing of Plasmodium falciparum. Science. 2012;338(6112):1348-1351.Love MS, Millholland MG, Mishra S, et al. Platelet Factor 4 Activity against P. falciparum and Its Translation to Nonpeptidic Mimics as Antimalarials. Cell host & microbe. 2012;12(6):815-823.Eslin DE, Zhang C, Samuels KJ, et al. Transgenic mice studies demonstrate a role for platelet factor 4 in thrombosis: dissociation between anticoagulant and antithrombotic effect of heparin. Blood. 2004;104(10):3173-3180. Disclosures No relevant conflicts of interest to declare.

Description: Key PointsMalarial parasite growth is impeded in erythropoietic protoporphyric erythrocytes because of decreased host cell ferrochelatase activity. A ferrochelatase competitive inhibitor prevents the growth of malarial parasites in normal red cells.

Description: Key Points AMPD3 activation reduces red blood cell half-life, which is associated with increased oxidative stress and phosphatidylserine exposure. AMPD3 activation causes malaria resistance through increased RBC turnover and increased RBC production.

Description: Many red cell polymorphisms are a result of selective pressure by the malarial parasite. Here we add another red cell disease to the panoply of erythrocytic changes that give rise to resistance to malaria. Erythrocytes from individuals with erythropoietic protoporphyria (EPP) have low levels of the final enzyme in the heme biosynthetic pathway, ferrochelatase. Cells from these patients are resistant to the growth of the human malarial parasite, Plasmodium falciparum. We first compared the growth and replication rates of P. falciparum cultured in red cells from EPP patients (n=4) and normal erythrocytes. There was a two to three-fold reduction in parasite growth in the patient cells. Next we sought to exclude the possible negative effects on the parasite due to elevated porphyrins and reduced cell hemoglobin. To do this we employed the EPP phenocopy, X-linked dominant protoporphyria (XLDPP), which has normal ferrochelatase activity. Cells from three individuals with XLDPP supported completely normal rates of parasite growth, proving that the EPP resistance phenomenon was due to the absence of ferrochelatase. We also tested the requirement of host ferrochelatase during malarial infection by using mice with a hypomorphic mutation in the murine ferrochelatase gene and the rodent malarial species, P. chabaudi. Mice homozygous for the mutation, Fechm1Pas, have 5% residual ferrochelatase activity compared to wild-type littermates 1. Following infection, we observed an almost two fold reduction in peak parasitemia levels and two to three times greater rates of survival in the homozygous mice. Host ferrochelatase is therefore also necessary to sustain a normal malarial infection in mice. To determine the requirement of parasite-expressed ferrochelase, we produced a P. berghei parasite line carrying a complete deletion of the ferrochelatase gene. This strain grew normally in wild-type mouse erythrocytes, indicating that parasite ferrochelatase is dispensable during the erythrocytic stage of infection. A complete absence of ferrochelatase in humans (and mice) is not compatible with life, and therefore testing parasite growth in complete knockout cells was not possible. Instead, we used a specific competitive inhibitor of the enzyme, N-methylprotoporphyrin (NMPP) to eliminate all ferrochelatase activity from the parasite and red cell. Treatment of P. falciparum cultures with NMPP resulted in potent cytocidal and growth inhibition effects against both antimalarial drug-sensitive and drug-resistant parasite lines. The activity of NMPP could be competitively removed by titration of the ferrochelatase substrate, protoporphyrin IX, proving that the effects of NMPP were due to specific enzyme inhibition and not off-target effects. Therefore ferrochelatase activity is also essential for the Plasmodium parasite. We conclude that the refractoriness of ferrochelatse-deficient red cells to Plasmodium is due to the parasite’s reliance on the host enzyme. Host ferrochelatase is probably utilized by the parasite for the biosynthesis of heme. In support of this hypothesis, others have observed that red cell ferrochelatase is imported by intraerythrocytic Plasmodium and enzymatic is retained 2,3. Finally, based on this collective data, we propose human ferrochelatase is a valid and novel “host-directed” target for an antimalarial therapy. Lyoumi S, Abitbol M, Andrieu V, et al. Increased plasma transferrin, altered body iron distribution, and microcytic hypochromic anemia in ferrochelatase-deficient mice. Blood. 2007;109(2):811-818.Bonday ZQ, Dhanasekaran S, Rangarajan PN, Padmanaban G. Import of host delta-aminolevulinate dehydratase into the malarial parasite: identification of a new drug target. Nat Med. 2000;6(8):898-903.Varadharajan S, Sagar BK, Rangarajan PN, Padmanaban G. Localization of ferrochelatase in Plasmodium falciparum. Biochem J. 2004;384(Pt 2):429-436. Disclosures No relevant conflicts of interest to declare.