Soulyemane Mboup

A genome-wide map of diversity in Plasmodium falciparum

Genetic variation allows the malaria parasite Plasmodium falciparum to overcome chemotherapeutic agents, vaccines and vector control strategies and remain a leading cause of global morbidity and mortality. Here we describe an initial survey of genetic variation across the P. falciparum genome. We performed extensive sequencing of 16 geographically diverse parasites and identified 46,937 SNPs, demonstrating rich diversity among P. falciparum parasites (π = 1.16 × 10−3) and strong correlation with gene function. We identified multiple regions with signatures of selective sweeps in drug-resistant parasites, including a previously unidentified 160-kb region with extremely low polymorphism in pyrimethamine-resistant parasites. We further characterized 54 worldwide isolates by genotyping SNPs across 20 genomic regions. These data begin to define population structure among African, Asian and American groups and illustrate the degree of linkage disequilibrium, which extends over relatively short distances in African parasites but over longer distances in Asian parasites. We provide an initial map of genetic diversity in P. falciparum and demonstrate its potential utility in identifying genes subject to recent natural selection and in understanding the population genetics of this parasite.

Distinct physiological states of Plasmodium falciparum in malaria-infected patients

Infection with the malaria parasite Plasmodium falciparum leads to widely different clinical conditions in children, ranging from mild flu-like symptoms to coma and death. Despite the immense medical implications, the genetic and molecular basis of this diversity remains largely unknown. Studies of in vitro gene expression have found few transcriptional differences between different parasite strains. Here we present a large study of in vivo expression profiles of parasites derived directly from blood samples from infected patients. The in vivo expression profiles define three distinct transcriptional states. The biological basis of these states can be interpreted by comparison with an extensive compendium of expression data in the yeast Saccharomyces cerevisiae. The three states in vivo closely resemble, first, active growth based on glycolytic metabolism, second, a starvation response accompanied by metabolism of alternative carbon sources, and third, an environmental stress response. The glycolytic state is highly similar to the known profile of the ring stage in vitro, but the other states have not been observed in vitro. The results reveal a previously unknown physiological diversity in the in vivo biology of the malaria parasite, in particular evidence for a functional mitochondrion in the asexual-stage parasite, and indicate in vivo and in vitro studies to determine how this variation may affect disease manifestations and treatment.

Genome-wide SNP genotyping highlights the role of natural selection in Plasmodium falciparum population divergence

BackgroundThe malaria parasite Plasmodium falciparum exhibits abundant genetic diversity, and this diversity is key to its success as a pathogen. Previous efforts to study genetic diversity in P. falciparum have begun to elucidate the demographic history of the species, as well as patterns of population structure and patterns of linkage disequilibrium within its genome. Such studies will be greatly enhanced by new genomic tools and recent large-scale efforts to map genomic variation. To that end, we have developed a high throughput single nucleotide polymorphism (SNP) genotyping platform for P. falciparum.ResultsUsing an Affymetrix 3,000 SNP assay array, we found roughly half the assays (1,638) yielded high quality, 100% accurate genotyping calls for both major and minor SNP alleles. Genotype data from 76 global isolates confirm significant genetic differentiation among continental populations and varying levels of SNP diversity and linkage disequilibrium according to geographic location and local epidemiological factors. We further discovered that nonsynonymous and silent (synonymous or noncoding) SNPs differ with respect to within-population diversity, inter-population differentiation, and the degree to which allele frequencies are correlated between populations.ConclusionsThe distinct population profile of nonsynonymous variants indicates that natural selection has a significant influence on genomic diversity in P. falciparum, and that many of these changes may reflect functional variants deserving of follow-up study. Our analysis demonstrates the potential for new high-throughput genotyping technologies to enhance studies of population structure, natural selection, and ultimately enable genome-wide association studies in P. falciparum to find genes underlying key phenotypic traits.

