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An adaptable two-color flow cytometric assay to quantitate the invasion of erythrocytes by Plasmodium falciparum parasites.

ABSTRACT: Plasmodium falciparum genotyping has recently undergone a revolution, and genome-wide genotype datasets are now being collected for large numbers of parasite isolates. By contrast, phenotyping technologies have lagged behind, with few high throughput phenotyping platforms available. Invasion of human erythrocytes by Plasmodium falciparum is a phenotype of particular interest because of its central role in parasite development. Invasion is a variable phenotype influenced by natural genetic variation in both the parasite and host and is governed by multiple overlapping and in some instances redundant parasite-erythrocyte interactions. To facilitate the scale-up of erythrocyte invasion phenotyping, we have developed a novel platform based on two-color flow cytometry that distinguishes parasite invasion from parasite growth. Target cells that had one or more receptors removed using enzymatic treatment were prelabeled with intracellular dyes CFDA-SE or DDAO-SE, incubated with P. falciparum parasites, and parasites that had invaded either labeled or unlabeled cells were detected with fluorescent DNA-intercalating dyes Hoechst 33342 or SYBR Green I. Neither cell label interfered with erythrocyte invasion, and the combination of cell and parasite dyes recapitulated known invasion phenotypes for three standard laboratory strains. Three different dye combinations with minimal overlap have been validated, meaning the same assay can be adapted to instruments harboring several different combinations of laser lines. The assay is sensitive, operates in a 96-well format, and can be used to quantitate the impact of natural or experimental genetic variation on erythrocyte invasion efficiency.

SUBMITTER: Theron M

PROVIDER: S-EPMC3047707 | biostudies-literature | 2010 Nov

REPOSITORIES: biostudies-literature

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An adaptable two-color flow cytometric assay to quantitate the invasion of erythrocytes by Plasmodium falciparum parasites.

Theron Michel M Hesketh Richard L RL Subramanian Sathish S Rayner Julian C JC

Cytometry. Part A : the journal of the International Society for Analytical Cytology 20101101 11

Plasmodium falciparum genotyping has recently undergone a revolution, and genome-wide genotype datasets are now being collected for large numbers of parasite isolates. By contrast, phenotyping technologies have lagged behind, with few high throughput phenotyping platforms available. Invasion of human erythrocytes by Plasmodium falciparum is a phenotype of particular interest because of its central role in parasite development. Invasion is a variable phenotype influenced by natural genetic variat ...[more]

PMID: 20872885

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Project description: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.

Project description:Remodeling the erythrocyte membrane and skeleton by the malarial parasite Plasmodium falciparum is closely associated with intraerythrocytic development. However, the mechanisms underlying this association remain unclear. In this study, we present evidence that erythrocytic α-spectrin, but not β-spectrin, was dynamically ubiquitinated and progressively degraded during the intraerythrocytic development of P. falciparum, from the ring to the schizont stage. We further observed an upregulated expression of P. falciparum phosphatidylinositol 3-kinase (PfPI3K) in the infected red blood cells during the intraerythrocytic development of the parasite. The data indicated that PfPI3K phosphorylated and activated erythrocytic ubiquitin-protein ligase, leading to increased α-spectrin ubiquitination and degradation during P. falciparum development. We further revealed that inhibition of the activity of PfPI3K impaired P. falciparum development in vitro and Plasmodium berghei infectivity in mice. These findings collectively unveil an important mechanism of PfPI3K-ubiquitin-mediated degradation of α-spectrin during the intraerythrocytic development of Plasmodium species. Proteins in the PfPI3K regulatory pathway are novel targets for effective treatment of severe malaria.ImportancePlasmodium falciparum is the causative agent of severe malaria that causes millions of deaths globally. The parasite invades human red blood cells and induces a cascade of alterations in erythrocytes for development and proliferation. Remodeling the host erythrocytic cytoskeleton is a necessary process during parasitization, but its regulatory mechanisms remain to be elucidated. In this study, we observed that erythrocytic α-spectrin is selectively degraded after P. falciparum invasion, while β-spectrin remained intact. We found that the α-spectrin chain was profoundly ubiquitinated by E3 ubiquitin ligase and degraded by the 26S proteasome. E3 ubiquitin ligase activity was regulated by P. falciparum phosphatidylinositol 3-kinase (PfPI3K) signaling. Additionally, blocking the PfPI3K-ubiquitin-proteasome pathway in P. falciparum-infected red blood cells reduced parasite proliferation and infectivity. This study deepens our understanding of the regulatory mechanisms of host and malarial parasite interactions and paves the way for the exploration of novel antimalarial drugs.

Dataset Information

An adaptable two-color flow cytometric assay to quantitate the invasion of erythrocytes by Plasmodium falciparum parasites.

Publications

An adaptable two-color flow cytometric assay to quantitate the invasion of erythrocytes by Plasmodium falciparum parasites.

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