Methods for analysis of deep sequencing data from mixtures of Plasmodium falciparum clones or stage-specific transcriptomes

Malaria is a life-threatening infectious disease caused by Plasmodium parasites transmitted to humans through bites of infected Anopheles mosquitos. An estimated 445,000 people die every year by an infection with Plasmodium parasites, most of them children living in sub-Saharan Africa. As a result of increased malaria control, the mortality was greatly reduced in the last decades. To develop new tools for elimination and to evaluate the impact of control, a good understanding of the epidemiology and biology of malaria parasites is required. Studies of infection and transmission dynamics of Plasmodium parasites were greatly improved by distinguishing individual parasite clones and monitoring their infection dynamics over time. In regions with high transmission of Plasmodium parasites, individuals are often infected with several clones concurrently. Individual parasites clones can be identified by genotyping. The current standard method used for genotyping is amplification of highly length-polymorphic merozoite surface protein 2 (msp2) or other antigen genes followed by sizing of the amplicon by capillary electrophoresis (CE). The sensitivity to detect low-abundant clones (minority clones) of msp2-CE genotyping is however limited, resulting in an underestimation of multiplicity of infection (MOI). A shortfall of this genotyping method is that frequency of individual clones within a sample cannot be determined. This urges the search for new genotyping methods that rely on sequencing of genomic fragments with extensive single nucleotide polymorphism (SNP). Improvement in next generation sequencing (NGS) technologies permitted the use of amplicon sequencing (Amp-Seq) in epidemiological studies. Genotyping by amplicon sequencing has a higher sensitivity to detect minority clones, can quantify the frequency of each clone within a sample, and allows the use of SNP polymorphic markers. In the frame of this thesis, a new Amp-Seq genotyping assay was developed, including known SNP polymorphic markers, and novel marker ‘cpmp’, as well as a bioinformatic analysis workflow. This genotyping assay was applied on field samples from a longitudinal study conducted in Papua New Guinea. A comparison to msp2-CE genotyping confirmed the higher sensitivity to detect minority clones by Amp-Seq genotyping method and showed a significant underestimation of MOI by classical size polymorphic marker. However, no significant increase in molecular force of infection (molFOI), i.e. number of new infections per individual per year, was observed. Quantification of the frequency of individual clones in longitudinal samples permitted to infer multi-locus haplotypes. Multi-locus haplotypes increased discriminatory power of genotyping and robustly distinguished new infections from those detected in an individual earlier. For calculating the density of clones from multi-clone infections the within-host clone frequency is multiplied by parasitaemia of this infection determined by quantitative PCR. Density of individual parasites clones in multi-clone infections over time is a new parameter for epidemiological studies. It will permit to study the dynamics, and thus fitness, of parasite clones exposed to within-host competition or to acquired natural immunity. NGS also gained great importance in gene expression studies of Plasmodium parasites in patient samples. Transcriptome studies are complicated by the mixture of different developmental stages present concurrently in samples collected from patients. Even in in vitro cultured samples after tight synchronisation or enrichment of a specific developmental stage, small fractions of other development stages are still found. This problem is of particular relevance for P. vivax, as the absence of continuous in vitro culture so far has hampered the study of isolated parasite stages. For example, the transcriptome of P. vivax gametocytes, one of the stages found in peripheral blood and infective to mosquitos, has not yet been described. A solution for differentiating mixed transcription may come from deconvolution methods, which either infer the stage proportion in samples or stage-specific transcriptome signatures. A large selection of different deconvolution methods has been developed for the analysis of heterogeneous tissues, e.g. cancer tissues or hematopoietic cell, but these methods have rarely been applied to mixed stages of malaria parasites. The best suited combination of normalisation and deconvolution methods for analysis of RNA sequencing (RNA-Seq) data from mixed-stage samples of Plasmodium parasites was evaluated based on experimentally mixed highly synchronised blood stages. Normalisation by count per million and deconvolution with a negative binomial regression model followed by selection of genes with significant fold change resulted in the best agreement with transcriptomes as observed in single stages. This strategy can easily be transferred to Plasmodium field samples with known stage proportions. This analysis performed in cultured parasites of defined mixed stages served as proof-of-concept and confirmed that identification of stage-specific genes is feasible also in field samples, notably in species that cannot be cultivated, such as P. vivax. NGS permits fundamentally new approaches to study Plasmodium parasites. This thesis presents a novel marker and data analysis platform for highly sensitive P. falciparum genotyping. Furthermore, a best practice workflow was identified to infer stage-specific gene expression from parasite infections consisting of mixed developmental stages. This provides a crucial tool for the analysis of gene expression data generated from Plasmodium field samples

