School of BioSciences - Theses
Permanent URI for this collection
Search Results
1 - 7 of 7
-
ItemNo Preview AvailableExploring the roles of bromodomain protein 2 (PfBDP2) and acetylated histone variant PfH2A.Z and PfH2B.Z in Plasmodium falciparum chromatin biology( 2022)The eukaryotic Plasmodium falciparum parasite employs multiple levels of gene regulation to alter its morphology throughout its complex life cycle to survive different environments it encounters in mosquitoes and humans. Thus, epigenetic mechanisms that control gene regulation are integral to the parasite’s survival. Histone lysine acetylation is the post-translational modification of histones, generally associated with eukaryotic transcriptional activation. This epigenetic modification is recognised by bromodomain, and bromodomain-containing proteins (BDPs) can recruit transcription factors to promote gene expression. In the P. falciparum genome, eight bromodomain-containing proteins were identified but only two have been functionally characterised. This thesis detailed the works done on the uncharacterised P. falciparum bromodomain protein 2 (PfBDP2). I explored cellular and epigenomic localisations of PfBDP2 in the asexual schizont-stage P. falciparum parasites via biochemical assays and native chromatin immunoprecipitation, which revealed that PfBDP2 is a nuclear protein expressed throughout the asexual cycle and is enriched within the 5' intergenic region of invasion genes in schizonts. PfBDP2 is also enriched within heterochromatin, particularly across the promoters of silent multigene families and across var gene introns. This points to PfBDP2 being a chromatin protein with dual epigenetic roles, associated with both gene expression and silencing. This thesis also reports a growth delay upon the conditional disruption of PfBDP2 expression in vitro, indicating its importance for normal growth during the asexual intraerythrocytic cycle. The inability to recover stable PfBDP2 knockouts following knockout induction suggests that PfBDP2 may be essential to blood-stage growth and could be druggable by novel antimalarials. Finally, this thesis also detailed the epigenomic localisation of two unique Apicomplexan histone variants, PfH2A.Z and PfH2B.Z, and their acetylated cognates, PfH2A.Zac and PfH2B.Zac, previously shown to be the binding targets of PfBDP2. PfSir2A is a histone deacetylase shown to anti-correlate with PfH2A.Z deposition at heterochromatin in the P. falciparum genome, fulfilling a critical role in the maintenance of chromatin structure. Using crosslinked chromatin immunoprecipitation, the genome-wide localisation of the histone variants was mapped in both wild-type and PfSir2A-KO parasites. There was an enrichment of both PfH2A.Zac and PfH2B.Zac across all euchromatic intergenic regions of P. falciparum genome in the wild-type parasites. This pattern was similar in PfSir2AKO parasites but differed in the aberrant, increased enrichment of the acetylated histone variants within heterochromatin, signalling a breakdown of heterochromatin-euchromatin boundaries. This indicates an important structural function of the two histone variants in maintaining chromatin boundaries in P. falciparum. Additionally, PfH2A.Zac and PfH2B.Zac appeared to be enriched at specific loci around var genes, most importantly var introns, suggesting a role in var gene regulation and lending further evidence to the acetylated histone variants being in vivo binding targets of PfBDP2.
