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Rosetting is an adhesion property of malaria parasites whereby infected erythrocytes bind to two or more uninfected erythrocytes, forming a so-called rosette. Rosetting of Plasmodium falciparum is associated with disease severity and high parasitaemia in sub-Saharan Africa, although currently the function of rosetting remains unknown. An early IFNg response elicited from the innate immune system is associated with resolution of malaria infection in mice. Published data suggests that optimal IFNg production may require contact between peripheral blood mononuclear cells and P. falciparum infected erythrocytes. The first part of this thesis investigates the hypothesis that rosetting is an immune evasion strategy to hide infected erythrocytes from detection by innate immune cells. Across five laboratory strains of P. falciparum rosetting was not associated with differential IFNg production when parasites were grown in group O blood. Reappraisal of the data with respect to blood group for one strain found that rosetting significantly reduced the IFNg response to parasites grown in group A blood (P=0.022, Wilcoxon signed-rank test), where it is known that rosettes are bigger and stronger. This is consistent with the hypothesis that rosetting is an immune evasion strategy and the first study to find evidence for a function of rosetting. Further work is needed in order to generalise this finding. The cytokine response to P. falciparum varies between people and this variation may be indicative of disease progression. In mice infected with malaria it is also apparent that parasite strain can determine the cytokine response of the host. It is unclear whether P. falciparum strains vary in their ability to induce cytokines. The second part of this thesis investigates variation in cytokine induction between P. falciparum strains. Across four laboratory strains of P. falciparum, IFNg production was significantly dependent on parasite strain (F3,178= 48.49, P<0.001). Production of GM-CSF, IL-1b, IL-6, IL-10 and TNFa significantly correlated with production of IFNg (P<0.001, Pearson correlation) and followed the same strain-dependent pattern. The ratio of pro-inflammatory cytokines to IL-10 was also dependent on parasite strain. These data provide strong evidence for P. falciparum strain-dependent cytokine responses which may be an important determinant of disease outcome. Phagocytosis by splenic macrophages is proposed to be the principle mechanism of parasitaemia control in malaria infection. CD36 mediated phagocytosis may by an important mechanism of non-opsonic parasite clearance. The final part of this thesis investigates the hypothesis that rosetting is an immune evasion strategy of P. falciparum in order to evade phagocytic clearance, in particular that mediated by CD36. Overall the data obtained were inconsistent. Phagocytosis was significantly reduced in rosetting versus non-rosetting parasites in some strains (e.g. R29; P=0.048, paired T test), whereas others showed no effect (e.g. Muz12; P=0.228, paired T test) or increased versus non-rosetting parasites (e.g. HB3, P=0.004, paired T test). The relationship between CD36 binding and phagocytosis was also unclear, and anti-CD36 antibody did not effectively block phagocytosis, suggesting the involvement of alternative mechanisms. Further experiments are needed to clarify these observations. Data presented in this thesis are suggestive that rosetting in non-group O blood may be an immune evasion strategy with regard to IFNg production by innate immune cells, mechanistically linking rosetting with enhanced parasitaemia and disease severity. Furthermore, parasite strain significantly affects cytokine production and may be a determinant of disease outcome. This thesis demonstrates the importance of continued research into the effect of parasite virulence on the immune response, with particular emphasis on rosetting.

Drug resistance is one of the most medically relevant forms of pathogen evolution. To date, vaccines have not failed with the same depressing regularity as drugs. Does that then make vaccines evolution-proof? In the face of vaccination, pathogens are thought to evolve in two ways: by evolving epitope changes at the antigenic target of vaccination (epitope evolution); or by evolving changes at other antigenic loci, some of which may involve virulence (virulence evolution). The fundamental difference between these two forms of evolution is that virulence evolution could lead to disease outcomes in unvaccinated people that are more severe than would have been seen prior to evolution. One of the theoretical assumptions of virulence evolution is that more virulent parasites will have a selective advantage over less virulent parasites in an immunized host, and are thus more likely to be transmitted. The assumption is that more virulent parasites may be competitively more superior in mixed infections, or may be better able to evade/modulate the host immune response. Thus, the aim of this thesis was to experimentally test whether more virulent parasites have a within-host selective advantage in an immunized host or whether vaccine efficacy is more likely to depend on genetic differences at the targeted sites of vaccination. I used clones (genotypes) of the rodent malaria Plasmodium chabaudi originally derived from wild-caught Thicket (Thamnomys rutilans) rats to infect laboratory mice and a rodent analogue of the candidate blood-stage malaria vaccine apical membrane antigen 1 (AMA-1). I found that within-host selection did not depend on parasite virulence, and that protective efficacy depended on genotype-specific differences at the vaccine target. Vaccine-induced protection was not enhanced by including a number of allelic variants. However, such genotype-specific responses were only observed when the vaccine was tested against genetically distinct P. chabaudi parasites. When one P. chabaudi genotype was serially passaged through naïve mice the derived line was more virulent and was subsequently less well controlled by vaccine-induced immunity. In other experiments I found within host competition not to be immune-mediated. Thus my results suggest that vaccination has the potential to select for more virulent parasites but that the selective advantage is likely to be independent of competition. The selective advantage may be attributable to the enhanced immune evasion of more virulent parasites. However, without genetic markers of virulence, the mechanisms that mediate this selection remain unknown. My thesis contributes towards a growing body of evidence that vaccines have the potential to differently alter the within-host parasite dynamics of particular pathogen genotypes and that the selection imposed is likely to be system specific, depending on the fine specificity of the vaccine-induced responses and the identity of infecting parasites. Although vaccine potency may not be enhanced by including more than one allelic variant of an antigen, multi-valent vaccines may be one of the best ways to avoid the inadvertent selection for more virulent malaria parasites.

