Patrick M. Lelliott

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.

Proliferation of East Antarctic Adélie penguins in response to historical deglaciation

BackgroundMajor, long-term environmental changes are projected in the Southern Ocean and these are likely to have impacts for marine predators such as the Adélie penguin (Pygoscelis adeliae). Decadal monitoring studies have provided insight into the short-term environmental sensitivities of Adélie penguin populations, particularly to sea ice changes. However, given the long-term nature of projected climate change, it is also prudent to consider the responses of populations to environmental change over longer time scales. We investigated the population trajectory of Adélie penguins during the last glacial-interglacial transition to determine how the species was affected by climate warming over millennia. We focussed our study on East Antarctica, which is home to 30 % of the global population of Adélie penguins.MethodsUsing mitochondrial DNA from extant colonies, we reconstructed the population trend of Adélie penguins in East Antarctica over the past 22,000 years using an extended Bayesian skyline plot method. To determine the relationship of East Antarctic Adélie penguins with populations elsewhere in Antarctica we constructed a phylogeny using mitochondrial DNA sequences.ResultsWe found that the Adélie penguin population expanded 135-fold from approximately 14,000 years ago. The population growth was coincident with deglaciation in East Antarctica and, therefore, an increase in ice-free ground suitable for Adélie penguin nesting. Our phylogenetic analysis indicated that East Antarctic Adélie penguins share a common ancestor with Adélie penguins from the Antarctic Peninsula and Scotia Arc, with an estimated age of 29,000 years ago, in the midst of the last glacial period. This finding suggests that extant colonies in East Antarctica, the Scotia Arc and the Antarctic Peninsula were founded from a single glacial refuge.ConclusionsWhile changes in sea ice conditions are a critical driver of Adélie penguin population success over decadal and yearly timescales, deglaciation appears to have been the key driver of population change over millennia. This suggests that environmental drivers of population trends over thousands of years may differ to drivers over years or decades, highlighting the need to consider millennial-scale trends alongside contemporary data for the forecasting of species’ abundance and distribution changes under future climate change scenarios.

Plasmodium products persist in the bone marrow and promote chronic bone loss

Plasmodium infection causes chronic inflammation and bone loss through Plasmodium product accumulation in the bone marrow. Plasmodium leftovers cause bone loss Individuals who recover from malarial infection may develop long-term consequences, such as bone loss and growth retardation. Lee et al. now report that Plasmodium by-products retained in the bone marrow lead directly to bone loss. Infection with a mutant Plasmodium that lacked the by-product hemozoin did not induce bone loss. Mechanistically, these products induced MyD88-dependent inflammatory responses in osteoclast and osteoblast precursors, resulting in bone resorption. Treating infected animals with alfacalcidol, a vitamin D3 analog, could prevent this bone loss, suggesting that combining bone therapies with antimalarial drugs may prevent bone loss in infected individuals. Although malaria is a life-threatening disease with severe complications, most people develop partial immunity and suffer from mild symptoms. However, incomplete recovery from infection causes chronic illness, and little is known of the potential outcomes of this chronicity. We found that malaria causes bone loss and growth retardation as a result of chronic bone inflammation induced by Plasmodium products. Acute malaria infection severely suppresses bone homeostasis, but sustained accumulation of Plasmodium products in the bone marrow niche induces MyD88-dependent inflammatory responses in osteoclast and osteoblast precursors, leading to increased RANKL expression and overstimulation of osteoclastogenesis, favoring bone resorption. Infection with a mutant parasite with impaired hemoglobin digestion that produces little hemozoin, a major Plasmodium by-product, did not cause bone loss. Supplementation of alfacalcidol, a vitamin D3 analog, could prevent the bone loss. These results highlight the risk of bone loss in malaria-infected patients and the potential benefits of coupling bone therapy with antimalarial treatment.