-
ItemEvolution of drug-resistance genes in the asymptomatic Plasmodium falciparum reservoir of infection in Ghana( 2019)Ghana is one of the 11 countries in the world with the highest malaria burden. Like many other African countries, the majority of individuals of all ages harbour asymptomatic Plasmodium falciparum infections, which sustain malaria transmission. Yet these infections are largely undiagnosed and untreated. Chloroquine (CQ) was the main drug for treating clinical malaria in Africa until it was replaced with artemisinin-based combination therapies (ACTs) in the early 2000s due to treatment failures. At the same time, sulphadoxine-pyrimethamine (SP) was adopted for intermittent preventative treatment in pregnancy (IPTp). In order to inform future malaria control strategies in Ghana, I investigated the asymptomatic P. falciparum reservoir in Bongo District (BD), where malaria transmission is both high and seasonal. To evaluate the reservoir of asymptomatic P. falciparum infections including antimalarial drug-resistance markers in BD, a cross-sectional Pilot survey of ~700 participants (≥ 1 year) was undertaken at the end of the dry season in June 2012. Following the completion of this Pilot investigation a larger serial cross-sectional study (~2,000 participants) involving six seasonally timed surveys was completed between 2012 and 2016. This study was designed to evaluate the impact of indoor residual spraying with insecticides (IRS) on the prevalence and diversity of asymptomatic P. falciparum infections in BD before, during, and after the IRS intervention. At the end of the dry season in 2012 I showed that 38.3% of the population across all ages (1-85 year) carried asymptomatic P. falciparum infections. The majority (>70%) of these infections harboured CQ sensitive alleles (Pfcrt K76 and Pfmdr1 N86) and/or alleles associated with reduced response to SP (Pfdhfr I51R59N108/Pfdhps G437) and/or the ACT partner-drug, lumefantrine (Pfmdr1 N86F184). There was no evidence of selection of multilocus haplotypes (i.e. Pfcrt- Pfmdr1- Pfdhfr- Pfdhps) with predicted resistance to both CQ and SP, nor was there any evidence of artemisinin resistance based on Pfk13 genotyping. To further understand this rebound of CQ sensitivity in BD further analyses of the microsatellite loci flanking Pfcrt and Pfmdr1 indicated that the CQ sensitive alleles spread through the asymptomatic parasite reservoir via soft selective sweeps. They may have expanded from CQ sensitive lineages that survived CQ drug pressure, i.e. before Ghana switched to ACTs. Following the completion of the 3-rounds of IRS in BD, undertaken between 2013 and 2014, both the prevalence and multiplicity of asymptomatic P. falciparum infections among children (1-10 years) reduced significantly compared to the pre-IRS surveys. Interestingly, despite these reductions, parasite diversity as assessed by msp2 heterozygosity remained high and stable from the pre-IRS through to the post-IRS surveys. My findings suggest that the asymptomatic P. falciparum reservoir in BD poses a threat to malaria elimination and plays a role in the evolution of antimalarial resistance in Ghana. Therefore, strategies combining IRS with population-wide antimalarial treatments, potentially using ACTs with CQ, would have to be deployed and sustained in BD. Nonetheless, continuous monitoring of the molecular markers of resistance and for changes in the parasite diversity will be crucial to inform elimination strategies in Ghana and Africa.
-
ItemGenetic epidemiology of the Plasmodium falciparum reservoir of infection in Bongo District, Ghana( 2018)Malaria remains a major global health problem, with Plasmodium falciparum being the causative agent of the majority of malaria-related deaths and clinical cases in humans. This burden is largely concentrated in West and Central Africa where >90% of cases and nearly all deaths occur. In these areas, a large parasite reservoir of infection exists in asymptomatic carriers posing a considerable challenge to malaria surveillance and control efforts. In areas of seasonal malaria transmission, infections that comprise this reservoir persist throughout the dry season, when transmission declines. Subsequently, these infections seed transmission during the next wet season when the mosquito population emerges. Thus, in recent years there has been an emphasis on describing, quantifying and examining the asymptomatic reservoir through epidemiological surveys, however our understanding is still limited. This thesis provides a comprehensive description of the genetic epidemiology of the asymptomatic P. falciparum reservoir of infection across all ages in Bongo District, Ghana, an area of seasonal transmission in West Africa. In Chapter 3, we examined the extent of genetic diversity by characterizing the population genetics of neutral microsatellite markers in the P. falciparum reservoir of infection among residents of all ages from Bongo. Significant linkage disequilibrium (LD) among neutral markers in the parasite genome occurred in the presence of high levels of genetic diversity and multiclonal infections in the population (Chapter 3, published manuscript). There was no association between the LD and geographical separation of parasite populations, or with linkage occurring in antigen-encoding var genes, suggesting it may be a result of past/current antimalarial drug usage in BD that has led to selection