A genetic screen for attenuated growth identifies genes crucial for intraerythrocytic development of Plasmodium falciparum

Abstract

A majority of the Plasmodium falciparum genome codes for genes with unknown functions, which presents a major challenge to understanding the parasite's biology. Large-scale functional analysis of the parasite genome is essential to pave the way for novel therapeutic intervention strategies against the disease and yet difficulties in genetic manipulation of this deadly human malaria parasite have been a major hindrance for functional analysis of its genome. Here, we used a forward functional genomic approach to study P. falciparum and identify genes important for optimal parasite development in the disease-causing, intraerythrocytic stages. We analyzed 123 piggyBac insertion mutants of P. falciparum for proliferation efficiency in the intraerythrocytic stages, in vitro. Almost 50% of the analyzed mutants showed significant reduction in proliferation efficiency, with 20% displaying severe defects. Functional categorization of genes in the severely attenuated mutants revealed significant enrichment for RNA binding proteins, suggesting the significance of post-transcriptional gene regulation in parasite development and emphasizing its importance as an antimalarial target. This study demonstrates the feasibility of much needed forward genetics approaches for P. falciparum to better characterize its genome and accelerate drug and vaccine development.

Figure 1. An in vitro proliferation assay for Plasmodium falciparum.

A flowchart of steps involved in the in vitro proliferation assay for Plasmodium falciparum intraerythrocytic stages.

Figure 2. Attenuation of intraerythrocytic growth rate in mutant Plasmodium falciparum clones.

(A) Categorization of piggyBac-transformed P. falciparum mutant clones as attenuated (with a reduced fold-change of >2 standard deviation (SD) from the mean of wild-type fold-change) and non-attenuated revealed attenuation of approximately 50% of the mutant clones. (B) Further sub-classification of the attenuated mutants as severely attenuated (with a reduced fold-change of >4 SD from the mean of WT fold-change) and moderately attenuated (with a reduced fold-change of 2>SD<4 from the mean of WT fold-change) showed severe attenuation of blood-stage growth in approximately 20% of the mutants.

Figure 3. Functional categorization of genes immediately flanking piggyBac insertion sites.

Functional distribution of all genes at piggyBac insertion loci did not show a difference between attenuated and non-attenuated mutants (A) however, following sub-classification of the attenuated mutants, genes involved in nucleic acid metabolism/nucleic acid binding/transcription were significantly enriched in the severely attenuated mutants (B).

Figure 4. Functional categorization of transcriptional units immediately flanking piggyBac insertion sites.

Functional distribution of transcriptional units (defined to include 1 kb sequence upstream to the coding sequence (CDS), the CDS and 0.3 kb downstream to the CDS) affected by piggyBac insertion did not reveal any differences between the attenuated and non-attenuated mutants as a whole (A) but showed significant enrichment of nucleic acid metabolism/nucleic acid binding/transcription genes in the severely attenuated mutants following sub-classification of the attenuated mutants (B).