Severe malaria is caused by the apicomplexan parasite
Despite decades of research, the distinct biology of these parasites has made it challenging to establish high-throughput genetic approaches to ...identify and prioritize therapeutic targets. Using transposon mutagenesis of
in an approach that exploited its AT-rich genome, we generated more than 38,000 mutants, saturating the genome and defining mutability and fitness costs for over 87% of genes. Of 5399 genes, our study defined 2680 genes as essential for optimal growth of asexual blood stages in vitro. These essential genes are associated with drug resistance, represent leading vaccine candidates, and include approximately 1000
-conserved genes of unknown function. We validated this approach by testing proteasome pathways for individual mutants associated with artemisinin sensitivity.
Genetic screening using random transposon insertions has been a powerful tool for uncovering biology in prokaryotes, where whole-genome saturating screens have been performed in multiple organisms. ...In eukaryotes, such screens have proven more problematic, in part because of the lack of a sensitive and robust system for identifying transposon insertion sites. We here describe quantitative insertion-site sequencing, or QIseq, which uses custom library preparation and Illumina sequencing technology and is able to identify insertion sites from both the 5' and 3' ends of the transposon, providing an inbuilt level of validation. The approach was developed using piggyBac mutants in the human malaria parasite Plasmodium falciparum but should be applicable to many other eukaryotic genomes. QIseq proved accurate, confirming known sites in >100 mutants, and sensitive, identifying and monitoring sites over a >10,000-fold dynamic range of sequence counts. Applying QIseq to uncloned parasites shortly after transfections revealed multiple insertions in mixed populations and suggests that >4000 independent mutants could be generated from relatively modest scales of transfection, providing a clear pathway to genome-scale screens in P. falciparum QIseq was also used to monitor the growth of pools of previously cloned mutants and reproducibly differentiated between deleterious and neutral mutations in competitive growth. Among the mutants with fitness defects was a mutant with a piggyBac insertion immediately upstream of the kelch protein K13 gene associated with artemisinin resistance, implying mutants in this gene may have competitive fitness costs. QIseq has the potential to enable the scale-up of piggyBac-mediated genetics across multiple eukaryotic systems.
The spread of Plasmodium falciparum multidrug resistance highlights the urgency to discover new targets and chemical scaffolds. Unfortunately, lack of experimentally validated functional information ...about most P. falciparum genes remains a strategic hurdle. Chemogenomic profiling is an established tool for classification of drugs with similar mechanisms of action by comparing drug fitness profiles in a collection of mutants. Inferences of drug mechanisms of action and targets can be obtained by associations between shifts in drug fitness and specific genetic changes in the mutants. In this screen, P. falciparum, piggyBac single insertion mutants were profiled for altered responses to antimalarial drugs and metabolic inhibitors to create chemogenomic profiles. Drugs targeting the same pathway shared similar response profiles and multiple pairwise correlations of the chemogenomic profiles revealed novel insights into drugs' mechanisms of action. A mutant of the artemisinin resistance candidate gene - "K13-propeller" gene (PF3D7_1343700) exhibited increased susceptibility to artemisinin drugs and identified a cluster of 7 mutants based on similar enhanced responses to the drugs tested. Our approach of chemogenomic profiling reveals artemisinin functional activity, linked by the unexpected drug-gene relationships of these mutants, to signal transduction and cell cycle regulation pathways.
Malaria remains one of the most devastating parasitic diseases worldwide, with 90% of the malaria deaths in Africa in 2013 attributable to
. The clinical symptoms of malaria include cycles of fever, ...corresponding to parasite rupture from red blood cells every 48 h. Parasite pathways involved in the parasite's ability to survive the host fever response, and indeed, the functions of ~40% of
genes as a whole, are still largely unknown. Here, we evaluated the potential of scalable forward-genetic screening methods to identify genes involved in the host fever response. We performed a phenotypic screen for genes linked to the parasite response to febrile temperatures by utilizing a selection of single-disruption
mutants generated via random
transposon mutagenesis in a previous study. We identified several mutants presenting significant phenotypes in febrile response screens compared to the wild type, indicating possible roles for the disrupted genes in this process. We present these initial studies as proof that forward genetics can be used to gain insight into critical factors associated with parasite biology.
Though the
genome sequence has been available for many years, ~40% of its genes do not have informative annotations, as they show no detectable homology to those of studied organisms. More still have not been evaluated via genetic methods. Scalable forward-genetic approaches that allow interrogation of gene function without any pre-existing knowledge are needed to hasten understanding of parasite biology, which will expedite the identification of drug targets and the development of future interventions in the face of spreading resistance to existing frontline drugs. In this work, we describe a new approach to pursue forward-genetic phenotypic screens for
to identify factors associated with virulence. Future large-scale phenotypic screens developed to probe other such interesting phenomena, when considered in parallel, will prove a powerful tool for functional annotation of the
genome, where so much remains undiscovered.