Implementing parasite genotyping into national surveillance frameworks: Feedback from control programmes and researchers in the Asia-pacific region

The Asia-Pacific region faces formidable challenges in achieving malaria elimination by the proposed target in 2030. Molecular surveillance of Plasmodium parasites can provide important information on malaria transmission and adaptation, which can inform national malaria control programmes (NMCPs) in decision-making processes. In November 2019 a parasite genotyping workshop was held in Jakarta, Indonesia, to review molecular approaches for parasite surveillance and explore ways in which these tools can be integrated into public health systems and inform policy. The meeting was attended by 70 participants from 8 malaria-endemic countries and partners of the Asia Pacific Malaria Elimination Network. The participants acknowledged the utility of multiple use cases for parasite genotyping including: quantifying the prevalence of drug resistant parasites, predicting risks of treatment failure, identifying major routes and reservoirs of infection, monitoring imported malaria and its contribution to local transmission, characterizing the origins and dynamics of malaria outbreaks, and estimating the frequency of Plasmodium vivax relapses. However, the priority of each use case varies with different endemic settings. Although a one-size-fits-all approach to molecular surveillance is unlikely to be applicable across the Asia-Pacific region, consensus on the spectrum of added-value activities will help support data sharing across national boundaries. Knowledge exchange is needed to establish local expertise in different laboratory-based methodologies and bioinformatics processes. Collaborative research involving local and international teams will help maximize the impact of analytical outputs on the operational needs of NMCPs. Research is also needed to explore the cost-effectiveness of genetic epidemiology for different use cases to help to leverage funding for wide-scale implementation. Engagement between NMCPs and local researchers will be critical throughout this process

A Method for Amplicon Deep Sequencing of Drug Resistance Genes in Plasmodium falciparum Clinical Isolates from India.

A major challenge to global malaria control and elimination is early detection and containment of emerging drug resistance. Next-generation sequencing (NGS) methods provide the resolution, scalability, and sensitivity required for high-throughput surveillance of molecular markers of drug resistance. We have developed an amplicon sequencing method on the Ion Torrent PGM platform for targeted resequencing of a panel of six Plasmodium falciparum genes implicated in resistance to first-line antimalarial therapy, including artemisinin combination therapy, chloroquine, and sulfadoxine-pyrimethamine. The protocol was optimized using 12 geographically diverse P. falciparum reference strains and successfully applied to multiplexed sequencing of 16 clinical isolates from India. The sequencing results from the reference strains showed 100% concordance with previously reported drug resistance-associated mutations. Single-nucleotide polymorphisms (SNPs) in clinical isolates revealed a number of known resistance-associated mutations and other nonsynonymous mutations that have not been implicated in drug resistance. SNP positions containing multiple allelic variants were used to identify three clinical samples containing mixed genotypes indicative of multiclonal infections. The amplicon sequencing protocol has been designed for the benchtop Ion Torrent PGM platform and can be operated with minimal bioinformatics infrastructure, making it ideal for use in countries that are endemic for the disease to facilitate routine large-scale surveillance of the emergence of drug resistance and to ensure continued success of the malaria treatment policy

The Malaria Cell Atlas: single parasite transcriptomes across the complete Plasmodium life cycle

Malaria parasites adopt a remarkable variety of morphological life stages as they transition through multiple mammalian host and mosquito vector environments. We profiled the single-cell transcriptomes of thousands of individual parasites, deriving the first high-resolution transcriptional atlas of the entire life cycle. We then used our atlas to precisely define developmental stages of single cells from three different human malaria parasite species, including parasites isolated directly from infected individuals. The Malaria Cell Atlas provides both a comprehensive view of gene usage in a eukaryotic parasite and an open-access reference dataset for the study of malaria parasites

Decoding the complexities of human malaria through systems immunology

The complexity of the Plasmodium parasite and its life cycle poses a challenge to our understanding of the host immune response against malaria. Studying human immune responses during natural and experimental Plasmodium infections can enhance our understanding of malaria-protective immunity and inform the design of disease-modifying adjunctive therapies and next-generation malaria vaccines. Systems immunology can complement conventional approaches to facilitate our understanding of the complex immune response to the highly dynamic malaria parasite. In this review, recent studies that used systems-based approaches to evaluate human immune responses during natural and experimental Plasmodium falciparum and Plasmodium vivax infections as well as during immunization with candidate malaria vaccines are summarized and related to each other. The potential for next-generation technologies to address the current limitations of systems-based studies of human malaria are discussed

