<i>P. berghei</i> telomerase subunit TERT is essential for parasite survival

Telomeres define the ends of chromosomes protecting eukaryotic cells from chromosome instability and eventual cell death. The complex regulation of telomeres involves various proteins including telomerase, which is a specialized ribonucleoprotein responsible for telomere maintenance. Telomeres of chromosomes of malaria parasites are kept at a constant length during blood stage proliferation. The 7-bp telomere repeat sequence is universal across different Plasmodium species (GGGTTT/CA), though the average telomere length varies. The catalytic subunit of telomerase, telomerase reverse transcriptase (TERT), is present in all sequenced Plasmodium species and is approximately three times larger than other eukaryotic TERTs. The Plasmodium RNA component of TERT has recently been identified in silico. A strategy to delete the gene encoding TERT via double cross-over (DXO) homologous recombination was undertaken to study the telomerase function in P. berghei. Expression of both TERT and the RNA component (TR) in P. berghei blood stages was analysed by Western blotting and Northern analysis. Average telomere length was measured in several Plasmodium species using Telomere Restriction Fragment (TRF) analysis. TERT and TR were detected in blood stages and an average telomere length of ~950 bp established. Deletion of the tert gene was performed using standard transfection methodologies and we show the presence of tert− mutants in the transfected parasite populations. Cloning of tert- mutants has been attempted multiple times without success. Thorough analysis of the transfected parasite populations and the parasite obtained from extensive parasite cloning from these populations provide evidence for a so called delayed death phenotype as observed in different organisms lacking TERT. The findings indicate that TERT is essential for P. berghei cell survival. The study extends our current knowledge on telomere biology in malaria parasites and validates further investigations to identify telomerase inhibitors to induce parasite cell death

Integration of parasite genetic information in malaria transmission modelling

Mathematical models of malaria transmission are increasingly used to quantify the impact of malaria control efforts and to assist in the development and costing of future initiatives such as the WHO Global Technical Strategy for Malaria 2016-2030. These models have highlighted both the progress made so far, but also how continued investment is needed to reach the milestones required. However, the increase in global malaria cases reported in 2018 suggests that new tools may be required to continue the gains made and to address the growing risk of antimalarial resistance threatening to reverse the recent declines in malaria burden. The proliferation of genetic sequencing and the publication of the Plasmodium falciparum reference genome in 2002 has facilitated a greater understanding of the genetic determinants of resistance and molecular tools are subsequently poised to become a routine tool for malaria control. Consequently, integrating parasite genetic information into established models of malaria transmission models can contribute to both our understanding of the drivers and optimum policies for addressing resistance and detailing the potential of molecular tools within malaria control. Plasmodium falciparum is known to have evolved several times in response to first line antimalarials. However, recent evidence has shown evolution to rapid diagnostic tests. The WHO has consequently issued guidance advising national malaria control programmes to conduct surveillance for pfhrp2/3 deletions. The timing of this policy recommendation and my previous work modelling pfhrp2 deletions necessitated a timely extension of our previous model to evaluate the implications of seasonality in malaria transmission on estimates of the prevalence of pfhrp2/3 deletions. Recent studies have suggested that malaria genotyping could be a useful tool for epidemiological surveillance. By developing an extended version of an established model of malaria transmission, which now models individual mosquitoes affording the full parasite life cycle to be represented, I characterise the potential utility of malaria genomics for inferring changes in transmission intensity. I conclude that although molecular tools could enable accurate estimation of malaria prevalence, greater attention needs to be placed on the chosen sampling scheme, recording patient metadata and developing the statistical toolkit for analysing polyclonal infected individuals. In 2015, health ministers in the Greater Mekong Subregion (GMS) adopted the WHO strategy for malaria elimination in the GMS 2016-2030. The strategy was developed to accelerate elimination in South-East Asia, which is currently the best approach to address the growing threat of artemisinin resistance and the emergence of multidrug resistant parasite lineages. In response, I demonstrate how the therapeutic lifespan of the five currently recommended artemisinin combination therapies can be prolonged by reducing antimalarial overprescription by ensuring that all suspected malaria fevers are tested before administering antimalarials. I conclude by comparing different cycling and mixing strategies before reviewing how each strategy can be improved to slow the spread of antimalarial resistance. Elimination in the GMS is undoubtedly an effective mechanism for preventing the spread of artemisinin resistance to Africa. However, if efforts to eliminate by 2030 have failed it will be imperative to understand the mechanisms with which resistance may continue to spread. To this extent, the capability of resistant strains to invade susceptible populations is evaluated using data from standard membrane feeding assays. Findings are incorporated in the transmission model to quantify the transmission advantage of artemisinin resistance at the population level.Open Acces

