title: Phenotypically and transcriptionally characterising hidden reservoirs of Plasmodium falciparum in paediatric severe malaria patients
Creators: name: Galloway, Lauren Jane
Publisher: name: University of Glasgow
Year: 2024

description: Malaria is a tropical infectious disease of global health importance which is caused by protozoan parasites of the genus Plasmodium. Most fatal malaria cases occur in children under five years old in sub-Saharan Africa where the dominant parasite species is Plasmodium falciparum. Recent studies have demonstrated that the haematopoietic spaces of the bone marrow (BM) represent an important hidden parasite reservoir of asexual and sexual stages, largely independent of classical vascular sequestration. Moreover, the spleen may represent a secondary haematopoietic organ with similar functions in extravascular parasite sequestration. The existence of hidden reservoirs has important implications for malaria eradication efforts as parasite reservoirs in these tissues may contribute to recrudescent infections, ongoing transmission, and the emergence of antimalarial resistance.

The aim of this thesis was to systematically investigate parasite sequestration in the BM and spleen in paediatric severe P. falciparum malaria cases, and the transcriptional pathways driving the switch from asexual to sexual stage development that may occur within these tissues. Specifically, this thesis aimed to determine whether the spleen harboured a significant parasite biomass compared to other tissues, to investigate the existence of an endosplenic life cycle, to determine the contribution of the spleen towards gametocyte formation and development in relation to the BM, and to deconvolute the transcriptional signatures of sexual commitment in two parasite strains with different sensitivities to environmental induction.

To evaluate the splenic reservoir, parasite distribution, density, and biomass were compared between the spleen, BM, lung, and peripheral circulation. Within the spleen, an enrichment of rings/early trophozoites was observed across all splenic compartments. While there were significantly higher parasite densities in the spleen compared to other tissues and peripheral circulation, there was no significant difference in the total parasite biomass between tissues. Furthermore, the parasite density in the spleen could be explained by the retention of peripherally circulating rings at a rate of 9.52%. Together, this suggests that in paediatric severe P. falciparum malaria, there is not a substantial hidden biomass of parasites in the spleen that is supported by an endosplenic life cycle. This contrasts with previous observations in chronic asymptomatic malaria in adults.

The spleen and BM were investigated for their contribution towards gametocyte formation and development. In the spleen, very few gametocytes and sexually committed schizonts were identified. In contrast, 13% of parasites identified in the BM were gametocytes, with 9% identified as sexually committed schizonts. This demonstrates the role of the BM in gametocyte formation and development.

To investigate the transcriptional signatures of asexual and sexually committed schizont populations which may exist in the BM, an in vitro single cell RNA sequencing (scRNAseq) experiment was performed. Sexual commitment was induced by growing parasites in medium depleted of the serum phospholipid lysophosphatidylcholine (LysoPC). Two parasite strains were used, Pf2004 and Dd2, with different sensitivities to LysoPC. In LysoPC depleted conditions, two transcriptionally distinct schizont populations were identified: i) an asexual population, which exhibited upregulation of cytoadherence associated genes and genes associated with purine metabolism, and ii) a sexually committed population, which exhibited upregulation of genes associated with sexual commitment and gene regulation. These two populations shared a metabolic signature in response to nutrient depletion. A final schizont population in steady state conditions exhibited higher expression of nutrient channel components and invasion related genes. In both asexual populations, a non-coding RNA (ncRNA), PF3D7_1370800, was identified. The expression patterns of PF3D7_1370800 in relation to sexual commitment markers, and the association of this ncRNA with asexual populations, suggests that this ncRNA may function in repressing sexual commitment; however, this remains unknown.

Overall, this thesis has revealed that the magnitude of the splenic parasite reservoir varies with disease severity. It has provided histological evidence of sexual commitment in the BM, suggesting that an endogenous asexual replication cycle in the BM contributes to gametocyte enrichment and development. In parallel, it has provided insight into the transcriptional signatures of sexually committed and asexual schizonts which are hypothesised to exist within the BM.