[Show abstract][Hide abstract] ABSTRACT: In regions with high malaria endemicity, the withdrawal of chloroquine (CQ) as first-line treatment of Plasmodium falciparum infections has typically led to the restoration of CQ susceptibility through the reexpansion of the wild-type (WT) allele K76 of the chloroquine resistance transporter gene (pfcrt) at the expense of less fit mutant alleles carrying the CQ resistance (CQR) marker K76T. In low-transmission settings, such as South America, drug resistance mutations can attain 100% prevalence, thereby precluding the return of WT parasites after the complete removal of drug pressure. In French Guiana, despite the fixation of the K76T allele, the prevalence of CQR isolates progressively dropped from >90% to <30% during 17 y after CQ withdrawal in 1995. Using a genome-wide association study with CQ-sensitive (CQS) and CQR isolates, we have identified a single mutation in pfcrt encoding a C350R substitution that is associated with the restoration of CQ susceptibility. Genome editing of the CQR reference strain 7G8 to incorporate PfCRT C350R caused a complete loss of CQR. A retrospective molecular survey on 580 isolates collected from 1997 to 2012 identified all C350R mutant parasites as being CQS. This mutation emerged in 2002 and rapidly spread throughout the P. falciparum population. The C350R allele is also associated with a significant decrease in piperaquine susceptibility in vitro, suggesting that piperaquine pressure in addition to potential fitness costs associated with the 7G8-type CQR pfcrt allele may have selected for this mutation. These findings have important implications for understanding the evolutionary dynamics of antimalarial drug resistance.
Proceedings of the National Academy of Sciences 08/2015; DOI:10.1073/pnas.1507142112 · 9.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Background
The human malaria parasite Plasmodium falciparum has a complex and multi-stage life cycle that requires extensive and precise gene regulation to allow invasion and hijacking of host cells, transmission, and immune escape. To date, the regulatory elements orchestrating these critical parasite processes remain largely unknown. Yet it is becoming increasingly clear that long non-coding RNAs (lncRNAs) could represent a missing regulatory layer across a broad range of organisms.
To investigate the regulatory capacity of lncRNA in P. falciparum, we harvested fifteen samples from two time-courses. Our sample set profiled 56 h of P. falciparum blood stage development. We then developed and validated strand-specific, non-polyA-selected RNA sequencing methods, and pursued the first assembly of P. falciparum strand-specific transcript structures from RNA sequencing data. This approach enabled the annotation of over one thousand lncRNA transcript models and their comprehensive global analysis: coding prediction, periodicity, stage-specificity, correlation, GC content, length, location relative to annotated transcripts, and splicing. We validated the complete splicing structure of three lncRNAs with compelling properties. Non-polyA-selected deep sequencing also enabled the prediction of hundreds of intriguing P. falciparum circular RNAs, six of which we validated experimentally.
We found that a subset of lncRNAs, including all subtelomeric lncRNAs, strongly peaked in expression during invasion. By contrast, antisense transcript levels significantly dropped during invasion. As compared to neighboring mRNAs, the expression of antisense-sense pairs was significantly anti-correlated during blood stage development, indicating transcriptional interference. We also validated that P. falciparum produces circRNAs, which is notable given the lack of RNA interference in the organism, and discovered that a highly expressed, five-exon antisense RNA is poised to regulate P. falciparum gametocyte development 1 (PfGDV1), a gene required for early sexual commitment events.
Electronic supplementary material
The online version of this article (doi:10.1186/s12864-015-1603-4) contains supplementary material, which is available to authorized users.
[Show abstract][Hide abstract] ABSTRACT: The widespread emergence of Plasmodium falciparum (Pf) strains resistant to frontline agents has fuelled the search for fast-acting agents with novel mechanism of action. Here, we report the discovery and optimization of novel antimalarial compounds, the triaminopyrimidines (TAPs), which emerged from a phenotypic screen against the blood stages of Pf. The clinical candidate (compound 12) is efficacious in a mouse model of Pf malaria with an ED99 <30 mg kg(-1) and displays good in vivo safety margins in guinea pigs and rats. With a predicted half-life of 36 h in humans, a single dose of 260 mg might be sufficient to maintain therapeutic blood concentration for 4-5 days. Whole-genome sequencing of resistant mutants implicates the vacuolar ATP synthase as a genetic determinant of resistance to TAPs. Our studies highlight the potential of TAPs for single-dose treatment of Pf malaria in combination with other agents in clinical development.