A Multidomain Adhesion Protein Family Expressed in Plasmodium falciparum Is Essential for Transmission to the Mosquito

The recent sequencing of several apicomplexan genomes has provided the opportunity to characterize novel antigens essential for the parasite life cycle that might lead to the development of new diagnostic and therapeutic markers. Here we have screened the Plasmodium falciparum genome sequence for genes encoding extracellular multidomain putative adhesive proteins. Three of these identified genes, named PfCCp1, PfCCp2, and PfCCp3, have multiple adhesive modules including a common Limulus coagulation factor C domain also found in two additional Plasmodium genes. Orthologues were identified in the Cryptosporidium parvum genome sequence, indicating an evolutionary conserved function. Transcript and protein expression analysis shows sexual stage–specific expression of PfCCp1, PfCCp2, and PfCCp3, and cellular localization studies revealed plasma membrane–associated expression in mature gametocytes. During gametogenesis, PfCCps are released and localize surrounding complexes of newly emerged microgametes and macrogametes. PfCCp expression markedly decreased after formation of zygotes. To begin to address PfCCp function, the PfCCp2 and PfCCp3 gene loci were disrupted by homologous recombination, resulting in parasites capable of forming oocyst sporozoites but blocked in the salivary gland transition. Our results describe members of a conserved apicomplexan protein family expressed in sexual stage Plasmodium parasites that may represent candidates for subunits of a transmission-blocking vaccine

Evolutionary analysis of the most polymorphic gene family in falciparum malaria

The var gene family of the human malaria parasite Plasmodium falciparum encode proteins that are crucial determinants of both pathogenesis and immune evasion and are highly polymorphic. Here we have assembled nearly complete var gene repertoires from 2398 field isolates and analysed a normalised set of 714 from across 12 countries. This therefore represents the first large scale attempt to catalogue the worldwide distribution of var gene sequences We confirm the extreme polymorphism of this gene family but also demonstrate an unexpected level of sequence sharing both within and between continents. We show that this is likely due to both the remnants of selective sweeps as well as a worrying degree of recent gene flow across continents with implications for the spread of drug resistance. We also address the evolution of the var repertoire with respect to the ancestral genes within the Laverania and show that diversity generated by recombination is concentrated in a number of hotspots. An analysis of the subdomain structure indicates that some existing definitions may need to be revised From the analysis of this data, we can now understand the way in which the family has evolved and how the diversity is continuously being generated. Finally, we demonstrate that because the genes are distributed across the genome, sequence sharing between genotypes acts as a useful population genetic marker

Multi-scale immune selection and the maintenance of structured antigenic diversity in the malaria parasite Plasmodium falciparum

The most virulent malaria parasite, Plasmodium falciparum, makes use of extensive antigenic diversity to maximise its transmission potential. Parasite genomes contain several highly polymorphic gene families, whose products are the target of protective immune responses. The best studied of these are the PfEMP1 surface proteins, which are encoded by the var multi-gene family and are important virulence factors. During infection, the parasite switches expression between PfEMP1 variants in order to evade adaptive immune responses and prolong infection. On the population level, parasites appear to be structured with respect to their var genes into non-overlapping repertoires, which can lead to high reinfection rates. This non-random structuring of antigenic diversity can also be found at the level of individual var gene repertoires and var genes themselves. However, not much is known about the evolutionary determinants which select for and maintain this structure at different ecological scales. In this thesis I investigate the mechanisms by which multi-scale immune selection and other ecological factors influence the evolution of structured diversity. Using a suite of theoretical frameworks I show that treating diversity as a dynamic property, which emerges from the underlying infection and transmission processes, has a major effect on the relationship between the parasite’s transmis- sion potential and disease prevalence, with important implications for monitoring control efforts. Furthermore, I show that an evolutionary trade-off between within-host and between-host fitness together with functional constraints on diversification can explain the structured diversity found at both the repertoire and parasite population level and might also account for empirically observed exposure-dependent acquisition of immunity. Together, this work highlights the need to consider evolutionary factors acting at different ecological scales to gain a more comprehensive understanding of the complex immune-epidemiology of P. falciparum malaria