at genes conferring drug resistance. In Chapter 4, we aimed to define the transmission dynamics of P. falciparum through the lens of var genes, encoding the major variant surface antigen of the malaria blood stages. We investigated the extent of var sequence diversity, seasonal and age-specific patterns of diversity, and temporal var dynamics by examining individuals across all ages harbouring asymptomatic microscopic and, for the first time, submicroscopic P. falciparum infections across two sequential transmission seasons. Over 42,300 var types were found to be circulating in the population, and this extensive diversity was uniquely structured into var repertoires that had minimal overlap regardless of season and infection complexity. Through the lens of var genes, we explain several key epidemiological features of P. falciparum malaria in endemic areas and demonstrate that the transmission system in BD is extremely complex. Despite this complexity, three key features of the molecular epidemiology emerge: (i) extremely high var sequence diversity, (ii) limited overlap of var repertoires in both seasons, and (iii) rapid turnover of repertoires but maintenance of certain var types between seasons. Using computational experiments, this highly diverse parasite population with essentially non-overlapping var repertoires was shown to explain age-specific patterns of immunity. Overall the data provide evidence to revise the epidemiological surveillance of malaria to consider parasite antigenic variation. This study is the first to describe the seasonal trends and temporal var transmission dynamics of P. falciparum. In conclusion, this PhD thesis provides a comprehensive description of the genetic epidemiology of the asymptomatic P. falciparum reservoir of infection across all ages. It has provided novel insights into the complexity of the P. falciparum transmission system under conditions of seasonal transmission through the lens of both neutral genetic markers and the genes encoding the major variant surface antigen of P. falciparum. The findings presented herein advance our current knowledge on the epidemiology of P. falciparum, the deadliest malaria-causing parasite, which ultimately challenge the paradigm of malaria surveillance and control.
-
ItemDrug targets in the apicoplast of malaria parasites( 2017)Human malaria is caused by six species of Plasmodium, and despite considerable effort, it remains as one of the deadliest of infectious diseases. African children under the age of five are the most vulnerable population. Drugs are our primary means of malaria control, but parasite drug resistance constantly erodes their efficacy. Thus, the search for new drugs and novel drug targets continues to be a high priority. Plasmodium has a plant-like plastid organelle known as the apicoplast. The apicoplast is no longer photosynthetic, but like plant plastids, it is derived from endosymbiotic bacteria and performs crucial metabolic functions. Apicoplast housekeeping and metabolic pathways are similar to those of prokaryotic bacteria, which offers distinct selectivity for inhibition and hence killing of the parasite. Knowledge of the apicoplast contents and functions are mostly derived from bioinformatics analysis of Plasmodium genome data. Many antibiotics, presumed to target the apicoplast, can kill the parasite, and several are already in use to treat malarial patients or in clinical trials but their actual targets have not yet been verified. In Chapter 2, I attempted to isolate the apicoplast (using flow cytometry from a parasite line in which the apicoplast is green fluorescent tagged) and define the apicoplast proteome using mass spectrometry. In total, I identified 732 proteins from isolated sub-cellular structures, which included some known and/or predicted apicoplast proteins but also many non-apicoplast proteins. I also found numerous mitochondrial proteins in my apicoplast fraction and confirmed that mitochondrial DNA was present in the sorted material. Mitochondria and apicoplasts are physically linked and exchange metabolites in Plasmodium, and this attachment appeared to survive the flow cytometry sorting, bringing mitochondria along with the apicoplasts. Although the apicoplast isolation and mass spectrometry protocol showed some promise, more work will be required to generate a stringent and robust apicoplast proteome. In Chapter 3, I used isopentenyl pyrophosphate (IPP) supplementation in conjunction with a battery of apicoplast viability assays to validate whether or not 22 presumed apicoplast targeting drugs do indeed have their primary target in the apicoplast. I confirmed a primary apicoplast target for nine antibiotics, all of which cause so-called delayed death drugs whereby they kill parasites at least 10 times more efficiently during the 2nd life cycle of drug application. These nine antibiotics appear to impact apicoplast housekeeping machineries on the basis of apicoplast degradation during drug treatment. IPP supplementation also rescued parasites from two IPP biosynthesis pathway inhibitors, but these drugs did not result in apicoplasts degradation. Moreover, these drugs caused immediate death making them potentially better suited to therapy for severe malaria, whereas the delayed death drugs would appear more suited to prophylaxis or use as partners to fast acting drugs. IPP supplementation assays confirmed that apicoplast fatty acid