piggyBac is an effective tool for functional analysis of the Plasmodium falciparum genome

Abstract

Background: Much of the Plasmodium falciparum genome encodes hypothetical proteins with limited homology to other organisms. A lack of robust tools for genetic manipulation of the parasite limits functional analysis of these hypothetical proteins and other aspects of the Plasmodium genome. Transposon mutagenesis has been used widely to identify gene functions in many organisms and would be extremely valuable for functional analysis of the Plasmodium genome.

Results: In this study, we investigated the lepidopteran transposon, piggyBac, as a molecular genetic tool for functional characterization of the Plasmodium falciparum genome. Through multiple transfections, we generated 177 unique P. falciparum mutant clones with mostly single piggyBac insertions in their genomes. Analysis of piggyBac insertion sites revealed random insertions into the P. falciparum genome, in regards to gene expression in parasite life cycle stages and functional categories. We further explored the possibility of forward genetic studies in P. falciparum with a phenotypic screen for attenuated growth, which identified several parasite genes and pathways critical for intra-erythrocytic development.

Conclusion: Our results clearly demonstrate that piggyBac is a novel, indispensable tool for forward functional genomics in P. falciparum that will help better understand parasite biology and accelerate drug and vaccine development.

Figure 1

Plasmid design for piggyBac mutagenesis of P. falciparum. A summary of different transposon and transposase plasmids tested in P. falciparum. Maximum transformation efficiency was obtained while using a dual promoter for transposase expression.

Figure 2

Distribution of piggyBac insertion sites in the P. falciparum genome. (a) A representation of the 14 P. falciparum chromosomes with piggyBac insertion loci (represented by red vertical lines) shows extensive distribution of piggyBac insertions through out the parasite genome. (b) Comparison of chromosomal distribution of piggyBac insertions to the percent genome content of each chromosome shows unbiased insertions into P. falciparum genome. Plot and curve fits of percent piggyBac insertions and percent chromosome size are depicted in the inset.

Figure 3

piggyBac insertions in the genome are random but preferentially occur in 5' untranslated regions. (a) Genomic transcription units were defined to include 2 kb of 5' UTR, the coding sequence, the introns and 0.5 kb of 3' UTR, based on previous studies in Plasmodium [48,49]. (b) Comparison of gene functions of all annotated genes in the genome (outer circle) to genes in piggyBac-inserted loci (inner circle) shows an equivalent distribution confirming random insertions in the parasite genome. (c) Comparison of stage-specific expression of all annotated genes (outer circle) to those in piggyBac-inserted loci (inner circle) validates the ability of piggyBac to insert in genes expressed in all parasite life cycle stages. (d) A comparison of piggyBac-inserted TTAA sequences to TTAA sequences randomly selected from the genome showed preferential insertion of piggyBac into 5' UTRs of genes (asterisk- χ² test, df 1, P = 1.5 × 10^-12) whereas a significantly lower number of insertions were observed in CDS and introns (double asterisks- χ² test, df 1, P = 1.09 × 10^-13).

Figure 4

piggyBac inserts into AT-rich regions of the P. falciparum genome. (a) Nucleotide composition analysis of the flanking sequences showed that piggyBac inserted TTAA sites preferentially occur in the middle of an AT-rich core of 10 nucleotides predominantly with 'T's upstream (χ² test, df 1, P = 6.3 × 10^-5) and 'A's downstream (χ² test, df 1, P = 2.07 × 10^-8) as compared to randomly selected genomic TTAA sequences. (b) A comparison of nucleotide composition of flanking sequences only in the 5' untranslated regions (UTRs) of piggyBac inserted and randomly selected TTAA sequences further confirms the specificity of piggyBac for AT-rich target sites.

Figure 5

A phenotype screen for attenuated blood-stage growth. (a) A schematic of mutant P. falciparum clones selected for growth rate analysis. Black vertical and horizontal arrows indicate the insertion site and orientation of the piggyBac transposon, respectively. The gene schematic, description and expression stages were all obtained from the PlasmoDB database at http://www.plasmodb.org. (b) Growth curves of 9 insertional mutant clones, were obtained by plotting parasite fold change against time. For the wild type (WT), an average of fold changes from three different NF54 clones was used. The order of samples, from top to bottom, indicates a decrease in parasite fold changes. (c) A bar-graph of fold changes in parasite numbers after 7 days of growth revealed a spectrum of attenuated growth phenotypes in several mutant clones when compared to the wild type clones. The error bars in (b) and (c) represent standard deviation from the mean of 3 measurements.