Rodent malaria parasites (RMPs) serve as tractable models for experimental genetics, and as valuable tools to study malaria parasite biology and host-parasitevector interactions. Plasmodium vinckei, one of four RMPs adapted to laboratory mice, is the most geographically widespread species and displays considerable phenotypic and genotypic diversity amongst its subspecies and strains. The phenotypes and genotypes of P. vinckei isolates have been relatively less characterized compared to other RMPs, hampering its use as an experimental model for malaria. Here, we have studied the phenotypes and sequenced the genomes and transcriptomes of ten P. vinckei isolates including representatives of all five subspecies, all of which were collected from wild thicket rats (Thamnomys rutilans) in sub-Saharan Central Africa between the late 1940s and mid 1960s. We have generated a comprehensive resource for P. vinckei comprising of five high-quality reference genomes, growth profiles and genotypes of P. vinckei isolates, and expression profiles of genes across the intra-erythrocytic developmental stages of the parasite. We observe significant phenotypic and genotypic diversity among P. vinckei isolates, making them particularly suitable for classical genetics and genomics-driven studies on malaria parasite biology. As part of a proof of concept study, we have shown that experimental genetic crosses can be performed between P. vinckei parasites to potentially identify genotype-phenotype relationships. We have also shown that they are amenable to genetic manipulation in the laboratory.

Biological rhythms are thought to have evolved to enable organisms to organise their activities according to the Earth’s predictable cycles, but quantifying the fitness advantages of rhythms is challenging and data revealing their costs and benefits are scarce. More difficult still is explaining why parasites that exclusively live within the bodies of other organisms have biological rhythms. Rhythms exist in the development and traits of parasites, in host immune responses, and in disease susceptibility. This raises the possibility that timing matters for how hosts and parasites interact and, consequently, for the severity and transmission of diseases. Despite their obvious importance in other fields, circadian rhythms are a neglected aspect of ecology and evolutionary biology. The ambitions of this thesis are to integrate chronobiology, parasitology and evolutionary theory with mathematical models to obtain a greater understanding about how and suggest why malaria parasites have rhythms as well as the effect of infection on host rhythms. First, I identify how malaria parasites lose their developmental rhythms in culture, when they lack any potential time cues from the host. Next, I characterise parasite rhythms inside the mammalian host in terms of synchrony and timing and demonstrate there is genotype by environment interactions for characteristics of parasite rhythms. Then, I investigate the effect that parasite infection has on host rhythms and show there is variation between parasite genotypes in their effect on host locomotor activity and body temperature rhythms during infections. Finally, I explore which host rhythms may be driving parasite synchrony and timing and demonstrate the importance of peripheral host rhythms for the timing of malaria parasite developmental rhythms. The data presented here provides novel and important information on the role of rhythms during disease and opens up a new arena for studying host-parasite coevolution.