In Vivo Transcriptome of Plasmodium falciparum Reveals Overexpression of Transcripts That Encode Surface Proteins

Infections with the human parasite Plasmodium falciparum continue to present a great challenge to global health. Fundamental questions regarding the molecular basis of virulence and immune evasion in P. falciparum have been only partially answered. Because of the parasites intracellular location and complex life cycle, standard genetic approaches to the study of the pathogenesis of malaria have been limited. The present study presents a novel approach to the identification of the biological processes involved in host-pathogen interactions, one that is based on the analysis of in vivo P. falciparum transcripts. We demonstrate that a sufficient quantity of P. falciparum RNA transcripts can be derived from a small blood sample from infected patients for whole-genome microarray analysis. Overall, excellent correlation was observed between the transcriptomes derived from in vivo samples and in vitro samples with ring-stage P. falciparum 3D7 reference strain. However, gene families that encode surface proteins are overexpressed in vivo. Moreover, this analysis has identified a new family of hypothetical genes that may encode surface variant antigens. Comparative studies of the transcriptomes derived from in vivo samples and in vitro 3D7 samples may identify important strategies used by the pathogen for survival in the human host and highlight, for vaccine development, new candidate antigens that were not previously identified through the use of in vitro cultures.

In vivo transcriptional profiling of Plasmodium falciparum

BackgroundBoth 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.MethodsA custom made oligonucleotide array with probes based on the P. falciparum 3D7 laboratory strain chromosome 2 sequence was used to detect in vivoP. 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.ResultsThe 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.ConclusionsWhole 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.

Mutations in Plasmodium falciparum dihydrofolate reductase and dihydropteroate synthase genes in Senegal

Senegal recently (2004) switched to sulfadoxine‐pyrimethamine (SP) with amodiaquine as first line therapy for malaria in response to increasing chloroquine resistance. In anticipation of emerging resistance to SP as a result of this change in drug pressure, we set out to define the baseline prevalence of SP‐associated mutations in the dhfr and dhps genes in Plasmodium falciparum using geographically diverse and longitudinally collected samples. A total of 153 blood samples were analysed from patients (5 years or older) with mild malaria after informed consent was obtained. Longitudinal samples were collected between 2000 and 2003 in Pikine, a suburb of Dakar. Geographically diverse site sampling was carried out in 2003. The mutation prevalence in DHFR codons 51, 59 and 108 is 65%, 61% and 78% in Pikine, 2003. The overall prevalence of the triple mutation that is associated with high‐level pyrimethamine resistance is 61%. The mutation prevalence rate in DHPS codons 436 and 437 is 21% and 40%, respectively. There is significant geographic variation in genotypic resistance, as samples from Pikine in 2003 had higher mutation prevalence in the pfdhfr and pfdhps genes compared to samples from Tambacounda (P < 0.015). In summary, this study demonstrates a high background prevalence of SP resistance mutations already present in P. falciparum in Senegal.

Erythrocyte invasion profiles are associated with a common invasion ligand polymorphism in Senegalese isolates of Plasmodium falciparum.

Plasmodium falciparum parasites use multiple ligand-receptor interactions to invade human erythrocytes. Variant expression levels of members of the PfRh and PfEBA ligand families are associated with the use of different erythrocyte receptors, defining invasion pathways. Here we analyse a major polymorphism, a large sequence deletion in the PfRh2b ligand, and erythrocyte invasion profiles in uncultured Senegalese isolates. Parasites vary considerably in their use of sialic acid-containing and protease-sensitive erythrocyte receptors for invasion. The erythrocyte selectivity index was not related to invasion pathway usage, while parasite multiplication rate was associated with enhanced use of a trypsin-resistant invasion pathway. PfRh2b protein was expressed in all parasite isolates, although the PfRh2b deletion was present in a subset (approximately 68%). Parasites with the PfRh2b deletion were found to preferentially utilize protease-resistant pathways for erythrocyte invasion. Sialic acid-independent invasion is reduced in parasites with the PfRh2b deletion, but only in isolates derived from blood group O patients. Our results suggest a significant role for PfRh2b sequence polymorphism in discriminating between alternative erythrocyte receptors for invasion and as a possible determinant of virulence.

