Brendan J. McMorran

A Novel ENU-mutation in Ankyrin-1 disrupts malaria parasite maturation in red blood cells of mice

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 (Ank1MRI23420) 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.

A flow cytometric assay to quantify invasion of red blood cells by rodent Plasmodium parasites in vivo

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.

The influence of host genetics on erythrocytes and malaria infection: is there therapeutic potential?

As parasites, Plasmodium species depend upon their host for survival. During the blood stage of their life-cycle parasites invade and reside within erythrocytes, commandeering host proteins and resources towards their own ends, and dramatically transforming the host cell. Parasites aptly avoid immune detection by minimizing the exposure of parasite proteins and removing themselves from circulation through cytoadherence. Erythrocytic disorders brought on by host genetic mutations can interfere with one or more of these processes, thereby providing a measure of protection against malaria to the host. This review summarizes recent findings regarding the mechanistic aspects of this protection, as mediated through the parasites interaction with abnormal erythrocytes. These novel findings include the reliance of the parasite on the host enzyme ferrochelatase, and the discovery of basigin and CD55 as obligate erythrocyte receptors for parasite invasion. The elucidation of these naturally occurring malaria resistance mechanisms is increasing the understanding of the host-parasite interaction, and as discussed below, is providing new insights into the development of therapies to prevent this disease.

In vivo assessment of rodent Plasmodium parasitemia and merozoite invasion by flow cytometry

During blood stage infection, malaria parasites invade, mature, and replicate within red blood cells (RBCs). This results in a regular growth cycle and an exponential increase in the proportion of malaria infected RBCs, known as parasitemia. We describe a flow cytometry based protocol which utilizes a combination of the DNA dye Hoechst, and the mitochondrial membrane potential dye, JC-1, to identify RBCs which contain parasites and therefore the parasitemia, of in vivo blood samples from Plasmodium chabaudi adami DS infected mice. Using this approach, in combination with fluorescently conjugated antibodies, parasitized RBCs can be distinguished from leukocytes, RBC progenitors, and RBCs containing Howell-Jolly bodies (HJ-RBCs), with a limit of detection of 0.007% parasitemia. Additionally, we outline a method for the comparative assessment of merozoite invasion into two different RBC populations. In this assay RBCs, labeled with two distinct compounds identifiable by flow cytometry, are transfused into infected mice. The relative rate of invasion into the two populations can then be assessed by flow cytometry based on the proportion of parasitized RBCs in each population over time. This combined approach allows the accurate measurement of both parasitemia and merozoite invasion in an in vivo model of malaria infection.

Erythrocytic Iron Deficiency Enhances Susceptibility to Plasmodium chabaudi Infection in Mice Carrying a Missense Mutation in Transferrin Receptor 1

ABSTRACT The treatment of iron deficiency in areas of high malaria transmission is complicated by evidence which suggests that iron deficiency anemia protects against malaria, while iron supplementation increases malaria risk. Iron deficiency anemia results in an array of pathologies, including reduced systemic iron bioavailability and abnormal erythrocyte physiology; however, the mechanisms by which these pathologies influence malaria infection are not well defined. In the present study, the response to malaria infection was examined in a mutant mouse line, Tfrc MRI24910 , identified during an N-ethyl-N-nitrosourea (ENU) screen. This line carries a missense mutation in the gene for transferrin receptor 1 (TFR1). Heterozygous mice exhibited reduced erythrocyte volume and density, a phenotype consistent with dietary iron deficiency anemia. However, unlike the case in dietary deficiency, the erythrocyte half-life, mean corpuscular hemoglobin concentration, and intraerythrocytic ferritin content were unchanged. Systemic iron bioavailability was also unchanged, indicating that this mutation results in erythrocytic iron deficiency without significantly altering overall iron homeostasis. When infected with the rodent malaria parasite Plasmodium chabaudi adami, mice displayed increased parasitemia and succumbed to infection more quickly than their wild-type littermates. Transfusion of fluorescently labeled erythrocytes into malaria parasite-infected mice demonstrated an erythrocyte-autonomous enhanced survival of parasites within mutant erythrocytes. Together, these results indicate that TFR1 deficiency alters erythrocyte physiology in a way that is similar to dietary iron deficiency anemia, albeit to a lesser degree, and that this promotes intraerythrocytic parasite survival and an increased susceptibility to malaria in mice. These findings may have implications for the management of iron deficiency in the context of malaria.

