In vivo transcriptional profiling of Plasmodium falciparum

BACKGROUND: Both host and pathogen factors contribute to disease outcome in Plasmodium falciparum infection. The feasibility of studying the P. falciparum in vivo transcriptome to understand parasite transcriptional response while it resides in the human host is presented. METHODS: A custom made oligonucleotide array with probes based on the P. falciparum 3D7 laboratory strain chromosome 2 sequence was used to detect in vivo P. falciparum transcripts. This study analyzed transcripts from total RNA derived from small blood samples of P. falciparum infected patients and compared the in vivo expression profile to the in vitro cultivated 3D7 strain transcriptome. RESULTS: The data demonstrated that in vivo transcription can be studied from a small blood sample, despite the abundance of human RNA. The in vivo transcriptome is similar to the 3D7 ring stage transcriptome, but there are significant differences in genes encoding a sexual stage antigen and surface proteins. CONCLUSIONS: Whole genome transcription analysis of P. falciparum can be carried out successfully and further studies in selected patient cohorts may provide insight into parasite in vivo biology and defense against host immunity

A Systematic Map of Genetic Variation in Plasmodium falciparum

Discovering novel genes involved in immune evasion and drug resistance in the human malaria parasite, Plasmodium falciparum, is of critical importance to global health. Such knowledge may assist in the development of new effective vaccines and in the appropriate use of antimalarial drugs. By performing a full-genome scan of allelic variability in 14 field and laboratory strains of P. falciparum, we comprehensively identified ≈500 genes evolving at higher than neutral rates. The majority of the most variable genes have paralogs within the P. falciparum genome and may be subject to a different evolutionary clock than those without. The group of 211 variable genes without paralogs contains most known immunogens and a few drug targets, consistent with the idea that the human immune system and drug use is driving parasite evolution. We also reveal gene-amplification events including one surrounding pfmdr1, the P. falciparum multidrug-resistance gene, and a previously uncharacterized amplification centered around the P. falciparum GTP cyclohydrolase gene, the first enzyme in the folate biosynthesis pathway. Although GTP cyclohydrolase is not the known target of any current drugs, downstream members of the pathway are targeted by several widely used antimalarials. We speculate that an amplification of the GTP cyclohydrolase enzyme in the folate biosynthesis pathway may increase flux through this pathway and facilitate parasite resistance to antifolate drugs

Identification and Functional Validation of the Novel Antimalarial Resistance Locus PF10_0355 in Plasmodium falciparum

The Plasmodium falciparum parasite's ability to adapt to environmental pressures, such as the human immune system and antimalarial drugs, makes malaria an enduring burden to public health. Understanding the genetic basis of these adaptations is critical to intervening successfully against malaria. To that end, we created a high-density genotyping array that assays over 17,000 single nucleotide polymorphisms (~1 SNP/kb), and applied it to 57 culture-adapted parasites from three continents. We characterized genome-wide genetic diversity within and between populations and identified numerous loci with signals of natural selection, suggesting their role in recent adaptation. In addition, we performed a genome-wide association study (GWAS), searching for loci correlated with resistance to thirteen antimalarials; we detected both known and novel resistance loci, including a new halofantrine resistance locus, PF10_0355. Through functional testing we demonstrated that PF10_0355 overexpression decreases sensitivity to halofantrine, mefloquine, and lumefantrine, but not to structurally unrelated antimalarials, and that increased gene copy number mediates resistance. Our GWAS and follow-on functional validation demonstrate the potential of genome-wide studies to elucidate functionally important loci in the malaria parasite genome.Bill & Melinda Gates FoundationEllison Medical FoundationExxon Mobil FoundationFogarty International CenterNational Institute of Allergy and Infectious Diseases (U.S.)Burroughs Wellcome FundDavid & Lucile Packard FoundationNational Science Foundation (U.S.). Graduate Research Fellowship Progra

A systematic map of genetic variation in Plasmodium falciparum.