Characterization of a Novel Sensing Mechanism Governing Antigenic Variation in P. Falciparum

Plasmodium falciparum expresses a multi-copy gene family called var in the intraerythroyctic stages of its life cycle in a mutually exclusive manner. var genes encode the chief antigenic and virulence determinant of P. falciparum, PfEMP1, and switching between active genes results in antigenic variation, allowing the parasite to evade the human immune system and cause chronic infections. The molecular mechanisms that control activation and silencing of individual var genes, as well as coordination of the switching process, presently remain incompletely defined. P. falciparum contains only ~60 var gene family members in its genome. Consequently, the question remains as to how this parasite can maintain an antigen-switch rate that allows for the emergence of a new variant when necessary, without rapidly exhausting all 60 members, to sustain chronic infections. The currently held paradigm proposes that antigenic variation follows an intrinsic, programed switching rate, operating independently of any external stimuli. In the following thesis I will present results suggesting the novel possibility that P. falciparum possesses cellular machinery capable of sensing changes in the environment of its host and is able to respond by altering antigen expression. It has been shown that changes in the transcription state of a var gene are controlled epigenetically. The methylation state of histone marks, deposited at active and silent var genes by histone methyltransferases (HMTs), play prominent roles in var gene regulation. Previously, Ukaegbu et al., 2015 showed that manipulating deposition of these marks had a striking impact on var gene expression. Metabolism and epigenetic control of gene expression are linked, as HMT activity is dependent on the intracellular concentrations of methyl donors, which can fluctuate based on nutrient availability. Various studies in other organisms have shown that there is a direct link between the level of intracellular S-adenosylmethionine (SAM), the principle methyl donor in biological methylation modifications, and histone methylation. I explored this connection between metabolism and var gene expression in P. falciparum. Parasites were cultured in growth media containing altered concentrations of nutrients involved in SAM metabolism. Bulk RNA was extracted from cultures, used as a template to synthesize cDNA, and analyzed by qPCR to determine the var gene expression at the population level. Conditions believed to increase SAM pools induced a coordinated switch to one particular var gene, var2csa, over time, phenocopying the results from Ukaegbu et al., 2015. This hypothesis was further tested by modifying expression of key enzymes involved in SAM metabolism. Once again, modifications thought to increase the intracellular level of SAM were found to induce a coordinated switch at the population level to var2csa. Conversely, modifications that lower the level of SAM did not induce expression of var2csa, but instead activated many vars at once across the population. These observations directly challenge the stochastic var switching paradigm by instead suggesting P. falciparum possesses the ability to sense environmental changes. After recognition of a pathogen, activated macrophages modify their microenvironments in various ways. I next tested the effect of two of these immune responses, depletion of amino acids and release of polyamines, on var expression of parasites in vitro. Both perturbations altered var expression, again specifically inducing var2csa. Taking these results together, I propose and discuss two possible models of antigenic variation in P. falciparum. The first centers on intracellular SAM metabolism in describing a promoter competition model governing var switching through var2csa. The second suggests that P. falciparum can sense when the host immune system first begins to recognize it via environmental cues resulting from antibody recognition, and respond by switching var gene expression. This would allow parasites to switch expression of var genes exactly when needed, allowing the most efficient utilization of their limited var gene repertoir

Fine mapping of susceptibility loci to malaria clinical episodes in a family-based cohort from Senegal