biosynthesis and photosynthesis are not valid drug targets in the red blood cell phase of the parasite. One drug, actinonin (which in bacteria abrogates a post-translational modification process), emerged as unique among those having primary targets in the apicoplast. Actinonin caused immediate death and apicoplast degradation. This unique combination suggested that it has a deadly impact on apicoplast biogenesis but does not target the housekeeping pathways perturbed by the nine delayed death antibiotics, which likely impact DNA replication and protein translation. In Chapter 4, I identified the likely target of actinonin through selection of drug resistance and genotyping. Taking my lead from published work on the related parasite Toxoplasma gondii showing that a putative apicoplast protease, TgFtsH1, is the target of actinonin, I was able to show by direct sequencing of select genes that actinonin likely targets an orthologue, PfFtsH1, on the basis of a point mutation in this gene. My genotyping also indicated that actinonin likely does not target post-translational modification of apicoplast synthesised proteins since these genes remained wild-type in the resistant line. My data provide the best evidence yet that actinonin targets PfFtsH1, and make localisation and determination of its role in the apicoplast a high priority to better understand how this unique drug lead works. In Chapter 5, I investigated the activity of verapamil, a drug used in human medicine but also known to be antimalarial. I showed that verapamil causes delayed death, killing parasites at drastically lower concentrations in the 2nd red blood cell cycle. Based on the strong pattern of delayed death observed for nine delayed death antibiotics characterised in Chapter 3 on apicoplast housekeeping, I hypothesised that verapamil would also inhibit apicoplast housekeeping. However, IPP supplementation assays showed that verapamil death cannot be rescued with IPP supplementation and does not detectably perturb the apicoplast, which refuted my initial hypothesis. I selected for verapamil resistance in P. falciparum and genotyped the resistant parasites but was not able to identify any mutation(s) definitely associated with resistance. This data leads to a new working hypothesis that verapamil has a target outside the apicoplast, perhaps impacting the cytosolic utilisation of apicoplast synthesised IPP to cause delayed death. In Chapter 6, I present a recap of my findings and some directions as to where I think this work could eventually lead and what types of investigations should be the highest priority in pursuing apicoplast drug targets to further combat malaria.
-
ItemDefining the roles of essential genes in the malaria parasite life cycle( 2017)The combination of drug resistance, lack of an effective vaccine and ongoing conflict and poverty mean that malaria remains a major global health crisis. Understanding metabolic pathways at all parasite life stages is important in prioritising and targeting novel anti-parasitic compounds. To overcome limitations of existing genetic tools to investigate all the parasite life stages, new approaches are vital. This project aimed to develop a novel genetic approach using post meiotic segregation to separate genes and bridge parasites through crucial life stages. The unusual heme synthesis pathway of the rodent malaria parasite, Plasmodium berghei, requires eight enzymes distributed across the mitochondrion, apicoplast and cytoplasm. Deletion of the ferrochelatase (FC) gene, the final enzyme in the pathway, confirms that heme synthesis is not essential in the red blood cell stages of the life cycle but is required to complete oocyst development in mosquitoes. The lethality of FC deletions in the mosquito stage makes it difficult to study the impact of these mutations in the subsequent liver stage. To overcome this, I combined locus-specific fluorophore expression with a genetic complementation approach to generate viable, heterozygous oocysts able to produce a mix of FC expressing and FC deficient sporozoites. In the liver stage, FC deficient parasites can be distinguished by fluorescence and phenotyped. Parasites lacking FC exhibited a severe growth defect from early to mid-stages of liver development in-vitro and could not infect naïve mice, confirming liver stage arrest. These results validate the heme pathway as a potential target for prophylactic drugs targeting liver stage parasites. Energy metabolism in malaria parasites varies remarkably over the parasite life cycle. Parasites depend solely on anaerobic glycolysis at blood stage but need Krebs cycle, the electron transport chain, and mitochondrial ATP synthase during mosquito stage development. Again, reverse genetic approaches to study the hepatic stage of Plasmodium have been thwarted because parasites with defects in energy pathways are unable to complete the mosquito stage. I used the genetic complementation approach established to study heme biosynthesis to bridge parasites lacking the β subunit of mitochondrial ATP synthase through mosquito stage and studied their development in the liver stage. ATPase knockouts were indistinguishable from wildtype in in-vitro liver stage assays of size, nuclear content, and merosome production. Robust progression to blood stage confirmed the dispensability of mitochondrial ATP synthesis in liver stages. I extended this approach to explore the essentiality of