Ankyrin-1 Gene Exhibits Allelic Heterogeneity in Conferring Protection Against Malaria

Allelic heterogeneity is a common phenomenon where a gene exhibits a different phenotype depending on the nature of its genetic mutations. In the context of genes affecting malaria susceptibility, it allowed us to explore and understand the intricate host–parasite interactions during malaria infections. In this study, we described a gene encoding erythrocytic ankyrin-1 (Ank-1) which exhibits allelic-dependent heterogeneous phenotypes during malaria infections. We conducted an ENU mutagenesis screen on mice and identified two Ank-1 mutations, one resulting in an amino acid substitution (MRI95845), and the other a truncated Ank-1 protein (MRI96570). Both mutations caused hereditary spherocytosis-like phenotypes and confer differing protection against Plasmodium chabaudi infections. Upon further examination, the Ank-1(MRI96570) mutation was found to inhibit intraerythrocytic parasite maturation, whereas Ank-1(MRI95845) caused increased bystander erythrocyte clearance during infection. This is the first description of allelic heterogeneity in ankyrin-1 from the direct comparison between two Ank-1 mutations. Despite the lack of direct evidence from population studies, this data further supported the protective roles of ankyrin-1 mutations in conferring malaria protection. This study also emphasized the importance of such phenomena in achieving a better understanding of host–parasite interactions, which could be the basis of future studies.

IFN-γ protects hepatocytes against Plasmodium vivax infection via LAP-like degradation of sporozoites

The first few days of exposure to Plasmodium sporozoites following a mosquito bite is a critical stage of malaria infection. From the bite site, sporozoites quickly migrate through the bloodstream and invade hepatocytes. Hepatocyte invasion occurs via invagination of the host cell plasma membrane, which parasites use to form a parasitophorous vacuole membrane (PVM) within the cell for their development. Single sporozoites within the PVM grow rapidly and replicate into thousands of merozoites. These are released into the blood stream and go on to invade red blood cells, initiating the cyclic, self-perpetuating blood stage of infection responsible for the clinical symptoms associated with malaria. Relatively few parasites reside within the host during the early liver stage compared with the latter blood stage; therefore, the hepatocyte stage of infection is particularly important and presents an attractive therapeutic target. However, our knowledge of Plasmodium sporozoite−hepatocyte interactions is very limited, and largely relies on rodent studies. The study of human Plasmodium sporozoites during the liver stage is extremely difficult, particularly in the case of Plasmodium vivax, which cannot be continuously cultured in vitro. Despite these experimental difficulties, in PNAS, Boonhok et al. (1) use a membrane feeding system with freshly isolated blood from P. vivax patients to infect mosquitoes and obtain sporozoites for study in a specially designed human hepatocyte cell line. With this, Boonhok et al. (1) provide an essential piece of information on the P. vivax and hepatocyte interaction by demonstrating that liver cells stimulated with interferon gamma (IFN-γ) can mount an intracellular immune response against P. vivax sporozoites that largely depends on a novel form of noncanonical autophagy. This mechanism is shown to be similar to microtubule-associated protein light chain 3 (LC3)-associated phagocytosis (LAP), a process in which LC3 is deposited directly on the pathogen-containing vacuole, leading to a …

Comparative population genomics reveals key barriers to dispersal in Southern Ocean penguins

The mechanisms that determine patterns of species dispersal are important factors in the production and maintenance of biodiversity. Understanding these mechanisms helps to forecast the responses of species to environmental change. Here, we used a comparative framework and genomewide data obtained through RAD‐Seq to compare the patterns of connectivity among breeding colonies for five penguin species with shared ancestry, overlapping distributions and differing ecological niches, allowing an examination of the intrinsic and extrinsic barriers governing dispersal patterns. Our findings show that at‐sea range and oceanography underlie patterns of dispersal in these penguins. The pelagic niche of emperor (Aptenodytes forsteri), king (A. patagonicus), Adélie (Pygoscelis adeliae) and chinstrap (P. antarctica) penguins facilitates gene flow over thousands of kilometres. In contrast, the coastal niche of gentoo penguins (P. papua) limits dispersal, resulting in population divergences. Oceanographic fronts also act as dispersal barriers to some extent. We recommend that forecasts of extinction risk incorporate dispersal and that management units are defined by at‐sea range and oceanography in species lacking genetic data.