Sources of Transcription Variation in Plasmodium falciparum
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ABSTRACT:
ORGANISM(S): Plasmodium falciparum
PROVIDER: GSE126876 | GEO | 2021/01/31
REPOSITORIES: GEO
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Project description:The determinants of transcriptional regulation in malaria parasites remain elusive. The presence of a well-characterized gene expression cascade shared by different Plasmodium falciparum strains could imply that transcriptional regulation and its natural variation do not contribute significantly to the evolution of parasite drug resistance. To clarify the role of transcriptional variation as a source of stain-specific diversity in the most deadly malaria species and to find genetic loci that dictate variations in gene expression, we examined genome-wide expression level polymorphisms (ELPs) in a genetic cross between phenotypically distinct parasite clones. Significant variation in gene expression is observed through direct co-hybridizations of RNA from different P. falciparum clones. Nearly 18% of genes were regulated by a significant eQTL. The genetic determinants of most of these ELPs resided in hotspots that are physically distant from their targets. The most prominent regulatory locus, influencing 269 transcripts, coincided with a Chromosome 5 amplification event carrying the drug resistance gene, pfmdr1, and 13 other genes. Drug selection pressure in the Dd2 parental clone lineage led not only to a copy number change in the pfmdr1 gene but also to increased copies of putative neighboring regulatory factors that, in turn, broadly influence the transcriptional network. Previously unrecognized transcriptional variation, controlled by polymorphic regulatory genes and possibly master regulators within large copy number variants, contributes to sweeping phenotypic evolution in drug-resistant malaria parasites. Keywords: Segregation patterns of gene expression levels. A total of 36 test parasite samples (Dd2, HB3, and progeny) from a single time point in the parasite life cycle (18 hours post RBC invasion) were co-hybridized to a common reference HB3 sample, each a single replicate.
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Project description:The combination of piperaquine and dihydroartemisinin has recently become the official first-line therapy in several Southeast Asian countries. The pharmacokinetic mismatching of these drugs, whose plasma half lives are ∼20 days and ∼1 hr respectively, implies that recrudescent or new infections emerging shortly after treatment cessation will encounter piperaquine as a monotherapy agent. This creates substantial selection pressure for the emergence of resistance. To elucidate potential resistance determinants, we subjected cloned Plasmodium falciparum Dd2 parasites to continuous piperaquine pressure in vitro (47 nM, ∼two-fold higher than the Dd2 IC(50) value). The phenotype of outgrowth parasites was assayed in two clones, revealing an IC(50) value against piperaquine of 2.1 μM and 1.7 μM, over 100-fold greater than the parent. To identify the genetic determinant of resistance, we employed comparative whole-genome hybridization analysis. Compared to the Dd2 parent, this analysis found (in both resistant clones) a novel single nucleotide polymorphism in pfcrt, deamplification of an 82 kb region of chromosome 5 (that includes pfmdr1), and amplification of an adjacent 63 kb region of chromosome 5. Continued propagation without piperaquine selection pressure resulted in derivation of "revertant" piperaquine-sensitive parasites. These retained the pfcrt polymorphism and further deamplified the chromosome 5 segment that encompasses pfmdr1; however, these two independently generated revertants both lost the neighboring 63 kb amplification. These results suggest that a copy number variation event on chromosome 5 (825600-888300) is associated with piperaquine resistance. Transgenic studies are underway with individual genes in this segment to evaluate their contribution to piperaquine resistance.
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Project description:The sexual stages are vital phases in malaria parasite transmission and are the targets of various interventions such as transmission blocking vaccines. The molecular mechanisms underlying sexual development, however, remain poorly understood. We report mappping of a determinant previously linked to a male gametocyte development defect in the P. falciparum Dd2 parasite to an 82 kb region on chromosome 12. In order to find a critical gene in this region, we compared gene expression pattern in sexual stage of the parasite between Dd2 and its normal gametocyte-producing ancestor W2 clones. The region contains a sexual stage specific gene (pfmdv 1) that is expressed substantially at a lower level in the Dd2 than in W2 parasite. Disruption of pfmdv 1 results in a dramatic reduction in mature gametocytes, especially male gametocytes, with the majority of sexually committed parasites arrested at stage-I. The pfmdv-1 knockout parasites show an enlarged nucleus, often with separation of the inner and outer nuclear membranes and presence of multi-membrane vesicles in red blood cell cytoplasm. Mosquito infectivity of the knockout parasites is also greatly reduced, but not completely lost, suggesting presence of compensatory mechanisms in the sexual development pathways. Data include Day 8 gametocytes of male defective Dd2 and parental W2 clones of Plasmodium falciparum. The series includes three biological repeats. Keywords: repeat sample
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