Concerns about malaria parasite resistance to treatment with artemisinin drugs (ARTs) have grown with findings of prolonged parasite clearance t
1/2s (>5 h) and their association with mutations in ...Plasmodium falciparum Kelch-propeller protein K13. Here, we describe a P. falciparum laboratory cross of K13 C580Y mutant with C580 wild-type parasites to investigate ART response phenotypes in vitro and in vivo. After genotyping >400 isolated progeny, we evaluated 20 recombinants in vitro: IC50 measurements of dihydroartemisinin were at similar low nanomolar levels for C580Y- and C580-type progeny (mean ratio, 1.00; 95% CI, 0.62–1.61), whereas, in a ring-stage survival assay, the C580Y-type progeny had 19.6-fold (95% CI, 9.76–39.2) higher average counts. In splenectomized Aotus monkeys treated with three daily doses of i.v. artesunate, t
1/2 calculations by three different methods yielded mean differences of 0.01 h (95% CI, −3.66 to 3.67), 0.80 h (95% CI, −0.92 to 2.53), and 2.07 h (95% CI, 0.77–3.36) between C580Y and C580 infections. Incidences of recrudescence were 57% in C580Y (4 of 7) versus 70% in C580 (7 of 10) infections (−13% difference; 95% CI, −58% to 35%). Allelic substitution of C580 in a C580Y-containing progeny clone (76H10) yielded a transformant (76H10C580Rev) that, in an infected monkey, recrudesced regularly 13 times over 500 d. Frequent recrudescences of ART-treated P. falciparum infections occur with or without K13 mutations and emphasize the need for improved partner drugs to effectively eliminate the parasites that persist through the ART component of combination therapy.
Severe malaria is caused by the apicomplexan parasite
Plasmodium
falciparum.
Despite decades of research the unique biology of these
parasites has made it challenging to establish high throughput ...genetic
approaches for identification of therapeutic targets. Using transposon
mutagenesis of
P. falciparum
in an approach that exploited its
AT-rich genome we generated >38,000 mutants, saturating the genome and
defining fitness costs for 95% of genes. Of 5,399 genes we found ~3,000
genes are essential for optimal growth of asexual blood-stages
in
vitro
. Our study defines ∼1000 essential genes, including
genes associated with drug resistance, vaccine candidates, and conserved
proteins of unknown function. We validated this approach by testing proteasome
pathways for individual mutants associated with artemisinin sensitivity.
Transposon mutagenesis of
Plasmodium falciparum
was used
to generate >38,000 mutants, saturating the genome and defining fitness
costs for 95% of genes. We functionally define the relative fitness cost of
disruption for 5,399 genes, and find that ~3,000 genes, ~62% of
the genome, are essential for optimal asexual blood-stage
in
vitro
growth. Our study defines ∼1000 essential genes,
including genes associated with drug resistance, leading vaccine candidates, and
hundreds of
Plasmodium-
conserved proteins of unknown function
that are now potential therapeutic intervention targets. We experimentally
validated the essentiality of proteasome pathways with drug studies of
individual mutants associated with artemisinin sensitivity. This study defines
high-priority targets and pathways and points the way for the future of
P. falciparum
high throughput genetics.
Saturation-scale mutagenesis of
Plasmodium falciparum
reveals a core set of genes essential for asexual blood-stage growth
in
vitro
.
Genetic screening using random transposon insertions has been a powerful tool for uncovering biology in prokaryotes, where whole-genome saturating screens have been performed in multiple organisms. ...In eukaryotes, such screens have proven more problematic, in part because of the lack of a sensitive and robust system for identifying transposon insertion sites. We here describe quantitative insertion-site sequencing, or QIseq, which uses custom library preparation and Illumina sequencing technology and is able to identify insertion sites from both the 5' and 3' ends of the transposon, providing an inbuilt level of validation. The approach was developed using piggyBac mutants in the human malaria parasitePlasmodium falciparum but should be applicable to many other eukaryotic genomes. QIseq proved accurate, confirming known sites in >100 mutants, and sensitive, identifying and monitoring sites over a >10,000-fold dynamic range of sequence counts. Applying QIseq to uncloned parasites shortly after transfections revealed multiple insertions in mixed populations and suggests that >4000 independent mutants could be generated from relatively modest scales of transfection, providing a clear pathway to genome-scale screens in P. Falciparum. QIseq was also used to monitor the growth of pools of previously cloned mutants and reproducibly differentiated between deleterious and neutral mutations in competitive growth. Among the mutants with fitness defects was a mutant with apiggyBac insertion immediately upstream of the kelch protein K13 gene associated with artemisinin resistance, implying mutants in this gene may have competitive fitness costs. QIseq has the potential to enable the scale-up ofpiggyBac-mediated genetics across multiple eukaryotic systems.
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