Article series: New technologies: methods and applications

Advances in molecular genetic systems in malaria

Journal name:
Nature Reviews Microbiology
Volume:
13,
Pages:
373–387
Year published:
DOI:
doi:10.1038/nrmicro3450
Published online

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.

At a glance

Figures

  1. Development of transfection technologies for Plasmodium parasites.
    Figure 1: Development of transfection technologies for Plasmodium parasites.

    Technologies that have been developed for Plasmodium falciparum (shown in blue boxes) and Plasmodium berghei (pink boxes) since transfection of a luciferase reporter was first reported in Plasmodium gallinaceum (green box) are shown. Technologies that were developed for both P. falciparum and P. berghei at similar times are shown in purple boxes. ATc, anhydrotetracycline; BSD, blasticidin; CAD, conditional aggregation domain; CAT, chloramphenicol acetyltransferase; CD, cytosine deaminase; DD, destabilization domain; DDD, DHFR degradation domain; DHFR, dihydrofolate reductase; DHODH, dihydroorotate dehydrogenase; GIMO, gene insertion/marker out; NEO, neomycin; PAC, puromycin; TetR, tetracycline repressor; TK, thymidine kinase; yFCU, yeast cytosine deaminase–uracil phosphoribosyl transferase fusion protein.

  2. Strategies to conditionally regulate gene expression in Plasmodium parasites.
    Figure 2: Strategies to conditionally regulate gene expression in Plasmodium parasites.

    a | Conditional deletion of a gene of interest (GOI) through diCre–lox recombination. Integration of a targeting construct by homologous recombination at the 5′ and 3′ UTR leads to replacement of the GOI with a codon-optimized version of the GOI (coGOI) and the human dihydrofolate reductase (DHFR) selectable marker, both of which are flanked by loxP sites. Parasites are then transfected with an episome that contains the blasticidin (bsd) selectable marker together with genes encoding two separate inactive polypeptides of Cre (Cre1 and Cre2) that are each fused to different rapamycin-binding proteins (FK506-binding protein (FKBP) and fibronectin binding protein (FBP)). Following the addition of rapamycin, the two Cre polypeptides form heterodimers (diCre), which restores recombinase activity and results in the excision of loxP-flanked sequences (in this case, both the coGOI and human DHFR). b | Transcriptional knockdown with the anhydrotetracycline (ATc)-inducible system. The targeting construct, which contains genes encoding a transcriptional transactivator domain (TRAD) and human DHFR, is integrated by homologous recombination at the 5′ UTR and amino terminus of the GOI. TRAD is placed under the transcriptional control of the promoter of the GOI, and the GOI is controlled by an inducible minimal promoter. This inducible promoter comprises seven tetracycline operator (TetO) sequences that are located immediately upstream of a minimal promoter element. In the absence of ATc, the expressed TRAD binds to the TetO sequences and induces transcription of the GOI. Addition of ATc prevents the TRAD from activating the minimal promoter and thus decreasing GOI transcription. c | Post-transcriptional knockdown using a ribozyme-based gene expression system. The targeting construct contains the glmS ribozyme, which is introduced into the genome by homologous recombination at the carboxyl terminus of the GOI; specifically, glmS is placed between the stop codon and the 3′ UTR downstream of the GOI. In addition, the construct contains a haemagglutinin epitope tag (HA) that is fused to the GOI and the gene encoding a selectable marker. Addition of glucosamine-6-phosphate (GlcN-6P) activates the ribozyme, which cleaves the mRNA and removes the 3′ UTR, leading to rapid degradation of the mRNA and a reduction in protein levels, as measured by HA levels. X indicates the regions where homologous recombination occurs. d | Post-translational knockdown. A mutant version of the human rapamycin-binding protein FKPB12, termed the destabilization domain (DD), is fused to the N terminus or C terminus of the target protein. Shield 1, which is a cell-permeable small-molecule ligand of FKPB12, binds to the DD, thereby stabilizing the protein. Removal of this ligand leads to the degradation of the protein of interest (POI). Thus, this system enables the rapid modulation of the expression level of the POI. X indicates the regions where homologous recombination occurs.

  3. New strategies for editing the Plasmodium spp. genome.
    Figure 3: New strategies for editing the Plasmodium spp. genome.