Applying next-generation sequencing to track falciparum malaria in sub-Saharan Africa

Next-generation sequencing (NGS) technologies are increasingly being used to address a diverse range of biological and epidemiological questions. The current understanding of malaria transmission dynamics and parasite movement mainly relies on the analyses of epidemiologic data, e.g. case counts and self-reported travel history data. However, travel history data are often not routinely collected or are incomplete, lacking the necessary level of accuracy. Although genetic data from routinely collected field samples provides an unprecedented opportunity to track the spread of malaria parasites, it remains an underutilized resource for surveillance due to lack of local awareness and capacity, limited access to sensitive laboratory methods and associated computational tools and difficulty in interpreting genetic epidemiology data. In this review, the potential roles of NGS in better understanding of transmission patterns, accurately tracking parasite movement and addressing the emerging challenges of imported malaria in low transmission settings of sub-Saharan Africa are discussed. Furthermore, this review highlights the insights gained from malaria genomic research and challenges associated with integrating malaria genomics into existing surveillance tools to inform control and elimination strategies

Transcriptomics of malaria host-pathogen interactions in primates

Malaria is a pernicious disease that has greatly impacted and continues to affect the human population. While much research has been performed to understand the underlying nature of this disease, gaps in the knowledge-base persist. In order to address these deficiencies, a multi-disciplinary, multi-institutional project has been funded to study the systems biology of the host pathogen interaction during malaria infection in both humans and non-human primates. In the course of investigating the transcriptome during two 100-day experiments in Macaca mulatta, this work elucidated many of the underlying molecular pathways of the host and parasite that are affected by antimalarial drugs, as well as through host-pathogen interactions. The malaria-disease-related host pathways are related to, not surprisingly, immune-associated signalling and hematopoesis, and the altered parasite pathways demonstrate an association between disease severity and parasite life stage abundance. Continuing integration of this research with other data-types collected during the course of these experiments will improve our understanding of malaria systems biology and improve targeted malaria therapies.Ph.D

Mechanism of Action Studies of Phenotypic Whole-cell Active Antimalarial Leads Through Target Identification