A novel ENU-induced ankyrin-1 mutation impairs parasite invasion and increases erythrocyte clearance during malaria infection in mice

Genetic defects in various red blood cell (RBC) cytoskeletal proteins have been long associated with changes in susceptibility towards malaria infection. In particular, while ankyrin (Ank-1) mutations account for approximately 50% of hereditary spherocytosis (HS) cases, an association with malaria is not well-established, and conflicting evidence has been reported. We describe a novel N-ethyl-N-nitrosourea (ENU)-induced ankyrin mutation MRI61689 that gives rise to two different ankyrin transcripts: one with an introduced splice acceptor site resulting a frameshift, the other with a skipped exon. Ank-1(MRI61689/+) mice exhibit an HS-like phenotype including reduction in mean corpuscular volume (MCV), increased osmotic fragility and reduced RBC deformability. They were also found to be resistant to rodent malaria Plasmodium chabaudi infection. Parasites in Ank-1(MRI61689/+) erythrocytes grew normally, but red cells showed resistance to merozoite invasion. Uninfected Ank-1(MRI61689/+) erythrocytes were also more likely to be cleared from circulation during infection; the “bystander effect”. This increased clearance is a novel resistance mechanism which was not observed in previous ankyrin mouse models. We propose that this bystander effect is due to reduced deformability of Ank-1(MRI61689/+) erythrocytes. This paper highlights the complex roles ankyrin plays in mediating malaria resistance.

Platelets Kill Intraerythrocytic Malarial Parasites and Mediate Survival to Infection

Platelets play a critical role in the pathogenesis of malarial infections by encouraging the sequestration of infected red blood cells within the cerebral vasculature. But platelets also have well-established roles in innate protection against microbial infections. We found that purified human platelets killed Plasmodium falciparum parasites cultured in red blood cells. Inhibition of platelet function by aspirin and other platelet inhibitors abrogated the lethal effect human platelets exert on P. falciparum parasites. Likewise, platelet-deficient and aspirin-treated mice were more susceptible to death during erythrocytic infection with Plasmodium chabaudi. Both mouse and human platelets bind malarial-infected red cells and kill the parasite within. These results indicate a protective function for platelets in the early stages of erythrocytic infection distinct from their role in cerebral malaria.

Platelet Factor 4 and Duffy Antigen Required for Platelet Killing of Plasmodium falciparum

Platelets Poison Parasites Activated platelets bound to malaria parasite–infected red blood cells were once thought to contribute to pathogenesis, but recently the platelets have been found to have a protective effect. McMorran et al. (p. 1348; see the Perspective by Engwerda and Good) extended this discovery to show that platelet activation releases intracellular granules containing a chemokine, PF4, which is internalized by Plasmodium falciparum–infected red cells. Subsequently, mature parasites within the cells die. The Duffy blood-group factor on red blood cells is known to act as a nonspecific receptor for chemokines, such as PF4, as well as a receptor for cell invasion by other species of malaria parasite. When the Duffy antigen was blocked by antibody treatment, platelets and PF4 were less able to kill the P. falciparum parasites within. Interaction of a platelet protein and a red cell protein enables platelets to attack malarial parasites inside red cells. Platelets restrict the growth of intraerythrocytic malaria parasites by binding to parasitized cells and killing the parasite within. Here, we show that the platelet molecule platelet factor 4 (PF4 or CXCL4) and the erythrocyte Duffy-antigen receptor (Fy) are necessary for platelet-mediated killing of Plasmodium falciparum parasites. PF4 is released by platelets on contact with parasitized red cells, and the protein directly kills intraerythrocytic parasites. This function for PF4 is critically dependent on Fy, which binds PF4. Genetic disruption of Fy expression inhibits binding of PF4 to parasitized cells and concomitantly prevents parasite killing by both human platelets and recombinant human PF4. The protective function afforded by platelets during a malarial infection may therefore be compromised in Duffy-negative individuals, who do not express Fy.

Host resistance to malaria: using mouse models to explore the host response

Malaria is a disease that infects over 500 million people, causing at least 1 million deaths every year, with the majority occurring in developing countries. The current antimalarial arsenal is becoming dulled due to the rapid rate of resistance of the parasite. However, in populations living in malaria-endemic regions there are many examples of genetic-based resistance to the severe effects of the parasite Plasmodium. Defining the genetic factors behind host resistance has been an area of great scientific interest over the last few decades; this review summarizes the current knowledge of the genetic loci involved. Perhaps the lessons learned from the natural variation in both the human populations and experimental mouse models of infection may pave the way for novel resistance-proof antimalarials.

The role of TNF in parasitic diseases: Still more questions than answers

The inhibition of TNF with therapeutic monoclonal antibodies or antibody/receptor fusion proteins in rheumatoid arthritis still constitutes the benchmark for a successful intervention in an ongoing auto-immune-inflammatory disease and underlines the importance of this cytokine. TNF plays a central role in the defence against intracellular infections and is responsible for the promotion of different aspects of the innate immune response such as inflammatory cell recruitment and cell differentiation. While this cytokine generally displays pro-inflammatory activities supporting the early stages of the inflammatory response, it has been demonstrated to be especially important during infection with intracellular pathogens and, consequently, leishmaniasis of TNF(-/-) mice ends fatally. However, the specific activities of TNF that confer protection are not yet fully understood. This review will summarize the current understanding of TNF function and signalling, and will discuss recent work in the models of malaria, toxoplasmosis, trypanosomiasis and leishmaniasis with particular emphasis on work with gene-deficient mouse models.