The blood stage of the plasmodium parasite life cycle is responsible for the clinical symptoms of malaria. Epidemiological studies have identified coincidental malarial endemicity and multiple red blood cell (RBC) disorders. Many RBC disorders result from mutations in genes encoding cytoskeletal proteins and these are associated with increased protection against malarial infections. However the mechanisms underpinning these genetic, host responses remain obscure. We have performed an N-ethyl-N-nitrosourea (ENU) mutagenesis screen and have identified a novel dominant (haploinsufficient) mutation in the Ank-1 gene ( Ank1 MRI23420 ) of mice displaying hereditary spherocytosis (HS). Female mice, heterozygous for the Ank-1 mutation showed increased survival to infection by Plasmodium chabaudi adami DS with a concomitant 30% decrease in parasitemia compared to wild-type, isogenic mice (wt). A comparative in vivo red cell invasion and parasite growth assay showed a RBC-autonomous effect characterised by decreased proportion of infected heterozygous RBCs. Within approximately 6–8 hours post-invasion, TUNEL staining of intraerythrocytic parasites, showed a significant increase in dead parasites in heterozygotes. This was especially notable at the ring and trophozoite stages in the blood of infected heterozygous mutant mice compared to wt (p<0.05). We conclude that increased malaria resistance due to ankyrin-1 deficiency is caused by the intraerythrocytic death of P. chabaudi parasites.

BackgroundMalaria treatments are becoming less effective due to the rapid spread of drug resistant parasites. Increased understanding of the host/parasite interaction is crucial in order to develop treatments that will be less prone to resistance. Parasite invasion of the red blood cell (RBC) is a critical aspect of the parasite life cycle and is, therefore, a promising target for the development of malaria treatments. Assays for analysing parasite invasion in vitro have been developed, but no equivalent assays exist for in vivo studies. This article describes a novel flow cytometric in vivo parasite invasion assay.MethodsExperiments were conducted with mice infected with erythrocytic stages of Plasmodium chabaudi adami strain DS. Exogenously labelled blood cells were transfused into infected mice at schizogony, and collected blood samples stained and analysed using flow cytometry to specifically detect and measure proportions of labelled RBC containing newly invaded parasites. A combination of antibodies (CD45 and CD71) and fluorescent dyes, Hoechst (DNA) and JC-1 (mitochondrial membrane potential), were used to differentiate parasitized RBCs from uninfected cells, RBCs containing Howell-Jolly bodies, leukocytes and RBC progenitors. Blood cells were treated ex vivo with proteases to examine the effects on in vivo parasite invasion.ResultsThe staining and flow cytometry analysis method was accurate in determining the parasitaemia down to 0.013% with the limit of detection at 0.007%. Transfused labelled blood supported normal rates of parasite invasion. Protease-treated red cells resulted in 35% decrease in the rate of parasite invasion within 30 minutes of introduction into the bloodstream of infected mice.ConclusionsThe invasion assay presented here is a versatile method for the study of in vivo red cell invasion efficiency of Plasmodium parasites in mice, and allows direct comparison of invasion in red cells derived from two different populations. The method also serves as an accurate alternative method of estimating blood parasitaemia.

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.The factors that determine red blood cell (RBC) lifespan and the rate of RBC aging have not been fully elucidated. In several genetic conditions, including sickle cell disease, thalassemia, and G6PD deficiency, erythrocyte lifespan is significantly shortened. Many of these diseases are also associated with protection from severe malaria, suggesting a role for accelerated RBC senescence and clearance in malaria resistance. Here, we report a novel, N-ethyl-N-nitrosourea-induced mutation that causes a gain of function in adenosine 59-monophosphate deaminase (AMPD3). Mice carrying the mutation exhibit rapid RBC turnover, with increased erythropoiesis, dramatically shortened RBC lifespan, and signs of increased RBC senescence/eryptosis, suggesting a key role for AMPD3 in determining RBC half-life. Mice were also found to be resistant to infection with the rodent malaria Plasmodium chabaudi. We propose that resistance to P. chabaudi is mediated by increased RBC turnover and higher rates of erythropoiesis during infection. (Blood. 2016; 128(9):1290-1301) IntroductionThe lifespan of red blood cells (RBC) is tightly regulated, lasting some 120 days in humans and 51 days in mice. 1 Senescent RBCs are cleared from the bloodstream by macrophages of the reticuloendothelial system and are replaced with new erythrocytes.2-4 This destruction is not random, and it is highly dependent on RBC age. 4 The factors determining the rate of RBC aging, and thereby RBC lifespan, have not been fully elucidated.In several genetic conditions, including pyruvate kinase deficiency, sickle cell disease, thalassemia, hereditary spherocytosis, and G6PD deficiency, 5,6 erythrocyte lifespan is significantly shortened. In most cases, cells show signs of increased senescence, including increased oxidative stress and phosphatidylserine (PS) exposure.2,7-9 Intriguingly, many of these diseases are also associated with protection from severe malaria, 10,11 which has led some authors to propose a role for RBC senescence and clearance in malaria resistance. 15,16 For example, the shortened lifespan of RBCs in pyruvate kinase-deficient individuals and associated pathology has been linked to reduced RBC ATP levels. Similarly, ATP loss contributes to pathology in sickle cell disease.17 Erythrocytic ATP levels are controlled by adenosine monophosphate deaminase (AMPD3), which converts AMP to inosine 59-monophosphate (IMP) and plays an important role in maintaining the adenylate energy charge or the ratio of ATP to AMP. Thus, ATP loss may be mediated through AMPD3, which converts AMP to IMP. In most cells, the conversion of ITP back to AMP by adenylsuccinate synthetase and adenylsuccinate lyase balances AMPD3 activity. However, in RBCs, the machinery for conversion of IMP back to AMP is absent, and to maintain AM...

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