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Stable knockout and complementation of receptor expression using in vitro cell line derived reticulocytes for dissection of host malaria invasion requirements

Timothy J Satchwell, Katherine E Wright, Katy L Haydn-Smith, Fernando Sánchez-Román Terán, Joseph Hawksworth, Jan Frayne, Ashley M Toye, Jake Baum

Preprint posted on December 13, 2018 https://www.biorxiv.org/content/early/2018/12/13/495853

Article now published in Nature Communications at http://dx.doi.org/10.1038/s41467-019-11790-w

CRISPR meets red blood cells – a new model system to illuminate the roles of host proteins in malaria parasite invasion

Selected by Alyson Smith

Why I think this study is interesting

This study turns over a new leaf in malaria research by applying a new model system to directly test the roles of red blood cell proteins in the parasite life cycle. Modern genetic technologies (e.g., CRISPR-Cas9) can now be employed to answer questions that were previously difficult or impossible to address. This and future studies can accelerate the development of therapies for this serious disease and answer other standing questions in basic and translational red blood cell biology.

Background

Parasites of the Plasmodium genus cause malaria by invading red blood cells and developing within them, ultimately causing red blood cell membrane rupture (1). Mature human red blood cells and reticulocytes (their immediate precursors in erythroid differentiation) lack nuclei, preventing genetic manipulation to probe the roles of host proteins in Plasmodium infection. While malaria researchers have used proteases, antibodies, and rare spontaneous mutations to study this process, our knowledge of host protein involvement remains incomplete.

To address this issue, scientists have developed in vitro culture systems using primary hematopoietic stem cells (CD34+ cells) or immortalized erythoid cell lines. However, CD34+ cells proliferate only briefly in an undifferentiated state and immortalized cell lines do not efficiently produce anucleate reticulocytes. These factors limit the efficiency of production of genetically modified reticulocytes to use in malaria invasion models.

Some of the authors of this study recently developed an immortalized human adult erythroid cell line (Bristol Erythroid Line Adult, or BEL-A) that can proliferate in an undifferentiated state and can be induced to terminally differentiate and form anucleate reticulocytes (2). In this study, the authors use this cell line and CRISPR-Cas9 to test the role of host proteins in P. falciparum invasion.

Key findings

Bristol Erythroid Line Adult (BEL-A)-derived reticulocytes are an effective model for P. falciparum invasion. 

The authors induced terminal differentiation in BEL-A cells and isolated anucleate reticulocytes. After infection with P. falciparum, these reticulocytes had similar parasitemia to native, mature red blood cells. The parasite appeared to grow, mature, and reinvade at similar rates in both systems.

Basigin (BSG) is required for P. falciparum invasion, but this activity does not require the BSG cytoplasmic tail. 

Previous work has implicated BSG (a host surface receptor) in the invasion process, and in other cell types the cytosplasmic tail of BSG plays a role in signaling. Using CRISPR-Cas9, the authors knocked out BSG without affecting BEL-A expansion, terminal differentiation, or expression of known P. falciparum receptors (GPA, GPC, band 3, CD55, and CD44). Both wild type BSG and BSG with the cytoplasmic tail truncated (expressed at close to endogenous levels) rescued P. falciparum invasion on the BSG KO background. These experiments confirm the role of BSG in invasion, but future work is required to find the BSG domain responsible for this activity.

Cyclophilin B (CypB) is not required for P. falciparum invasion.

A recent study suggested that CypB forms a multiprotein complex (including BSG) that is required for invasion. Using CRISPR-Cas9, the authors found that CypB KO BEL-A-derived reticulocytes had similar parasitemia to unmodified reticulocytes, suggesting that CypB is not required for invasion.

Flow cytometry can detect parasitemia in BEL-A-derived reticulocytes.

In mature red blood cells – which lack DNA and mRNA – parasitemia can be quickly measured using nucleic acid-binding dyes to detect parasite DNA by flow cytometry. In this study, the mRNA in the BEL-A-derived reticulocytes and contaminating DNA from nucleated reticulocyte precursors and unruptured schizonts created significant background in this assay. Despite this, the authors detected differences in parasitemia between BSG KO and wild type reticulocytes. The authors recommend using light microscopy to confirm phenotypes detected in flow cytometry.

Future directions

This study opens the door to future work to elucidate the roles of host proteins in P. falciparum invasion, development and egress. Because BEL-A-derived reticulocytes express receptors for other Plasmodium species (P. vivax and P. knowlesi), this system can be expanded to study these species as well. Because flow cytometry can detect changes in parasitemia in this system, it can be adapted for high-throughput screens to test the roles of host proteins and develop new therapeutics.

Despite the utility of BEL-A-derived reticulocytes as a model system, they likely fail to reproduce all aspects of native Plasmodium infection. The malaria community will have to devise methods to improve this system and verify its results. Beyond malaria research, the genetic toolkit available in other human cell lines can now be applied to expand our understanding of reticulocyte and erythroid biology and pathology.

References

  1. Koch M & Baum J (2016) The mechanics of malaria parasite invasion of the human erythrocyte – towards a reassessment of the host cell contribution. Cell Microbiol 18(3):319-329.
  2. Trakarnsanga, K., Griffiths, R.E., Wilson, M.C., Blair, A., Satchwell, T.J., Meinders, M., Cogan, N., Kupzig, S., Kurita, R., Nakamura, Y., et al. (2017). An immortalized adult human erythroid line facilitates sustainable and scalable generation of functional red cells. Nature communications 8, 14750.

Tags: crispr, malaria, parasite invasion, red blood cells

Posted on: 8th January 2019

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