[Show abstract][Hide abstract] ABSTRACT: Identifying the source of resurgent parasites is paramount to a strategic, successful intervention for malaria elimination.
Although the malaria incidence in Panama is low, a recent outbreak resulted in a 6-fold increase in reported cases. We hypothesized
that parasites sampled from this epidemic might be related and exhibit a clonal population structure. We tested the genetic
relatedness of parasites, using informative single-nucleotide polymorphisms and drug resistance loci. We found that parasites
were clustered into 3 clonal subpopulations and were related to parasites from Colombia. Two clusters of Panamanian parasites
shared identical drug resistance haplotypes, and all clusters shared a chloroquine-resistance genotype matching the pfcrt haplotype of Colombian origin. Our findings suggest these resurgent parasite populations are highly clonal and that the high
clonality likely resulted from epidemic expansion of imported or vestigial cases. Malaria outbreak investigations that use
genetic tools can illuminate potential sources of epidemic malaria and guide strategies to prevent further resurgence in areas
where malaria has been eliminated.
The Journal of Infectious Diseases 05/2015; 211(7):1087-96. DOI:10.1093/infdis/jiu575 · 5.78 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Plasmodium vivax, one of the five species of Plasmodium parasites that cause human malaria, is responsible for 25-40% of malaria cases worldwide. Malaria global elimination efforts will benefit from accurate and effective genotyping tools that will provide insight into the population genetics and diversity of this parasite. The recent sequencing of P. vivax isolates from South America, Africa, and Asia presents a new opportunity by uncovering thousands of novel single nucleotide polymorphisms (SNPs). Genotyping a selection of these SNPs provides a robust, low-cost method of identifying parasite infections through their unique genetic signature or barcode. Based on our experience in generating a SNP barcode for P. falciparum using High Resolution Melting (HRM), we have developed a similar tool for P. vivax. We selected globally polymorphic SNPs from available P. vivax genome sequence data that were located in putatively selectively neutral sites (i.e., intergenic, intronic, or 4-fold degenerate coding). From these candidate SNPs we defined a barcode consisting of 42 SNPs. We analyzed the performance of the 42-SNP barcode on 87 P. vivax clinical samples from parasite populations in South America (Brazil, French Guiana), Africa (Ethiopia) and Asia (Sri Lanka). We found that the P. vivax barcode is robust, as it requires only a small quantity of DNA (limit of detection 0.3 ng/μl) to yield reproducible genotype calls, and detects polymorphic genotypes with high sensitivity. The markers are informative across all clinical samples evaluated (average minor allele frequency > 0.1). Population genetic and statistical analyses show the barcode captures high degrees of population diversity and differentiates geographically distinct populations. Our 42-SNP barcode provides a robust, informative, and standardized genetic marker set that accurately identifies a genomic signature for P. vivax infections.
[Show abstract][Hide abstract] ABSTRACT: Host nutrition can affect the outcome of parasitic diseases through metabolic effects on host immunity and/or the parasite. Here we show that modulation of mouse immunometabolism through brief restriction of food intake (dietary restriction, DR) prevents neuropathology in experimental cerebral malaria (ECM). While no effects are detected on parasite growth, DR reduces parasite accumulation in peripheral tissues including the brain, and increases clearance in the spleen. Leptin, a host-derived adipokine linking appetite, energy balance and immune function, is required for ECM pathology and its levels are reduced upon DR. Recombinant leptin abrogates DR benefits, while pharmacological or genetic inhibition of leptin signalling protects against ECM. DR reduces mTORC1 activity in T cells, and this effect is abrogated upon leptin administration. Furthermore, mTORC1 inhibition with rapamycin prevents ECM pathology. Our results suggest that leptin and mTORC1 provide a novel mechanistic link between nutrition, immunometabolism and ECM pathology, with potential therapeutic implications for cerebral malaria.
[Show abstract][Hide abstract] ABSTRACT: Complex malaria infections are defined as those containing more than one genetically distinct lineage of Plasmodium parasite. Complexity of infection (COI) is a useful parameter to estimate from patient blood samples because it is associated with clinical outcome, epidemiology and disease transmission rate. This manuscript describes a method for estimating COI using likelihood, called COIL, from a panel of bi-allelic genotyping assays.