O parasita da malária, P. falciparum, mata na ordem de um milhão de crianças Africanas em cada ano, e esta é uma pequena fracção do número de pessoas infectadas em todo o mundo. A evolução clínica de uma infecção por este parasita depende em certa medida, da constituição genética do indivíduo infectado. O papel dos factores genéticos que regulam a gravidade da infecção da malária tem sido repetidamente demonstrado em humanos e animais. Os estudos de associação são realizados com o objectivo de identificar os genes implicados na causalidade do resultado da infecção. Foi detectado anteriormente, linkage no cromossoma humano 5p15 ao número de ataques de Plasmodium falciparum (PFA) em Dielmo, uma aldeia senegalesa [48]. Posteriormente, e antes deste estudo, um levantamento usando um ensaio "GoldenGate" da Illumina, com cerca de 1.450 SNPs foi realizada na região de Linkage com o fenótipo PFA. A análise foi realizada com três programas estatísticos baseados na família: Merlin, QTDT e FBAT/PBAT. Estes programas identificaram três genes candidatos associados com o fenótipo PFA: três SNPs (rs4867417, rs7714218 e rs11959398), localizados no gene PDZD2, um SNP (rs11134099) no gene ADAMTS16, e outro (rs3777320) localizado no gene SEMA5A. O objectivo deste estudo foi investigar estas associações. Os SNPs das regiões destes genes candidatos foram escolhidos por sequenciação de exões situados na região candidata ou por análise bioinformática utilizando dados do HapMap da população Yoruba. O estudado para genotipagem foi através das análises de pré-design ou “Custom” dos SNPs (Applied Biosystems). Os dados foram incluídos num banco de dados e a verificação dos erros de transmissão mendeliana foi efectuada. As análises estatísticas foram realizadas utilizando dois programas de associação familiar, PBAT e QTDT. Foram utilizados diferentes modelos de transmissão de alelos e foi definido como limite de significância p-value = 10-3. As análises de SNPs dos genes PDZD2 e ADAMTS16 não confirmaram a associação, mas encontrou-se associação significativa com SNPs do gene SEMA5A. Um SNP (rs3777325) foi significativamente associado com o fenótipo PFA usando ambos os programas (p-value= - 6.49x10-4 usando o programa PBAT e p-value = 2.0x10-4 usando o programa QTDT). A análise de haplótipos de dois SNPs adjacentes (rs4541632 e rs1018956), também mostrou uma associação significativa do haplótipo GC (p-value= -6.82x10-5) utilizando o programa PBAT. Este estudo confirma que o locus de susceptibilidade para o fenótipo PFA está localizado no gene SEMA5A. Mais estudos serão necessários para replicar essa associação e identificar o polimorfismo causal.The malaria parasite, P. falciparum, kills on the order of a million African children each year, and this is a small fraction of the number of infected individuals world-wide. The clinical outcome of an infection by this parasite depends to some extent on the genetic makeup of the infected individual. The role of genetic factors that regulate the severity of malaria infection has been repeatedly demonstrated in humans and animals. Association studies are conducted with the aim of identifying the causal genes implicated in the outcome of infection. Linkage was previously detected on human chromosome 5p15 controlling the number of Plasmodium falciparum attacks (PFA) in Dielmo, a Senegalese village [48]. Subsequently, and prior to this present study, a fine mapping study using a "GoldenGate assay” from Illumina, with about 1450 SNPs was performed in this region of linkage with PFA phenotype. Analysis was performed with three statistical family-based programs: Merlin, QTDT, and FBAT/PBAT. These programs identified three candidate genes associated with PFA phenotype: three SNPs (rs4867417, rs7714218, and rs11959398) located in PDZD2, one SNP (rs11134099) in ADAMTS16, and one (rs3777320) in SEMA5A. The aim of this present study was to investigate these associations. Novel SNPs in the candidate regions of these genes were selected either by sequencing exons located in these candidate regions or by bioinformatics analysis using HapMap data from Yoruba population. SNPs were studied using either Pre-design or Custom SNP genotyping assay (Applied Biosystems). Data were included in an Access Database and checked for error of Mendelian transmission. Statistical analyses were performed using two family-based association programs, PBAT and QTDT. We used different models of allele transmission and defined p=10-3 as significance threshold. The analyses did not confirm the association with SNPs of PDZD2 or ADAMTS16, but did find significant association with SNPs of SEMA5A. One SNP (rs3777325) was significantly associated with PFA phenotype using both programs (p-value= -6.49x10-4 using the PBAT program and p-value=2.0x10-4 using the QTDT program). A haplotype analysis of two adjacent SNPs (rs4541632 and rs1018956) also showed a significant association of the haplotype GC (p-value= -6.82x10-5) using the PBAT program. This work confirms that a susceptibility locus to PFA phenotype is located inside SEMA5A. Further studies will be necessary to replicate this association and identify the causal polymorphism