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  • Review Article
  • Published:

Advances in molecular genetic systems in malaria

Nature Reviews Microbiology volume 13, pages 373–387 (2015)Cite this article

Subjects

Key Points

  • The ability to genetically manipulate the malaria parasite to discover the functions of its genes has provided a powerful technique to gain detailed insight into Plasmodium spp. biology and to identify and characterize potential targets for antimalaria intervention.

  • The human malaria species Plasmodium falciparum and the rodent malaria species Plasmodium berghei have been the species predominantly used for gene manipulation studies. Although the genetic systems for these species vary in their efficiency and their approach and whether they are used in vitro or in vivo, their results can often be very complementary.

  • As a result of the haploid nature of malaria parasites, genes that have an important role in the blood stages of Plasmodium spp. cannot be genetically targeted for disruption using conventional knockout approaches, as this would lead to parasite death or severe growth defects. This has forced the development of conditional systems to rapidly regulate gene expression.

  • An array of conditional systems that are capable of regulating gene expression at the genome, transcript or protein level are now available for Plasmodium spp.. Each system has advantages and drawbacks, which should be considered carefully before choosing a system to regulate a gene of interest.

  • A variety of methods can be used to edit the Plasmodium genome. These include the use of piggyBac transposons that integrate randomly into the genome, or more precise editing tools such as the CRISPR–Cas9 system, zinc-finger nucleases and integrases.

  • Improvements to transfection efficiency and investment in the development of more robust genetic tools and phenotypic assays should lead to large-scale forward-genetic screens being possible in Plasmodium spp..

Abstract

Robust tools for analysing gene function in Plasmodium parasites, which are the causative agents of malaria, are being developed at an accelerating rate. Two decades after genetic technologies for use in Plasmodium spp. were first described, a range of genetic tools are now available. These include conditional systems that can regulate gene expression at the genome, transcriptional or protein level, as well as more sophisticated tools for gene editing that use piggyBac transposases, integrases, zinc-finger nucleases or the CRISPR–Cas9 system. In this Review, we discuss the molecular genetic systems that are currently available for use in Plasmodium falciparum and Plasmodium berghei, and evaluate the advantages and limitations of these tools. We examine the insights that have been gained into the function of genes that are important during the blood stages of the parasites, which may help to guide the development and improvement of drug therapies and vaccines.

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Figure 1: Development of transfection technologies for Plasmodium parasites.
Figure 2: Strategies to conditionally regulate gene expression in Plasmodium parasites.
Figure 3: New strategies for editing the Plasmodium spp. genome.

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Acknowledgements

The authors apologize to those whose work could not be cited owing to length restrictions. They would like to acknowledge support from the National Health and Medical Research Council, Australia and from the Victorian State Government Operational Infrastructure Support Scheme, Australia.

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Authors and Affiliations

  1. Deakin University, Waurn Ponds, 3216, Victoria, Australia

    Tania F. de Koning-Ward

  2. Macfarlane Burnet Institute for Medical Research and Public Health, Melbourne, 3004, Victoria, Australia

    Paul R. Gilson & Brendan S. Crabb

  3. Monash University, Clayton, 3800, Victoria, Australia

    Paul R. Gilson & Brendan S. Crabb

  4. The University of Melbourne, Parkville, 3010, Victoria, Australia

    Brendan S. Crabb

Authors
  1. Tania F. de Koning-Ward
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  3. Brendan S. Crabb
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Correspondence to Tania F. de Koning-Ward, Paul R. Gilson or Brendan S. Crabb.

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Glossary

Artemisinin

A rapid-acting antimalarial drug isolated from the plant Artemisia annua. Artemisinin-based combination therapies (ACTs) are recommended by the World Health Organisation as the first-line treatment for uncomplicated Plasmodium falciparum malaria.

Immunogens

Antigens that are capable of inducing an immune response.

Transfection

The process of introducing nucleic acids into cells.

Schizonts

Mature forms of malaria parasites that are present in the liver and blood, which in blood contain ∼12–16 individual merozoites.

Single-crossover recombination

A homologous recombination event that leads to the insertion of the entire vector backbone and duplication of targeting sequences.

Negative selection

A process used to deplete parasites that express a negative-selectable marker from a population, in order to enrich parasites that contain the desired genomic integration event.

Double-crossover recombination

A homologous recombination event that results in the replacement of a DNA sequence that is flanked by two targeting sequences.

Parasitophorous vacuole

A vacuole in the host cell, in which Plasmodium parasites reside and develop.

Cryptic polyadenylation sites

A processing site that is not normally used for the addition of a polyadenylic acid tail to mRNA.

Merosomes

Structures containing hundreds of infectious merozoites that are surrounded by a membrane that is derived from the hepatocyte host cell.

Ubiquitylation

A post-translational enzymatic modification involving the attachment of ubiquitin to a protein substrate.

Dominant negative transgenes

A gene that when expressed in trans causes an adverse effect on the normal, wild-type gene product that is expressed in the same cell.

Maurer's cleft

Single-membrane-bound structure that is present in the cytoplasm of erythrocytes that are infected with Plasmodium falciparum and that functions in transport of proteins from the parasite to the surface of the erythrocyte.

Viral 2A ribosomal skipping peptide

A peptide derived from foot-and-mouth disease virus 2A that, when introduced as a linker between two proteins, allows autonomous intraribosomal self-processing of the resulting polyproteins that are expressed from a single polycistronic mRNA transcript.

Ring-form parasites

The feeding stages of blood-stage parasites that show a ring-like morphology in Giemsa-stained blood smears.

Lambda Red recombination system

This tool enables targeted genetic changes to DNA in Escherichia coli expressing the lambda Red recombinase. This system has been used in conjunction with Gateway technology to convert Plasmodium berghei genomic DNA clones that have been maintained in E. coli into gene-targeting vectors.

Gateway cloning system

A molecular methodology that enables the transfer of DNA fragments between plasmids using attP recombination sequences and a mixture of commercial clonase enzymes.

Two-photon microscopy

A fluorescence imaging technique that absorbs two photons of infrared light to provide deeper tissue penetration, which enables living tissues to be imaged to a greater depth than conventional confocal microscopy.

Non-essential genes

Genes that can be deleted in the parasite without causing parasite death under certain conditions. These include genes that do not impart moderate or severe growth defects when mutated. However, genes that are termed 'non-essential' may in fact be essential to parasite growth when tested under different environmental conditions.

Hypnozoites

Dormant forms of Plasmodium spp. parasites that are present in the liver.

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de Koning-Ward, T., Gilson, P. & Crabb, B. Advances in molecular genetic systems in malaria. Nat Rev Microbiol 13, 373–387 (2015). https://doi.org/10.1038/nrmicro3450

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