COIL assumes that distinct parasite lineages in complex infections are unrelated and that genotyped loci do not exhibit significant linkage disequilibrium. Using the population minor allele frequency (MAF) of the genotyped loci, COIL uses the binomial distribution to estimate the likelihood of a COI level given the prevalence of observed monomorphic or polymorphic genotypes within each sample.
COIL reliably estimates COI up to a level of three or five with at least 24 or 96 unlinked genotyped loci, respectively, as determined by in silico simulation and empirical validation. Evaluation of COI levels greater than five in patient samples may require a very large collection of genotype data, making sequencing a more cost-effective approach for evaluating COI under conditions when disease transmission is extremely high. Performance of the method is positively correlated with the MAF of the genotyped loci. COI estimates from existing SNP genotype datasets create a more detailed portrait of disease than analyses based simply on the number of polymorphic genotypes observed within samples.
The capacity to reliably estimate COI from a genome-wide panel of SNP genotypes provides a potentially more accurate alternative to methods relying on PCR amplification of a small number of loci for estimating COI. This approach will also increase the number of applications of SNP genotype data, providing additional motivation to employ SNP barcodes for studies of disease epidemiology or control measure efficacy. The COIL program is available for download from GitHub, and users may also upload their SNP genotype data to a web interface for simple and efficient determination of sample COI.
[Show abstract][Hide abstract] ABSTRACT: This paper demonstrates the enrichment of reticulocytes by centrifuging whole blood through aqueous multiphase systems (AMPSs)—immiscible phases of solutions of polymers that form step-gradients in density. The interfaces of an AMPS concentrate cells; this concentration facilitates the extraction of blood enriched for reticulocytes. AMPS enrich reticulocytes from blood from both healthy and hemochromatosis donors. Varying the osmolality and density of the phases of AMPS provides different levels of enrichment and yield of reticulocytes. A maximum enrichment of reticulocytemia of 64 ± 3 % was obtained from donors with hemochromatosis. When used on peripheral blood from normal donors, AMPS can provide a higher yield of enriched reticulocytes and a higher proportion of reticulocytes expressing CD71 than differential centrifugation followed by centrifugation over Percoll. Blood enriched for reticulocytes by AMPS could be useful for research on malaria. Several species of malaria parasites show a preference to invade young erythrocytes and reticulocytes; this preference complicates in vitro cultivation of these species in human blood. Plasmodium knowlesi malaria parasites invade normal human blood enriched for reticulocytes by AMPSs at a rate 2.2 times greater (p-value < 0.01) than they invade unenriched blood. Parasite invasion in normal blood enriched by AMPS was 1.8 times greater (p-value < 0.05) than in blood enriched to a similar reticulocytemia by differential centrifugation followed by centrifugation over Percoll. The enrichment of reticulocytes that are invaded by malaria parasites demonstrates that AMPSs can provide a label-free method to enrich cells for biological research.
American Journal of Hematology 01/2015; 90(1). DOI:10.1002/ajh.23860 · 3.48 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Background
Drug resistance remains a major public health challenge for malaria treatment and eradication. Individual loci associated with drug resistance to many antimalarials have been identified, but their epistasis with other resistance mechanisms has not yet been elucidated.ResultsWe previously described two mutations in the cytoplasmic prolyl-tRNA synthetase (cPRS) that confer resistance to halofuginone. We describe here the evolutionary trajectory of halofuginone resistance of two independent drug resistance selections in Plasmodium falciparum. Using this novel methodology, we discover an unexpected non-genetic drug resistance mechanism that P. falciparum utilizes before genetic modification of the cPRS. P. falciparum first upregulates its proline amino acid homeostasis in response to halofuginone pressure. We show that this non-genetic adaptation to halofuginone is not likely mediated by differential RNA expression and precedes mutation or amplification of the cPRS gene. By tracking the evolution of the two drug resistance selections with whole genome sequencing, we further demonstrate that the cPRS locus accounts for the majority of genetic adaptation to halofuginone in P. falciparum. We further validate that copy-number variations at the cPRS locus also contribute to halofuginone resistance.Conclusions
We provide a three-step model for multi-locus evolution of halofuginone drug resistance in P. falciparum. Informed by genomic approaches, our results provide the first comprehensive view of the evolutionary trajectory malaria parasites take to achieve drug resistance. Our understanding of the multiple genetic and non-genetic mechanisms of drug resistance informs how we will design and pair future anti-malarials for clinical use.