Discovering novel genes involved in immune evasion and drug resistance in the human malaria parasite, Plasmodium falciparum, is of critical importance to global health. Such knowledge may assist in the development of new effective vaccines and in the appropriate use of antimalarial drugs. By performing a full-genome scan of allelic variability in 14 field and laboratory strains of P. falciparum, we comprehensively identified approximately 500 genes evolving at higher than neutral rates. The majority of the most variable genes have paralogs within the P. falciparum genome and may be subject to a different evolutionary clock than those without. The group of 211 variable genes without paralogs contains most known immunogens and a few drug targets, consistent with the idea that the human immune system and drug use is driving parasite evolution. We also reveal gene-amplification events including one surrounding pfmdr1, the P. falciparum multidrug-resistance gene, and a previously uncharacterized amplification centered around the P. falciparum GTP cyclohydrolase gene, the first enzyme in the folate biosynthesis pathway. Although GTP cyclohydrolase is not the known target of any current drugs, downstream members of the pathway are targeted by several widely used antimalarials. We speculate that an amplification of the GTP cyclohydrolase enzyme in the folate biosynthesis pathway may increase flux through this pathway and facilitate parasite resistance to antifolate drugs

The Heat Map Shows the Base 2 Logarithm of the Ratio of the Normalized, Background Subtracted Probe Signal for the Listed Isolate (18.02, Dd2, 7G8 or HB3), Relative to the Sequenced Isolate, 3D7

<p>All probes on the array were selected to be unique within 3D7. Each horizontal bar represents a single gene on the right arm of Chromosome 12. Dark probes, with low signal intensities, do not match any region in the genome of the listed isolate, while light probes, displaying high signal intensities, such as those near the GTP-cyclohydrolase gene <i>(PFL1155w)</i> in Dd2, HB3, and 7G8 contain matches to multiple regions of the genome. The signal intensity and the consecutive nature of the pattern suggest that strains 7G8, HB3, and Dd2 contain more than one copy of the GTP-cyclohydrolase gene <i>PFL1155w.</i> An arrow represents the location of the GTP-cyclohydrolase<i>PFL1155w</i> gene on Chromosome 12.</p

Example of a Deleted Region

<p>Hybridization levels were calculated with the MOID algorithm as previously described [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0020057#ppat-0020057-b059" target="_blank">59</a>]. The figure shows the signal for 26 genes beginning with <i>PFB0010w</i> and ending with <i>PFB0120w</i> for three strains (41.02, 3D7, and C188). The background level (zero) was determined by analyzing the signal of non-P. falciparum genes on the array. Data for all genes and all strains are available from <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0020057#ppat-0020057-st012" target="_blank">Table S12</a>.</p

Linkage Disequilibrium surrounding <i>pfcrt (MAL7P1.27)</i> in FCB and Dd2 on Chromosome 7

<p>Scores were generated by calculating the probability of observing the same genotype by chance over a moving 40-kbp window (with the probability of observing the same genotype for any one SFP by chance placed at 0.33). The plot shows the ratio between the probability and the maximum possible probability for regions with at least four SFPs, with 1 indicating the best possible score. The position of antigenic variation clusters <i>(vars, stevors,</i> or <i>rifins),</i> are shown in blue and are marked. SFPs mapping to these genes were excluded from the calculations because our data indicate that mitotic recombination may be occurring in these genes. <i>pfcrt,</i> which is located between bases 307,926 and 311,020 on Chromosome 7, is shown as a black triangle. The trough at <i>pfcrt</i> is likely to be due to the strong selective pressure on <i>pfcrt</i>, and is consistent with the observation that FCB and Dd2 have different alleles of <i>pfcrt</i> even though published SNP data also shows disequilibrium in surrounding regions [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0020057#ppat-0020057-b005" target="_blank">5</a>]. Data for other chromosomes are shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0020057#ppat-0020057-sg001" target="_blank">Figure S1</a>.</p