    a | Site-specific integration into the Plasmodium spp. genome using the Bxb1 integrase system. Two plasmids are transfected, one of which contains the gene encoding the Bxb1 integrase, which catalyses recombination between an incoming attP site that is present on the second plasmid containing the desired (trans)gene of interest (GOI) and a chromosomal attB site that has already been engineered into a gene that is not essential for blood-stage growth (neg). Recombination between the attP and attB sites produces asymmetric attL and attR sites that cannot recombine. b | Editing of the Plasmodium spp. genome using zinc-finger nucleases (ZFNs). A donor plasmid encoding a ZFN pair (ZFNL and ZFNR) that has been co-expressed from a single promoter using a viral 2A ribosomal skipping peptide is transfected into Plasmodium parasites, together with a plasmid containing the GOI with a specific mutation in the GOI cDNA. Following expression, dimerization of ZFNR and ZFNL results in the assembly of an artificial enzyme, which induces a double-strand break in the genome at the site homologous to sequences that are included in the ZFNs. Subsequently, the breaks are repaired by homologous-directed repair using homologous regions of the donor plasmid as the template (in this case, the 5′ and 3′ UTRs). This leads to the replacement of the GOI with a cDNA version of the GOI, which contains the desired mutation (for example, mutations that confer drug resistance). c | CRISPR–Cas9 genome editing requires expression of both the Cas9 endonuclease and the single-guide RNA (sgRNA). Cas9 is expressed in the parasite from an episome that contains the yeast dihydroorotate dehydrogenase (dhodh) drug-selectable marker. The sgRNA, which is placed under the transcriptional control of the 5′ UTR and 3′ UTR of the U6 polymerase III promoter (5′ U6 and 3′ U6, respectively), is incorporated into the targeting construct, which contains the human dihydrofolate reductase (DHFR) selectable marker and is flanked by targeting sequences (in this case GOI sequences) to drive homologous integration into the genome. The sgRNA must comprise 20 nucleotides that match the target DNA site, as well as a Cas9-binding domain (not shown) to guide the Cas9 endonuclease to the target DNA site, where it induces double-strand breaks. These breaks are subsequently repaired by homologous recombination. d | Random gene insertional mutagenesis in the Plasmodium spp. genome using the piggyBac transposon system. Two plasmids are transfected into Plasmodium parasites: pXL–Bacll–DHFR, which contains the human DHFR selectable marker flanked by two inverted terminal repeats (ITR1 and ITR2) of the piggyBac element; and pHTH, which contains a sequence encoding the piggyBac class II integrase that precisely excises the piggyBac element to randomly target a tetranucleotide target site (TTAA) in the genome of the parasite. Insertions can occur in a random gene (rg), as indicated in the figure, or can flank a protein-coding sequence. After a library of parasite clones has been obtained, the piggyBac insertions and their flanking genes can be identified by PCR. bsd, blasticidin selectable marker; gDNA, genomic DNA.

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Affiliations

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

    • Tania F. de Koning-Ward
  2. Macfarlane Burnet Institute for Medical Research and Public Health, Melbourne, Victoria 3004, Australia.

    • Paul R. Gilson &
    • Brendan S. Crabb
  3. Monash University, Clayton, Victoria 3800, Australia.

    • Paul R. Gilson &
    • Brendan S. Crabb
  4. The University of Melbourne, Parkville, Victoria 3010, Australia.

    • Brendan S. Crabb

Competing interests statement

The authors declare no competing interests.

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Author details

  • Tania F. de Koning-Ward

    Tania F. de Koning-Ward obtained her Ph.D. in microbiology from the University of Melbourne, Australia, in 1996. After postdoctoral research at Leiden University in the Netherlands, and at the Walter and Eliza Hall Institute of Medical Research, Victoria, Australia, in which she utilized both rodent and human malaria transfection systems, she established her own research programme at the School of Medicine at Deakin University, Waurn Ponds, Victoria, Australia, as an associate professor. Her research interests include understanding at a molecular level how malaria parasites remodel their host cells to ensure their success as pathogens and identifying new strategies for targeting malaria parasites. She is currently a National Health and Medical Research Council of Australia career development fellow.
    Tania F. de Koning-Ward's homepage

  • Paul R. Gilson

    Paul R. Gilson obtained his Ph.D. in evolutionary botany from the School of Botany, University of Melbourne, Australia. After postdoctoral appointments that involved working on plants and protozoa, he began his parasitology work on malaria at the Walter and Eliza Hall Institute of Medical Research, Victoria, Australia, in 2003. In 2008, he moved to the Burnet Institute, Melbourne, Australia, to co-head a malaria research team with Brendan S. Crabb. His main interests include understanding how malaria parasites invade human erythrocytes and then modify their host cells to grow rapidly and avoid the immune system of the human host. This work is being done with the long-term goal of developing improved drug therapies and vaccines.
    Paul R. Gilson's homepage

  • Brendan S. Crabb

    Brendan S. Crabb obtained his Ph.D. in molecular microbiology from The University of Melbourne, Australia. He is now the Director and CEO of the Burnet Institute in Melbourne, Australia, and co-head of a malaria research team with Paul Gilson. He is also the Immediate-Past President of the Association of Australian Medical Research Institutes. He has a particular interest in infectious disease and in health issues in the developing world, and his primary research interests include the development of a malaria vaccine and the identification of new treatments for this disease. He is the current Chair of the MVI/PATH Malaria Vaccine Science Portfolio Advisory Committee. Previously, he was a Senior Principal Research Fellow of the National Health and Medical Research Council of Australia and an International Research Fellow of the Howard Hughes Medical Institute in the USA. He was the editor-in-chief of the International Journal for Parasitology from 2006 to 2009, and he was awarded a Companion of the Order of Australia in 2015 for his contributions to medical research and global health.
    Brendan S. Crabb's homepage

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