Exploration of the malaria parasite lifecycle stages and strains circulating in natural infections at single-cell resolution
Malaria is still a major global public health problem, resulting in an unacceptably high annual mortality of over half million. Extensive diversity in malaria parasites is a key challenge in developing effective interventions. Many infections constitute several strains, and this complicates malaria studies since it is hard to disentangle strains using traditional bulk approaches. There are also parasites of different stages circulating in natural infections, but there is very limited understanding of these stages, especially gametocytes, which are overwhelmingly underrepresented in a typical infection. The recent development of single-cell RNA sequencing (scRNAseq) offers a great opportunity to study malaria parasites in natural infections and opens new avenues of exploration previously inaccessible to bulk approaches. In Chapter 1, I delve deeper into the motivations for using scRNAseq in natural infections and the general background knowledge on malaria infections and technologies necessary to contextualise my findings. I then detail my findings, some of which are novel, in the remaining chapters. In Chapter 2, I explore the stages circulating in asymptomatic infections, finding early asexuals and late gametocytes as previously observed. However, the high scRNAseq resolution reveals that the female and male gametocytes each constitute two transcriptomically distinct forms. One of the male and female gametocyte forms is characterized by low expression compared to the other male and female pair, and I label these as LE (low expression) gametocytes. I hypothesise that these are older, dormant gametocytes. In Chapter 3, I investigate strain dynamics in one asymptomatic infection. I estimate around eight strains in this infection and find that the distribution of strains in the asexual stage does not correlate with that in the sexual stage. Close relatedness patterns between the strains in this infection suggest that they were introduced by the same mosquito. Using this information together with the imbalanced distribution of strains in the asexual and sexual stage, I speculate on differential investment in sexual development between strains. In Chapter 4, I replicate observations from the previous chapters across more donors and go on to assess the differential gene expression (DE) between different strains within the same infection for the asexual, male and female stages. I find many genes to be differentially expressed, with GO analysis implicating these genes in key processes necessary for survival in the respective stage. Asexual stage DE genes are mainly involved in interaction with the human host, female DE genes are involved in crystalloid components which are necessary for development in the mosquito and male DE genes are implicated in functions necessary for mitotic replication which is essential for sexual reproduction. I also perform DE between asexual parasites from asymptomatic and symptomatic individuals and between individuals with different haemoglobin genotypes, finding genes previously observed to be associated with these phenotypes in literature. Finally, in Chapter 5, I discuss some of the key limitations, and potential areas of improvement, of this study regarding the protocol and study design. I propose a future longitudinal study design to best understand sequestration dynamics of asexual parasites in natural infections, genes involved in the transition of parasites from the asexual to the gametocytes stage, the nature of LE gametocytes and the genes involved in the transition of parasites through the mosquito transmissive stages.
Lifecycle stages, Malaria, Mali, Natural infections, Plasmodium falciparum, single-cell RNA sequencing, Strains
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