Adaptive phenotypic plasticity, the ability of a genotype to give rise to different phenotypes in different environments, evolves to allow organisms to fine-tune their life-history traits according to the varying conditions they encounter during their lives. Reproductive investment - the manner in which organisms divide their resources between survival and reproduction - is well studied in evolutionary ecology because it is a key determinant of fitness. However, whilst plasticity in reproductive effort is well understood for free-living multicellular taxa (such as insects, birds, and mammals), the application of evolutionary theory for plasticity and life history strategies to unicellular parasites and pathogens is lacking. In this thesis, I use empirical and theoretical approaches to uncover how differential resource allocation to non-replicating, sexual stages (gametocytes) versus asexually replicating stages can be harnessed by the rodent malaria parasite Plasmodium chabaudi to maximise its fitness across the often very variable conditions it encounters during infections. Differential allocation between those stages is equivalent to the fundamental life-history trade-off between survival and reproduction because gametocytes are responsible for between-host transmission (i.e. reproduction of the infection) whereas asexual parasites mediate host exploitation and within-host survival. A suite of within-host models reveal that malaria parasites could gain considerable fitness benefits in the face of low levels of drug treatment if they reduce their investment into gametocyte production ("reproductive restraint"), thereby assuring the continuity of the infection and capitalising on opportunities for future transmission. In contrast, high levels of drug treatment typically select parasites to commit all of their resources to gametocyte production ("terminal investment"), to escape a host that does not offer much opportunity for future transmission. My experiments reveal that P. chabaudi increases both its reproductive investment and its asexual replication rate in anaemic hosts (i.e. host that have a low density of red blood cells), suggesting that parasites profit from host anaemia and can afford high investment in gametocytes ("affluent investment"). I also uncover plasticity in a number of traits that underpin asexual replication rate, including invasion preference for different ages of red blood cells, but it is plasticity in the number of progeny (merozoites) per infected cell that is the main contributor to asexual replication rate. My experiments also reveal genetic variance in plasticity of the life-history traits investigated, which has profound implications for their evolution. Furthermore, plastic modification of these traits is associated with minimal costs or constraints, so that parasites can rapidly match life-history traits appropriately to the within-host environment. Severe anaemia is one of the deadliest symptoms of malaria, so observing that virulence and infectiousness increases in anaemic hosts has also fundamental clinical implications. Finally, the empirical and theoretical observations of affluent investment, reproductive restraint and terminal investment match theoretical predictions of how organisms should behave in varying environments, confirming P. chabaudi as a useful model system to test life-history theory.

This thesis entitled “Mechanism of Anticancer and Antimalarial Action of a Modulator of Heat Shock Proteins” describes the successful elucidation of the mechanism of anticancer and antimalarial action of 15-Deoxyspergualin (DSG). DSG, a relatively well known immunosuppressant and antitumor molecule has been demonstrated to kill the malaria parasite in vitro and in vivo (Midorikawa et al., 1997; Midorikawa et al., 1998). A highly polar molecule, DSG binds the carboxy terminal “EEVD” motif of heat shock proteins, Hsp70 and Hsp90, enhances the ATPase activity of Hsp70 (Nadler et al., 1992; Nadler et al., 1998), and modulates several seemingly unrelated cellular processes. DSG has also been demonstrated to inhibit protein synthesis and polyamine synthesis in cells (Kawada et al., 2002; Hibasami et al., 1991), and previously speculated to inhibit malaria parasite growth by inhibiting polyamine synthesis. The grim situation with regard to malaria infection and mortality, principally an offshoot of the emergence of chloroquine resistant strains of the causative agent of malaria - Plasmodium falciparum, calls for intense efforts towards developing efficacious antimalarial agents with few side effects. DSG, having been used already in graft rejection cases in man and demonstrated to potently inhibit malaria in mice (Midorikawa et al., 1997), offers promise in this regard. It was, therefore, of interest to solve the mystery of its mechanism of antimalarial action. Chapter 1 surveys literature related to DSG mechanism of action and presents the thesis objective. Chapter 1 also gives an overview of heat shock proteins and their role in cancer, and the biology of the malaria parasite (Plasmodium falciparum), the working of the principal metabolic pathways existing in it, and a description of processes related to the intriguing, relict plastid present in apicomplexans. The metabolic processes previously speculated to be targeted by DSG, and those later found to be involved in DSG mechanism of action – polyamine synthesis and transport, protein synthesis and apicoplast processes are dealt with in more detail. Though DSG has been speculated to kill the malaria parasite by inhibiting polyamine synthesis, that DSG could clear malaria infection in Plasmodium berghei infected mice did not corroborate with the observation that inhibitors of polyamine biosynthesis are incapable of inhibiting the malaria parasite in vivo probably because the parasites make do with polyamines salvaged from