[Show abstract][Hide abstract] ABSTRACT: As the intensity of malaria transmission has declined, Plasmodium falciparum parasite populations have displayed decreased clonal diversity resulting from the emergence of many parasites with common
genetic signatures (CGS). We have monitored such CGS parasite clusters from 2006 to 2013 in Thiès, Senegal, using the molecular
barcode. The first, and one of the largest observed clusters of CGS parasites, was present in 24% of clinical isolates in
2008, declined to 3.4% of clinical isolates in 2009, and then disappeared. To begin to explore the relationship between the
immune responses of the population and the emergence and decline of specific parasite genotypes, we have determined whether
antibodies to CGS parasites correlate with their prevalence. We measured (i) antibodies capable of inhibiting parasite growth
in culture and (ii) antibodies recognizing the surfaces of infected erythrocytes (RBCs). IgG obtained from volunteers in 2009
showed increased reactivity to the surfaces of CGS-parasitized erythrocytes over IgG from 2008. Since P. falciparum EMP-1 (PfEMP-1) is a major variant surface antigen, we used var Ups quantitative reverse transcription-PCR (qRT-PCR) and sequencing with degenerate DBL1α domain primers to characterize
the var genes expressed by CGS parasites after short-term in vitro culture. CGS parasites show upregulation of UpsA var genes and 2-cysteine-containing PfEMP-1 molecules and express the same dominant var transcript. Our work indicates that the CGS parasites in this cluster express similar var genes, more than would be expected by chance in the population, and that there is year-to-year variation in immune recognition
of surface antigens on CGS parasite-infected erythrocytes. This study lays the groundwork for detailed investigations of the
mechanisms driving the expansion or contraction of specific parasite clones in the population.
Infection and Immunity 11/2014; 83(1). DOI:10.1128/IAI.01979-14 · 4.16 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Antimalarial drug resistance is the biggest threat to the current malaria eradication agenda (MaLERA) based on oral artemisinin-based combination therapies (ACTs). ACTs are the standard of care for uncomplicated malaria, but reports of clinical treatment failures (defined as a reduced parasite clearance rate or persistence of parasites on the third day of treatment) of artesunate (AS) / mefloquine (MQ) combinations are rapidly emerging from South-East Asia. The Thai Plasmodium falciparum C2A clone is a Multiple Drug Resistant (MDR) isolate obtained from a patient in Thailand in 1992 before deployment of this ACT, first introduced in 1994 and adopted in 2005, though, artemisinins had been in use in western Cambodia since the late 70's (Witkowski et al, 2013). In a previous in vivo adaptation study in Aotus monkeys, we observed that even in the absence of MQ pressure, C2A appeared to gradually loose its susceptibility to MQ as passage levels increased. In order to further characterize this observation we assessed the efficacy of MQ and AS alone or in combination in infected splenectomized Aotus monkeys. Our results confirmed the previous findings of resistance to oral MQ at 40 mg / Kg given once or to oral AS at 33 mg / Kg x 3 days alone or in combination with MQ at 40 mg / kg once. Strikingly, in the AS alone group, clearance occur in 1 / 2 animals on day 7 post-treatment (PT) but recrudesce on day 8 PT (slow clearance phenotype) and persisted in the other one until day 10 PT when it was finally rescue treated. As a comparison, AS at 20 mg / Kg orally x 3 days against infections of the Vietnam CQ resistant FVO strain, clears infection within 1-2 days PT in spleen intact Aotus. Only when AS was administered IV at 20 mg / Kg x 3 days in combination with oral MQ at 40 mg / Kg, infections were cured in 4 / 5, but cleared and recrudesce in 1 / 5 rescue treated animals. Additional in vitro assays of C2A demonstrated reduced susceptibility to MQ, CQ, artemisinin, dihydro-arteminsinin, and atovaquone-proguanil, arteether and AS compared to the sensitive D6 strain, the in vitro adapted TM90-C2A line or passage III Aotus adapted C2A when compared to the later monkey passage X. Analysis of drug resistance loci and copy number variation is underway to determine the genetic basis for the observed drug resistance profile. We anticipate that these studies will shed light on the early spread and evolution of multi drug resistance and reduced artemisinin susceptibility in South-East Asia.
