RBC barcoding allows for the study of erythrocyte population dynamics and P. falciparum merozoite invasion.

Martha A Clark,Morgan M Goheen,Nicholas A Spidale,Anthony Fulford,Raj S Kasthuri,Carla Cerami

doi:10.1371/journal.pone.0101041

Martha A Clark, Morgan M Goheen + Show 4 more

Open Access

https://doi.org/10.1371/journal.pone.0101041

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Journal: PloS one	Publication Date: Jul 1, 2014
Citations: 16	License type: CC BY 4.0

Affiliation: University of North Carolina at Chapel Hill

Abstract

Plasmodium falciparum invasion of host erythrocytes is essential for the propagation of the blood stage of malaria infection. Additionally, the brief extracellular merozoite stage of P. falciparum represents one of the rare windows during which the parasite is directly exposed to the host immune response. Therefore, efficient invasion of the host erythrocyte is necessary not only for productive host erythrocyte infection, but also for evasion of the immune response. Host traits, such as hemoglobinopathies and differential expression of erythrocyte invasion ligands, can protect individuals from malaria by impeding parasite erythrocyte invasion. Here we combine RBC barcoding with flow cytometry to study P. falciparum invasion. This novel high-throughput method allows for the (i) direct comparison of P. falciparum invasion into different erythrocyte populations and (ii) assessment of the impact of changing erythrocyte population dynamics on P. falciparum invasion.

Highlights

Malaria is responsible for significant morbidity and mortality in the developing world, causing an estimated 250 million infections and 1 million deaths annually[1]
Barcoding red blood cells (RBCs) with CellTrace dyes DDAO and Violet allows for the direct comparison of P. falciparum invasion into different RBC populations To develop a P. falciparum RBC invasion assay that would allow for direct comparison of invasion of P. falciparum into two different RBC populations, we utilized a fluorescent RBC staining approach that permits the definitive detection of two distinct RBC populations by flow cytometry
SYBR Green I staining allows for the identification of parasitized RBCDDAO and pRBCViolet by both microscopy (Figure 1D & 1E) and flow cytometry (Figure 1G & 1H)

Summary

Introduction

Malaria is responsible for significant morbidity and mortality in the developing world, causing an estimated 250 million infections and 1 million deaths annually[1]. Following the asymptomatic liver stage, merozoites are released into the bloodstream where they infect host red blood cells (RBCs). It is the RBC stage of infection that is responsible for all symptoms of disease. Upon entry into the host RBC the malaria parasite progresses through a 48 hour lifecycle. At the end of the 48 hour intra-erythrocytic lifecycle the host RBC is ruptured and newly formed merozoites are released into the serum to invade new host RBCs [2]. Invasion occurs within 10 minutes of RBC rupture and the 48 hour intra-erythrocyte cycle begins again [3]. Successful invasion of the host RBC is essential for propagation of the parasite. Merozoite invasion represents a promising point of attack for antimalarial drugs and vaccines [4]

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Conclusion