upstream mitochondrial electron transport and Krebs cycle during the liver stage. I speculate that energy metabolism in the liver stage resembles that in the blood stage, relying predominantly on glycolysis for ATP production. There are numerous genetic tools to manipulate the blood stage malaria parasite genome in general, but existing genetic tools to generate viable parasites with defects in blood stage essential genes are limited. To overcome this limitation, I have developed a novel strategy in which I first insert a complementary copy of the essential gene-of-interest, and then delete the endogenous gene, and then take advantage of meiosis and segregation during the mosquito stage to create haploid knockout sporozoites. I genotype the parasites along the way by fluorescence microscopy. As proof of principle, I created complemented knockouts of the blood stage essential 1-deoxy-D-xylulose-5- phosphate reductoisomerase (DXR) gene, crossed these with wildtype parasites, and then tracked the progeny through in-vitro and in-vivo liver development. Precomplementation proved difficult, perhaps due to inappropriate expression of important metabolic genes. Additionally, problems with apparent silencing of the fluorophore tags compromised my ability to genotype cross progeny preventing any firm conclusion on the function of isoprenoid precursor pathway of liver stage parasites. Nevertheless, my success in generating a blood stage essential gene knockout via precomplementation provides encouragement that this novel reverse genetic strategy can be implemented to investigate the role of blood stage-essential genes in sporozoite and liver stages of malaria parasites.
-
ItemAlternative splicing and stage differentiation in apicomplexan parasites( 2017)Alternative splicing is the phenomenon by which single genes code for multiple mRNA isoforms. This is common in metazoans, with alternative splicing observed in over 90% of human genes (Wang et al., 2008). However, the full extent of alternative splicing in apicomplexans has been previously under-reported. Here, I address this deficiency by transcriptomic analysis of two apicomplexan parasites: Toxoplasma gondii, which causes toxoplasmosis; and Plasmodium berghei, which is a murine model for human malaria. I identified apicomplexan homologues to SR (serine-arginine–rich) proteins, which are alternative-splicing factors in humans. I then localised a homologue, which I named TgSR3, to a subnuclear compartment in T. gondii. Conditional overexpression of TgSR3 was deleterious to growth. I detected perturbation of alternative splicing by qRT-PCR. Parasites were sequenced with RNA-seq, and 2000 genes were identified as constitutively alternatively spliced. Overexpression of TgSR3 perturbed alternative splicing in over 1000 genes. Previously, computational tools were poorly suited to compacted parasite genomes, making these analyses difficult. I alleviated this by writing a program, GeneGuillotine, which deconvolutes RNA-seq reads mapped to these genomes. I wrote another program, JunctionJuror, which estimates the amount of constitutive alternative splicing in single samples. Most alternative splicing in humans is tissue specific (Wang et al., 2008; Pan et al., 2008). However, unicellular parasites including Apicomplexa lack tissue. Nevertheless, I have shown that alternative splicing can still be common. I hypothesised that the tissue-specific alternative splicing of metazoans is analogous to stage-specific alternative splicing in unicellular organisms. I purified female and male gametocytes of P. berghei and sequenced these stages, with the aim of investigating alternative splicing and its relationship to stage differentiation. As a reference point, I first established the wild-type differences between female and male gametocytes. I detected a trend towards downregulation of transcripts in gametocytes compared to asexual erythrocytic stages, with this phenomenon more marked in female gametocytes. I was also able to identify many female- and male-specific genes, some previously-characterised, and some novel. My findings were further placed in an evolutionary context. Sex-specific genes were well conserved within the Plasmodium genus, but relatively poorly conserved outside this clade, suggesting that many Plasmodium sex-related genes evolved within this genus. This trend is least pronounced in male-specific genes, which suggests that sexual development of male gametocytes may have preferentially evolved from genes already present in organisms outside this genus. I then analysed these transcriptomes, now focusing on changes in alternative splicing. While non-gendered gametocyte differentiation is modulated by known transcription factors such as AP2-G (Sinha et al., 2014), I provide evidence that alternative splicing adds another level of regulation, which is required for differentiation into specific genders. I ablated a Plasmodium SR-protein homologue, which I named PbSR-MG. By transcriptomic analysis, I show that it regulates alternative splicing, predominantly in male gametocytes. Ablation was also associated with a drastic reduction in the viability of male gametocytes. Hence, I have shown that alternative splicing is common in apicomplexan parasites, is regulated by specific genes, and acts on specific targets. Alternative splicing is important for parasite viability and fundamental to stage differentiation in Plasmodium.
