Ousmane Sarr

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.

Identification and functional validation of the novel antimalarial resistance locus PF10_0355 in Plasmodium falciparum.

The Plasmodium falciparum parasites 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.

A Molecular Epidemiological Study of var Gene Diversity to Characterize the Reservoir of Plasmodium falciparum in Humans in Africa

Background The reservoir of Plasmodium infection in humans has traditionally been defined by blood slide positivity. This study was designed to characterize the local reservoir of infection in relation to the diverse var genes that encode the major surface antigen of Plasmodium falciparum blood stages and underlie the parasites ability to establish chronic infection and transmit from human to mosquito. Methodology/Principal Findings We investigated the molecular epidemiology of the var multigene family at local sites in Gabon, Senegal and Kenya which differ in parasite prevalence and transmission intensity. 1839 distinct var gene types were defined by sequencing DBLα domains in the three sites. Only 76 (4.1%) var types were found in more than one population indicating spatial heterogeneity in var types across the African continent. The majority of var types appeared only once in the population sample. Non-parametric statistical estimators predict in each population at minimum five to seven thousand distinct var types. Similar diversity of var types was seen in sites with different parasite prevalences. Conclusions/Significance Var population genomics provides new insights into the epidemiology of P. falciparum in Africa where malaria has never been conquered. In particular, we have described the extensive reservoir of infection in local African sites and discovered a unique var population structure that can facilitate superinfection through minimal overlap in var repertoires among parasite genomes. Our findings show that var typing as a molecular surveillance system defines the extent of genetic complexity in the reservoir of infection to complement measures of malaria prevalence. The observed small scale spatial diversity of var genes suggests that var genetics could greatly inform current malaria mapping approaches and predict complex malaria population dynamics due to the import of var types to areas where no widespread pre-existing immunity in the population exists.

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.

A flow cytometry-based assay for measuring invasion of red blood cells by Plasmodium falciparum

Variability in the ability of the malaria parasite Plasmodium falciparum to invade human erythrocytes is postulated to be an important determinant of disease severity. Both the parasite multiplication rate and erythrocyte selectivity are important parameters that underlie such variable invasion. We have established a flow cytometry‐based method for simultaneously calculating both the parasitemia and the number of multiply‐infected erythrocytes. Staining with the DNA‐specific dye SYBR Green I allows quantitation of parasite invasion at the ring stage of parasite development. We discuss in vitro and in vivo applications and limitations of this method in relation to the study of parasite invasion. Am. J. Hematol., 2010.

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.

Molecular analysis of erythrocyte invasion in Plasmodium falciparum isolates from Senegal.

ABSTRACT The human malaria parasite, Plasmodium falciparum, utilizes multiple ligand-receptor interactions for the invasion of human erythrocytes. Members of the reticulocyte binding protein homolog (PfRh) family have been shown to be critical for directing parasites to alternative erythrocyte receptors that define invasion pathways. Recent studies have identified gene amplification, sequence polymorphism, and variant expression of PfRh paralogs as mechanisms underlying discrimination between pathways for invasion. In this study, we find considerable heterogeneity in the invasion profiles of clonal, uncultured P. falciparum parasite isolates from a low-transmission area in Senegal. Molecular analyses revealed minimal variation in protein expression levels of the PfRh ligands, PfRh1, PfRh2a, and PfRh2b, and an absence of gene amplification in these isolates. However, significant sequence polymorphism was found within repeat regions of PfRh1, PfRh2a, and PfRh2b. Furthermore, we identified a large sequence deletion (∼0.58 kb) in the C-terminal region of the PfRh2b gene at a high prevalence in this population. In contrast to findings of earlier studies, we found no associations between specific sequence variants and distinct invasion pathways. Overall these data highlight the importance of region-specific elaborations in PfRh sequence and expression polymorphisms, which has important implications in our understanding of how the malaria parasite responds to polymorphisms in erythrocyte receptors and/or evades the immune system.