63th Annual Meeting of the American Society of Tropical Medicine and Hygiene, New Orleans, LA, USA; 11/2014
[Show abstract][Hide abstract] ABSTRACT: During the latter half of the natural 48-h intraerythrocytic life cycle of human Plasmodium falciparum infection, parasites sequester deep in endothelium of tissues, away from the spleen and inaccessible to peripheral blood. These late-stage parasites may cause tissue damage and likely contribute to clinical disease, and a more complete understanding of their biology is needed. Because these life cycle stages are not easily sampled due to deep tissue sequestration, measuring in vivo gene expression of parasites in the trophozoite and schizont stages has been a challenge.
We developed a custom nCounter® gene expression platform and used this platform to measure malaria parasite gene expression profiles in vitro and in vivo. We also used imputation to generate global transcriptional profiles and assessed differential gene expression between parasites growing in vitro and those recovered from malaria-infected patient tissues collected at autopsy.
We demonstrate, for the first time, global transcriptional expression profiles from in vivo malaria parasites sequestered in human tissues. We found that parasite physiology can be correlated with in vitro data from an existing life cycle data set, and that parasites in sequestered tissues show an expected schizont-like transcriptional profile, which is conserved across tissues from the same patient. Imputation based on 60 landmark genes generated global transcriptional profiles that were highly correlated with genome-wide expression patterns from the same samples measured by microarray. Finally, differential expression revealed a limited set of in vivo upregulated transcripts, which may indicate unique parasite genes involved in human clinical infections.
Our study highlights the utility of a custom nCounter® P. falciparum probe set, validation of imputation within Plasmodium species, and documentation of in vivo schizont-stage expression patterns from human tissues.
Genome Medicine 11/2014; 6(11):110. DOI:10.1186/s13073-014-0110-6 · 4.94 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Background. The emergence and spread of drug resistance to current antimalarial therapies remains a pressing concern, escalating the need for compounds that demonstrate novel modes of action. Diversity-Oriented Synthesis (DOS) libraries bridge the gap between conventional small molecule and natural product libraries, allowing the interrogation of more diverse chemical space in efforts to identify probes of novel parasite pathways.
Methods. We screened and optimized a probe from a DOS library using whole-cell phenotypic assays. Resistance selection and whole-genome sequencing approaches were employed to identify the cellular target of the compounds.
Results. We identified a novel macrocyclic inhibitor of Plasmodium falciparum with nanomolar potency and identified the reduction site of cytochrome b as its cellular target. Combination experiments with reduction and oxidation site inhibitors showed synergistic inhibition of the parasite.
Conclusions. The cytochrome b oxidation center is a validated antimalarial target. We show that the reduction site of cytochrome b is also a druggable target. Our results demonstrating a synergistic relationship between oxidation and reduction site inhibitors suggests a future strategy for new combination therapies in the treatment of malaria.
The Journal of Infectious Diseases 10/2014; 211(7). DOI:10.1093/infdis/jiu565 · 5.78 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Here, we describe medicinal chemistry that was accelerated by a diversity-oriented synthesis (DOS) pathway, and in vivo studies of our previously reported macrocyclic antimalarial agent that derived from the synthetic pathway. Structure-activity relationships that focused on both appendage and skeletal features yielded a nanomolar inhibitor of P. falciparum asexual blood-stage growth with improved solubility and microsomal stability and reduced hERG binding. The build/couple/pair (B/C/P) synthetic strategy, used in the preparation of the original screening library, facilitated medicinal chemistry optimization of the antimalarial lead.
[Show abstract][Hide abstract] ABSTRACT: Whole-cell high-throughput screening of the AstraZeneca compound library against the asexual blood stage of Plasmodium falciparum (Pf) led to the identification of amino imidazoles, a robust starting point for initiating a hit-to-lead medicinal chemistry effort. Structure–activity relationship studies followed by pharmacokinetics optimization resulted in the identification of 23 as an attractive lead with good oral bioavailability. Compound 23 was found to be efficacious (ED90 of 28.6 mg·kg–1) in the humanized P. falciparum mouse model of malaria (Pf/SCID model). Representative compounds displayed a moderate to fast killing profile that is comparable to that of chloroquine. This series demonstrates no cross-resistance against a panel of Pf strains with mutations to known antimalarial drugs, thereby suggesting a novel mechanism of action for this chemical class.