Prevalence of Plasmodium falciparum pfcrt polymorphisms and in vitro chloroquine sensitivity in Senegal

Mutations in pfcrt K76T are associated with chloroquine resistance in Plasmodium falciparum. Previous studies of K76T mutations in Senegal reported the association of T76 with in vitro-resistant isolates, but this mutation was also prevalent in chloroquine-sensitive isolates. This suggests involvement of additional genetic loci in modulating chloroquine resistance. Additional pfcrt polymorphisms at codons A220S, Q271E, N326S and R371I have been found in chloroquine-resistant isolates. We wanted to test if sequential acquisition of mutations at these codons leads to in vitro chloroquine resistance. Stepwise accumulation of mutations was not detected, rather there was almost complete linkage between the pfcrt K76T mutation and polymorphisms in these codons. Therefore these additional polymorphisms do not enhance the correlation between pfcrt T76 and chloroquine resistance in Senegal. These data suggest that in vitro chloroquine resistance requires the genetic background of the pfcrt K76T mutation and additional mutations in genetic loci outside the pfcrt gene.

Correction: A Systematic Map of Genetic Variation in Plasmodium falciparum.

In PLoS Pathogens, volume 2, issue 6: DOI: 10.1371/journal.ppat.0020057 In the Materials and Methods section, the following Supporting Information files were mislabeled: Table S7 should be Table S10, Table S8 should be Table S11, and Table S9 should be Table S12. The corrected citations in text follow: To identify potential amplifications in 3D7, we compared the list of genes showing a 1.5-fold or greater change in 3D7 relative to each of the Senegal isolates (n = 5) as described above. This returned some genes that were likely to be highly polymorphic in individual Senegal strains, but such deletions were not held in common by all. Only two genes were shared by all the Senegal strains examined. These two genes were for GTP cyclohydrolase (PFL1155w) and P. falciparum 11–1 protein PF10_0374, the gene11–1 product, which is highly expressed during gametocytogenesis. Examination of the ratios for these genes (Table S10) is consistent with a 3D7 amplification (generally from 1.5- to 4-fold changes) rather than a Senegal deletion where ratios show a 20-fold difference in signal. Quantitative real-time PCR analysis further confirmed a probable amplification in 3D7 for GTP cyclohydrolase (Table S11). In contrast, gene deletions were identified as follows. The custom-designed Affymetrix malaria full-genome array consists of 2,397 probes for 100 viral genes that serve as background controls [14]. Intensities from these probes represent the level of cross-hybridization for a deleted gene. A new probe-to-gene map was generated to include both sense and antisense probes, and the MOID algorithm [59] was applied to assign “present” and “absent” calls to each gene. Based on all the background control data collected in this study, this analysis, similar to that described in Le Roch et al. [14], shows that a deleted gene has only a 2% chance to be misclassified as “present” if it is required to have both an intensity level of E > 10 and a Kolmogorov-Smirnov test of log10P of less than −0.5. Excluding genes with fewer than six probes and the highly variable var, rifin, and stevor genes, we found a total of 33 genes being called “absent” in at least two out of the three hybridizations for each strain (Table S12). Additionally, the Supporting Information file in the legend text for Table S7 was mislabeled. Table 5 should be Table S8. The corrected citation in text follows: Aside from those deleted genes (underlined), no distinct differences were observed between the classes of highly variable genes within the laboratory strains compared with the Senegal strains (Table S8). Indeed, no significant differences were observed in the variation within immunogens (p = 0.87) and protein biosynthesis (p = 0.45) genes between the two groups of isolates, but variation in multi-gene families was significant (p = 6.37E−58).