-
ItemInvestigating apicoplast membrane transporters of the malaria parasites Plasmodium berghei and P. falciparum( 2017)The malaria-causing parasite, genus Plasmodium, contains a unique non-photosynthetic plastid known as the apicoplast. The apicoplast is an essential organelle bound by four membranes. Although membrane transporters are attractive drug targets, only two transporters have been characterised in the apicoplast membranes. The aim of this thesis was to characterise membrane transporters of the apicoplast to ultimately identify novel drug targets to kill or perturb the malaria parasite. I selected 28 candidate genes and performed a genetic screen in P. berghei, which is amenable to medium throughput molecular genetics, to determine the blood stage essentiality and cellular localisation of these potential membrane transporters. My approach was successful, identifying eight blood stage essential genes in P. berghei, three of which are apicoplast-localised. I also found 20 blood stage dispensable P. berghei genes, four of which are apicoplast targeted. Interestingly, three apicoplast-localised candidates do not contain a canonical targeting signal, which led me to investigate novel apicoplast targeting mechanisms. Protein features required for the targeting of the only previously known leaderless apicoplast membrane transporter, PfoTPT, were dissected, and an N-terminal tyrosine was found to be necessary but not sufficient for apicoplast localisation. Similar conserved tyrosines were observed in all three novel leaderless apicoplast putative membrane transporters identified in this thesis, suggesting that this residue could be an important component of leaderless apicoplast targeting. Genes essential at the blood stage are potentially good drug targets because they are required in the medically important stage of malaria and perturbation can be assumed to kill the parasite. To further characterise my shortlisted essential apicoplast membrane transporter candidates, I switched my study organism to P. falciparum, which offers the best systems for the inducible knockdown of essential apicoplast genes. I created inducible ribozyme-mediated knockdown parasite lines for two P. falciparum putative membrane transporters shown to be essential in P. berghei, one of which produced ambiguous results. The advantage of doing inducible apicoplast knockdowns in P. falciparum cultures is that the loss of apicoplast-produced isopentenyl diphosphate (IPP) can be supplemented by adding IPP to the culture medium, thereby enabling lethal knockdowns to be maintained. I found that knockdown of PfDMT2, an essential apicoplast putative membrane transporter in P. berghei, was only viable when parasites were supplemented with IPP. Knockdown of PfDMT2 resulted in complete loss of the apicoplast, and these apicoplast-minus parasites were reliant on exogenous IPP to survive. PfDMT2 is therefore a crucial apicoplast membrane transporter and an excellent candidate for therapeutic intervention. Five novel apicoplast putative membrane transporters were identified, three of which are non-canonically targeted to the organelle. Additionally, two novel apicoplast putative membrane proteins were identified. This work has significantly contributed to the apicoplast transportome and will provide a platform for future studies to better understand apicoplast biology and whether it can be exploited to kill or perturb the malaria parasite.