Medical Biology - Theses
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ItemComplement evasion mechanisms of the deadly human pathogen Plasmodium falciparum( 2016)The human complement system is a front-line defence system against invading pathogens. It has over 50 different protein components that are involved either in pathogen clearance or in the regulation of complement. The two main mechanisms of clearance are direct membrane lysis or opsonisation leading to enhanced phagocytosis. Despite the presence of this potent system, many pathogens thrive in human serum due to the evolution of complement evasion strategies. One common evasion strategy involves pathogens recruiting host regulators of complement activation to down- regulate complement attack on their surfaces. Merozoites, the invasive stage of malaria parasites are exposed to serum after egress from the host red blood cell. In this thesis, we examined whether merozoites recruit human regulators of complement activation to their surface to evade complement-mediated destruction. We found that merozoites recruit the human regulators Factor H, Factor H-like 1 and C1 esterase inhibitor to their surface. Factor H and Factor H-like 1 are recruited by an interaction between the merozoite surface protein Pf92, a member of the six cysteine family of merozoite surface proteins, and the complement control protein modules 5-6 of the Factor H and Factor H-like 1 proteins. When bound to the merozoite surface, Factor H and Factor H- like 1 retain cofactor activity, a key function that allows them to down-regulate the alternative pathway of complement activation. Deletion of the Pf92 gene resulted in a loss of Factor H and Factor H-like 1 recruitment and an increased susceptibility of merozoites to immune destruction. We also showed that C1 esterase inhibitor is recruited to the merozoite surface by an interaction between PfMSP3.1, a member of the MSP3 family of merozoite surface proteins, and the C1 esterase inhibitor serpin domain. Bound C1 esterase inhibitor retained the ability to complex with complement activating proteases C1s, MASP1 and MASP2, allowing it to down-regulate both the classical and lectin pathways of complement on the merozoite surface. Deletion of the PfMSP3.1 gene led to a loss of C1 esterase inhibitor recruitment and an increase in complement deposition on merozoites. However, this resulted in enhanced merozoite invasion in the presence of active complement rather than merozoite destruction. Overall, the ability of merozoites to sequester host complement regulators has important implications for the immune evasion strategy of malaria parasites amid a growing body of evidence for an important role of complement in protection.
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ItemThe molecular epidemiology of malaria in Solomon Islands( 2016)Historically, Solomon Islands in the Southwest Pacific has endured considerable P. falciparum and P. vivax burden. In the last 20 years, it has achieved 90% reduction in malaria cases through sustained, intensified malaria interventions (long lasting insecticide nets, indoor residual sprays and artemisinin-combination therapy) and is aiming for elimination by 2030. In 2012 and 2013, we conducted two cross-sectional surveys (study 1, all age, n=3501; study 2, age 0.5-12 years, n=1078) in Ngella, an area of low to moderate transmission. We aimed to investigate the natural reservoir and local epidemiology of P. vivax and P. falciparum. The contrast was striking. In the 2012 survey, only five clonal P. falciparum infections were identified from a single village and had the same msp2 genotpye. P. vivax prevalence was found to be moderately high (PCR, 13.4%), with predominantly afebrile, submicroscopic infections. The P. vivax infections displayed high genetic complexity (by genotyping with msp1F3 and MS16) and considerable spatial heterogeneity among and within different Ngella regions, and even at sub-village level with some households disproportionately harboring more infected co-inhabitants than others. In the 2013 study, a further seven P. falciparum infections were found in multiple locations, indicating that transmission of this species is continuing but at very low levels and infections are predominantly asymptomatic. To investigate the transmission scenario of the two species in more detail, we undertook population genetics analyses. We typed the five 2012 P. falciparum infections at 10 polymorphic microsatellite loci and 323 P. vivax infections at nine microsatellite loci. The five P. falciparum infections also clonal by this panel of 10 markers. Subsequent analyses of diversity (FST, GST, Jost’s D) and structure (Bayesian clustering) for P. vivax, revealed a genetically diverse population, but spatially fragmented, even among villages 6-15km apart. This indicates that whilst P. vivax may be more difficult to eliminate than P. falciparum, local parasite populations of both species have been affected by control interventions. A noteworthy epidemiological result from the 2012 survey was that living in a household with at least one other P. vivax carrier increased the risk of P. vivax infection, suggesting possible intra-household transmission. Subsequent analysis of genetic relatedness of P. vivax infections within households vs. among households indicated supported this hypothesis. Isolates from the same household were more genetically related than isolates from different households, and a high level of genetic kinship was retained among households located up to 100 meters of each other. Associations of P. vivax infection with human genetic factors known to confer protection against infection (α-thalassemia and Southeast Asian ovalocytosis, SAO) have been investigated in a second cross-sectional study conducted in 2013 in children aged 6 months to 12 years of age. SAO was not found in Ngella, whereas approximately a third of 1078 subjects were found to harbor the α-thalassemia alleles. The findings presented in this thesis will be discussed in the context of factors which may impact on follow-up elimination strategies in Solomon Islands, the Southwest Pacific and elsewhere in the endemic world where both P. falciparum and P. vivax are co-endemic.
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ItemMerozoite surface protein 1: insights into complex formation and function in erythrocyte invasion( 2016)The invasion of Plasmodium falciparum into host erythrocytes during the parasites’ asexual blood stage is a critical step in the perpetuation of symptomatic infection of malaria in the human host. Merozoites, the invasive form of the parasite, express a glycosylphosphatidylinositol-anchored 190 kDa Merozoite Surface Protein 1 (MSP1) on the surface. This abundant, essential protein exists in a large complex that includes other peripheral Merozoite Surface Proteins (MSPs). Together, these large macromolecular complexes are thought to mediate the initial stages of invasion. MSPs are of great interest to the field as they are exposed to the host immune system and also contribute directly to the invasion process. Therefore, there is a strong consideration for considering them as therapeutic targets. The majority of the work assessing these molecules as potential vaccine candidates has been performed with single MSP antigens and vaccine trials on these MSPs have shown variable results. A main concern arising from these trials is the fact that these antigens are often found in complex with other antigens and therefore have regions that are masked when found on the parasite surface. In order to address this, the work presented in this thesis utilises parasite-derived complexes to understand how peripheral MSPs: MSP3, MSP6, MSPDBL1, MSPDBL2 and MSP7 utilise MSP1 as a platform to be presented on the merozoite surface to form an array of complexes with different functional roles. In addition, multiple forms of erythrocyte binding complexes were found to have overlapping functions in invasion. Complexes that are involved in erythrocyte binding were characterised, where two components, MSPDBL1 and MSPDBL2 were shown to mediate erythrocyte binding directly. Overall, this study has identified and validated the presence of multiple Merozoite Surface Protein 1 complexes that are involved in mediating the interaction of the merozoite to receptors on the red blood cell surface, which is a vital process for successful invasion of parasites into host erythrocytes. Together, these findings have provided valuable insights into the complexity of MSP1 complexes and have contributed to the most complete model for the molecular arrangements that occur on the parasite surface to date.