Probing the chemical biology involved in the haem detoxification pathway of the human malaria parasite, plasmodium falciparum

Garnie, Larnelle

Probing the chemical biology involved in the haem detoxification pathway of the human malaria parasite, plasmodium falciparum

Thesis / Dissertation

2025

Publisher

University of Cape Town

Department

Department of Chemistry

Faculty

Faculty of Science

Abstract

A biological pathway that is critical to the survival of Plasmodium falciparum, the infectious agent responsible for most malaria-related deaths in humans, is the haem detoxification pathway. P. falciparum parasites in the asexual blood stage take up haemoglobin from the red blood cell (RBC) cytosol to make space for its growth and to prevent premature RBC lysis by maintaining the osmotic pressure within the RBC. The haemoglobin is degraded in the digestive vacuole (DV) to release peptides that are further broken down to amino acids, a nutrient source for the growing parasite. This process releases toxic haem which is then oxidised and detoxified by incorporation into insoluble haemozoin crystals. Given that antimalarials like chloroquine (CQ), amodiaquine (AQ) and piperaquine (PPQ) target this process, the pathway has received tremendous recognition within the drug discovery and development arena as a valuable, druggable target. However, the pathway involves multiple processes that are equally important and present as druggable targets. Thus, to better understand this pathway in wild-type NF54 parasites, a mathematical model has been previously developed to describe the entire haem detoxification pathway. To improve and validate this model, a crucial set of experimental input data for various processes was identified. The most important time- dependent parameters were (1) changes in DV lumen volume, (2) uptake of RBC cytosol into the DV, (3) protease enzyme concentration within the DV, (4) basal levels of haemoglobin, haem and haemozoin and (5) the cellular effects of perturbing the pathway with inhibitors. As such, this thesis addresses the gap in the required experimental data relating to these parameters in anticipation of improving the model in future and to gain deeper insight into the biology underpinning the haem detoxification pathway. To measure changes in the DV lumen volume and uptake, a method using confocal microscopy was optimised and coupled to image analysis techniques. Untreated parasites were allowed to reinvade RBCs pre-loaded with pHrodo™ dextran beads, which fluoresce only at acidic pH. Z-stacks of these parasites were acquired at various timepoints using an LSM 880 Airyscan microscope (Zeiss, GmbH) and analysed in ImageJ to calculate the growth of the lumen and uptake over the trophozoite to schizont life stages. It was found that in both NF54 and Dd2 the lumen grew following a Gompertz growth curve during the trophozoite phase, and then abruptly collapsed during the schizont stage. The uptake into the DV followed a similar pattern. To study the protease enzyme levels over time, the immunoblotting technique was used to measure changes in levels of plasmepsin (PM) I and IV as well as binding immunoglobulin protein (BiP) which was used as a control for total protein. To achieve this objective, untreated trophozoite cell lysates were collected from the early trophozoite to late schizont stages, and the proteins separated on an SDS-page. Once transferred onto a PVDF membrane, the protein bands were probed with anti-PM I, anti-PM IV and anti- BiP antibodies. The relative levels of PM I and PM IV were found to increase over time but were observed to be constant when normalised to BiP, indicating that these protease enzyme levels increase in accordance with the overall increase in protein content as the parasite grows. However, when normalised to the DV lumen volume, a larger apparent concentration of PM I and PM IV was observed in the early trophozoite stages before reaching a steady state in the mid-to-late trophozoite stages. Using a cellular fractionation approach, the basal levels of haemoglobin, haem, haemozoin and total Iron (Fe) were studied in untreated NF54 parasites from the early trophozoite to late schizont stages. These studies revealed that the percent of each haem species remained constant over the period studied, while the absolute levels displayed differing trends. The findings indicated that there is a tight regulation of the haem detoxification pathway. To determine the effects of perturbing the haem detoxification pathway, a set of endocytosis and protease inhibitors were selected and screened for their suitability for use as a tool compound. The evaluation cascade used to determine the suitability of the compounds involved screening their β-haematin formation inhibition activity, parasite growth inhibition activity and observed trends when subjected to dose-dependent cellular fractionation assays. Through this, an endocytosis inhibitor, 2,3-butanedione monoxime (BDM), a protease inhibitor, trans-epoxysuccinyl-L-leucylamido(4- guanidino)butane (E-64), and a haemozoin formation inhibitor, CQ, were chosen. These were then used to treat parasites over 24 hours at a fixed concentration to observe the trends over time. Endocytosis inhibition was characterised by a decrease in haem, haemozoin and total Fe absolute levels, while the percent of haem and haemozoin remained constant. Protease inhibition was characterised by an increase in haemoglobin levels concomitant with a decrease in haemozoin levels. Previously established trends for haemozoin formation inhibition characterised by an increase in haem levels coupled with a decrease in haemozoin levels, were observed. Altogether, the datasets related to untreated parasites provide a more holistic overview of DV physiology and complexity. These data confirm previous findings concerning the early trophozoite stages and offer new insights into the schizont stage. Importantly, this thesis describes the use of the cellular fractionation assay to identify and classify inhibitors in classes other than haemozoin formation inhibition. Additionally, the inhibitors were studied over time using the same assay. In its entirety the data collected will enhance model predictions and approximations, as well as validate the model in the future. More substantially, the insights advance the understanding of the processes within the DV and the haem detoxification pathway, providing a valuable foundation for deeper exploration of DV physiology.

Keywords

Chemical biology

Reference:

Collections

PhD / Doctoral

Full item page