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Human Malaria Parasite Mutagenesis Highlights Essential Genes, Potential Drug Targets

NEW YORK (GenomeWeb) – A team from the US and the UK have used a saturation mutagenesis experiment technique to tally up the essential gene repertoire of the malaria parasite Plasmodium falciparum.

The researchers used high-throughput transposon mutagenesis, coupled with quantitative insertion site sequencing, to systematically alter P. falciparum genes. The approach produced 38,000 distinct insertions in the P. falciparum genome, in and around almost 5,400 nuclear protein-coding genes. By retracing the distribution of these mutations, and their growth consequences during the parasite's asexual blood stage they identified 2,680 essential genes, highlighting potential drug targets. The results were published online today in Science.

"The complete characterization of the essential genome with high-throughput saturation mutagenesis will help open new frontiers for antimalarial therapeutic research," corresponding authors John Adams and Rays Jiang, with the University of South Florida's global health department, and Wellcome Trust Sanger Institute malaria researcher Julian Rayner, and their colleagues wrote. They noted that the same strategy "opens the way for new systematic functional screens for other phenotypes, such as transmission and cytoadherence."

The P. falciparum parasite has a notoriously adenine- and thymine-rich genome with more than 81 percent AT content, the team explained, which complicates aspects of CRISPR-Cas9-based genome editing but is well suited to piggyBac mutagenesis. They noted that this sequence bias produces the transposon's thymine- and adenine-based, four-nucleotide target site roughly once every 70 P. falciparum bases, on average.

The team used a high-throughput transfection mutagenesis method that relied on a robot to transfect the wild-type P. falciparum stain NF54 with piggyBac transposons in 96-well plates. The approach yielded roughly 38,000 mutants, each containing a distinct piggyBac insertion identified by Illumina sequencing.

All told, these mutations affected 5,399 different protein-coding genes in P. falciparum, the team reported. Through a series of follow-up analyses, the group looked at the distribution of coding and non-coding sequence insertions, the predicted consequences on biological processes and gene expression patterns, and mutant parasite growth potential in competitive growth screens.

In the process, the researchers classified some 2,680 parasite genes as essential for optimal growth in vitro during the blood stage of infection. They noted that the essential gene set, which spans some 1,000 conserved genes with unknown functions, will likely provide fodder for future drug target- and drug resistance studies.

"Using our genetic analysis tools, we're able to determine the relative importance of each gene for parasite survival," Adams said in a statement. "This understanding will help guide future drug development efforts targeting those essential genes."

For example, when the researchers looked at the proteasome degradation process, they found that 54 of the 72 genes in that pathway were classified as essential based on results from the mutagenesis screen, suggesting the pathway may prove useful as a drug target. Conversely, their results suggest that a proteasome inhibitor could boost the sensitivity of mutant malaria-causing parasites with known sensitivity to the artemisinin malaria drug. 

The authors argued that such results provide "additional support for the association between the ART (artemisinin) mechanism of action and proteasome function."

In a related study published last year in Cell, a Sanger Institute-led team did a functional screen of the mouse malaria parasite  P. berghei, using barcoded knockout mutant parasites to assess the growth consequences of systematically lopping out almost 2,600 P. berghei genes.

In a commentary article in Science accompanying the new study, University of Washington malaria researchers Pradipsinh Rathod and John White wrote that the list of essential P. falciparum genes may contain "our best hopes for identifying good targets for the most clinically relevant part of the parasite life cycle," adding, "Even if the malaria research community, within a decade or two, finds that only [10 percent] of the 2,680 identified essential malaria genes are high-value targets for drug development, this screening approach will be considered successful." 

Given the challenges that have plagued malaria research in the past, as well as the rarity of authenticated, effective drug targets, the duo cautioned that "modest expectations" are likely in order. Still, they called the new study "a powerful start for identifying rare, high-value, potentially druggable processes in human malaria parasites."