the host (Assaraf et al., 1984; Bitonti et al., 1987). On the other hand, DSG is known to bind heat shock proteins, and inhibit protein synthesis, and heat shock proteins are speculated to be involved in the activation of HRI (heme regulated inhibitor), a type of eIF2á kinase that phosphorylates the eukaryotic initiation factor, eIF2á in conditions of heme deficiency or other cellular stress. eIF2á phosphorylation leads to stalling of protein synthesis. It seemed likely that if HRI is activated upon sequestration of heat shock proteins by DSG, it would culminate in protein synthesis inhibition and ultimately, cell death. With the intention to investigate this line of thought, the PlasmodB database was mined for proteins essential to the existence of heme dependent protein synthesis in Plasmodium falciparum. Two Hsp70 proteins from Plasmodium falciparum, one with the carboxy terminal “EEVD” motif implicated in DSG binding, and one without, and an Hsp70 interacting protein were cloned and expressed in their recombinant form in Escherichia coli. The preliminary characterization of these heat shock proteins described in Chapter 2 revealed that they were functionally active. DSG did not inhibit either the chaperone activity of the Hsp70s or the interaction of Hsp70 with Hip, but stimulated their ATPase activity as anticipated. Chapter 3 gives a complete picture of the mechanism of protein synthesis inhibition by DSG in the standard protein synthesis system – reticulocyte lysate. The experiments carried out revealed that DSG inhibits protein synthesis precisely through the mechanism envisaged, i.e. through phosphorylation of HRI following sequestration of Hsp70. Experiments involving exogenous addition of heat shock protein to in vitro translation reactions confirmed this hypothesis. Moreover, DSG inhibited protein synthesis in cancer cells in vivo, too, and HRI knockdown cells were not affected by DSG. Interestingly, the Hsp70 levels in various cancer cell lines inversely correlated with the inhibitory activity of DSG, and modulation of Hsp70 levels through standard methods altered DSG inhibition of protein synthesis in these cells. It was thus confirmed that DSG did indeed inhibit mammalian cells through the pathway envisaged. Its previously reported antitumor property is probably through this outlined mechanism of interference with protein regulation. In the malaria parasite, too, DSG inhibited protein synthesis through eIF2 alpha phosphorylation following Hsp70 sequestration as outlined in Chapter 4. However, while the concentration of DSG required for inhibition of malaria parasite growth was in the nanomolar range, high micromolar concentrations of DSG were required to effect protein synthesis inhibition in the malaria parasite, indicating that yet another target for DSG existed in the malaria parasite. With protein synthesis no longer a candidate target of DSG, I looked into the previously implicated polyamine synthesis pathway. In the event of DSG inhibiting polyamine transport in addition to polyamine biosynthesis, it would be expected to clear malaria infection in vivo contrary to other inhibitors of polyamine biosynthesis. In Chapter 5, evidence for the polyamine synthesis pathway in the malaria parasite is provided. Experiments involving incorporation of radiolabeled precursors in the malaria parasite and in mammalian cells, however, revealed that only high micromolar concentrations of DSG inhibit polyamine synthesis. Polyamine transport was also studied in considerable detail in malaria parasite infected red blood cells. Though infected red blood cells demonstrated different kinetic parameters, implying that new polyamine transporters were employed by the parasite on the red blood cell upon infection, DSG did not potently inhibit polyamine transport, either. The mystery of the target of DSG in the malaria parasite was, however, close to solution, when the growth inhibition of the malaria parasite by DSG was studied carefully. DSG invoked “delayed death” – a phenomenon wherein death is invoked only one cycle after incubation with the inhibitor. “Delayed death” is typical of inhibitors that target apicoplast processes (Fichera and Roos, 1997). DSG did not inhibit either fatty acid synthesis or prokaryotic protein synthesis – processes that occur in the apicoplast, but effected a decrease in the amount of nucleus encoded proteins that are targeted to the apicoplast, suggesting that it inhibited the trafficking of nucleus encoded proteins to the apicoplast. Confocal microscopy of parasites transfected with GFP fusion protein confirmed these findings, and is described in Chapter 6. The thesis ends with a summary of the findings in Chapter 7. Apicoplast processes have always been considered to harbor immense potential in the development of antimalarial agents, thanks to the absence of an equivalent organelle and hence pathways, in the human host. Trafficking of nucleus encoded proteins to the apicoplast has remained unexplored however. The work done in this thesis not only serves to demystify DSG with regard to its mechanism of action, but also paves the way for further studies in this area of intracellular trafficking, which could help in the development of more efficacious antimalarial agents. It also adds a new dimension to previous work conducted with regard to the anticancer action of DSG. Appendix 1 revolves around inhibitors which target various apicoplast processes. Apicoplast processes have been conventionally linked to the intriguing but unfortunate (with respect to clinical application) “delayed death”. Results presented in this section demonstrate that not all apicoplast processes invoke “delayed death”. Inhibition of apicoplast processes such as fatty acid biosynthesis and heme synthesis evoke rapid death. Inhibitors designed to target these processes could, therefore, be highly efficacious.