As one of the deadliest organisms on the planet, the malaria protozoan parasite, in particular Plasmodium falciparum, owes much of its pathogenicity to its unique genomic makeup. This parasite species has a genome that is overwhelmingly skewed toward A-T base pairs (>80%), often making it difficult to study and intractable to basic molecular biological techniques. However now, a team of scientists led by investigators at the University of South Florida has exploited this quirk in the genetic makeup of P. falciparum to create 38,000 mutant strains and then determine which of the organism's genes are essential to its growth and survival. Findings from the new study were published recently in Science (“Uncovering the Essential Genes of the Human Malaria Parasite Plasmodium falciparum by Saturation Mutagenesis”).
P. falciparum is responsible for about half of all malaria cases and 90% of all malaria deaths. This new information about the parasite's critical gene repertoire could help investigators prioritize targets for future antimalarial drug development.
“Our study exploits the AT-richness of the P. falciparum genome, which provides numerous piggyBac transposon insertion targets within both gene coding and noncoding flanking sequences, to generate more than 38,000 P. falciparum mutants,” the authors wrote. “At this level of mutagenesis, we could distinguish essential genes as nonmutable and dispensable genes as mutable.”
The complete genetic sequence of P. falciparum was determined more than a decade ago, but the functions of most of its genes remain unknown, and until now only a few hundred mutant strains had been created in the lab. The difficulties in manipulating P. falciparum stem in part from the extremely high percentage of adenine and thymine in its genome. Standard methods for creating mutants rely on more variation in gene sequences and so do not work on P. falciparum.
In the current study, the researchers created mutated versions of nearly all the parasite's 6000 genes with a technique that preferentially targets areas rich in adenine and thymine, thus exploiting the very feature that had foiled previous attempts at genetic manipulation.
“We calculated mutagenesis index scores (MISs) and mutagenesis fitness scores (MFSs) in order to functionally define the relative fitness cost of disruption for 5399 genes,” the authors noted. “A competitive growth phenotype screen confirmed that MIS and MFS were predictive of the fitness cost for in vitro asexual growth. Genes predicted to be essential included genes implicated in drug resistance—such as the “K13” Kelch propeller, mdr, and dhfr-ts—as well as targets considered to be a high value for drugs development, such as pkg and cdpk5. The screen revealed essential genes that are specific to human Plasmodium parasites but absent from rodent-infective species, such as lipid metabolic genes that may be crucial to transmission commitment in human infections.”
The computational analysis utilized was able to distinguish nonessential genes (those that could be mutated) from essential, nonmutable ones. About 2600 were identified as indispensable for growth and survival during the parasite's asexual blood stage. These included ones associated with P. falciparum's ability to resist antimalaria drugs, highlighting them as high-priority targets for new or improved antimalarial compounds.
“Saturation-scale mutagenesis allows prioritization of intervention targets in the genome of the most important cause of malaria. The identification of more than 2680 essential genes, including ~1000 Plasmodium-conserved essential genes, will be valuable for antimalarial therapeutic research,” the authors concluded.