Chemotherapy has remained the backbone of malaria control and prevention. Over the past century, potent antimalarial drugs with different mechanisms of action have been successfully developed and used to treat malaria. However, the ability of the most virulent species, P. falciparum, to resist these available antimalarial chemotypes and compromise their potency has raised the importance of using combination therapies and developing new, safe, and efficacious molecules with novel modes of action for the treatment of malaria. Phenotypic whole-cell screening, followed by medicinal chemistry optimization efforts, identified the pyrido[1,2-a]benzimidazole compounds KP68 and KP124 and the benzimidazole compound DM253 as efficacious antimalarial leads. However, the essential details of their mechanism of action against P. falciparum remain unresolved. This thesis employs ‘omics-based techniques with support from fluorescence live-cell imaging, compound docking, and heme fractionation studies to generate insights into the action of these compounds against P. falciparum. The previous mechanism of action studies on these antimalarial chemotypes has focused mainly on the inhibition of hemozoin biocrystallization in the acidic digestive vacuole of the parasite. However, the intrinsic fluorescence properties of KP68 and KP124 were used to comprehensively study the subcellular accumulation of these compounds in an infected erythrocyte. Using the inherent fluorescence properties of these compounds is advantageous because accurate localization due to the compounds is observed with no KP68 PfNF54 IC50 = 0.03 µM PfK1 IC50 = 0.04 µM in vivo P. berghei (p.o) 4x50 mg/kg = 98.0% 3/3 malaria infected mice cured KP124 PfNF54 IC50 = 0.14 µM PfK1 IC50 = 0.13 µM DM253 PfNF54 IC50 = 0.012 µM PfK1 IC50 = 0.040 µm in vivo P. berghei (p.o) 4x50 mg/kg = 99.52 Mean survival days = 14 days influence from an external fluorophore. On the other hand, DM253 required the attachment of an external fluorophore for live-cell imaging. As such, a novel fluorescent derivative was designed and synthesized with guidance from extensive structure-activity relationship studies previously conducted in this series. 7-Nitrobenz-2-oxa-1,3-diazole (NBD) was identified as an appropriate external fluorophore and was attached to the compounds investigated. The spacer chain length between the compounds and the fluorophore was varied to find suitable fluorescent derivatives that would appropriately represent the parent compounds. The photophysical and physicochemical properties of all fluorescent compounds were evaluated. Although the fluorescent derivatives lost antiplasmodium potency relative to their parent compounds, all but NBD-labelled KP124 retained antiplasmodium activity in the chloroquine-sensitive strain of P. falciparum. Furthermore, a detergent-mediated assay indicated that all fluorescently labelled derivatives retained activity against βhematin formation compared to the parent molecules. These results suggest that except for KP124-NBD, all fluorescent compounds and the fluorescent analogues were suitable for live-cell fluorescence accumulation studies. Live-cell imaging showed selective accumulation of all fluorescent compounds within P. falciparum-infected red blood cells. Different accumulation patterns were observed when using the inherent fluorescence of the structurally related KP68 and KP124. KP124 was observed to colocalize in the parasite's digestive vacuole and associate with hemozoin crystals, whiles KP68 which differs from KP124 by the replacement of the imidazole[1,2- a:4,5-b′]dipyridine core with the benzimidazole core, as well as the presence of chloro substituents, showed no accumulation in the parasite's digestive vacuole. Quantitative colocalization studies of parasite cells co-stained with KP124, DM253-NBD, and LysoTracker Red demonstrated an excellent colocalization between these signals. This indicates a preference for these compounds in the parasite's acidic compartment. Furthermore, the quantitative analysis also revealed that none of the compounds localized in the nucleus, eliminating the nucleus as a site of action for these compounds. To mitigate the limitations of resolution, Airyscan and super-resolution structured-illumination microscopy (SR-SIM) were employed. Fluorescence imaging using the ER-Tracker Red revealed a broad colocalization between KP124 and DM253-NBD and the tracker dye, suggesting that both compounds accumulate in the endoplasmic reticulum (ER). However, no significant amounts of KP68 were localized in the ER. The mitochondrion was, however, implicated in the action of KP68. Although colocalization was not observed between the MitoTracker Deep Red and KP68, significant amounts of the compound localize around the mitochondrion membrane. Finally, all compounds were assessed in the cellular heme fractionation assay. Results from this assay indicate that inhibition of hemozoin formation is a mechanism of action for KP124 but not for KP68 and DM253. The recent growth in genomics and genetics has provided powerful tools for mode of action studies. In vitro resistance selections represent one of the genomics tools for target deconvolution of hit and lead molecules. The mechanism of resistance of the pyrido[1,2- a]benzimidazoles was investigated through resistance selection. Whole-genome sequencing of the mutant clones generated from KP68 under drug pressure showed a single nucleotide polymorphism in the mitochondrion carrier protein. It also revealed a few copy number variations, including the deamplification of the mitochondrialprocessing peptidase and the P. falciparum multidrug resistance transporter PfMDR1.This result, coupled with the significant amounts of KP68 observed to accumulate around the parasite's mitochondrion, confirms the mitochondrion as an organelle of interest in the compound's mode of action in P. falciparum. Furthermore, no cross-resistance was observed between KP68 and chloroquine, suggesting that both compounds may act through different resistance mechanisms and possibly different mechanisms of action. Similarly, no cross-resistance was observed between the mutant clones generated for KP68 and KP124, meaning that the parasite's mode of resistance and the action of both compounds may be mediated through different mechanisms. This also confirms the livecell imaging and heme fractionation assay results, which support hemozoin inhibition as a mode of action for KP124 but not KP68. Finally, chemical proteomics was employed to identify the protein binding partners of these antimalarial compounds in P. falciparum. Here, drug-labelled matrices were used to capture protein binding partners of KP68 and KP124 from P. falciparum cell lysates. Several protein binding partners specific to these compounds were detected from the parasite lysate prepared and identified by mass spectrometry and proteomic analysis. Out of the many proteins identified as protein binding partners for KP68, the high molecular weight EMP1-trafficking protein, PfEMP1 is of significant interest. This is because it is essential for the parasite's survival and has been implicated in the action of other antimalarials such as dihydroartemisinin. These results suggest that these compounds may impact different parasite pathways and processes. Besides hemozoin formation, KP124 has also been implicated in interfering with the parasite's protein synthesis. Overall, this work has developed new tools that have aided in understanding the mechanistic details of these compounds. The observations described here, and further studies using the techniques and approaches to target deconvolution discussed here may facilitate the identification of novel targets for treating malaria. Also, once chemically validated, the protein targets identified in this work can serve as suitable starting points for target-based antimalarial drug discovery efforts