The development of axenic in vitro growth models of the human malaria parasite Plasmodium falciparum, has been pivotal in accelerating knowledge of this very important human pathogen. Despite the importance of this pathogen, there have been very few studies relating to the metabolism of the parasite. Furthermore, much of the preceding studies have been undertaken using culture conditions that do not accurately represent the physiological environment of the human host. There is a need to address whether different nutrient environments would trigger a parasite response at the systems level promoting a metabolic rewiring that would have an effect in progeny generation or life cycle progression. Because of its robustness, reproducibility and suitability for footprinting studies, NMR spectroscopy was chosen as the analytic technique for the study. One of the disadvantages of NMR is limited availability of software for identification and quantification of metabolites. This was taken as an opportunity to develop a pipeline using free, open-source programming framework. This tool was used to find unique and discriminatory metabolic profiles for both uninfected and P. falciparum infected red blood cells at various life-cycle stages using cell extracts and extracellular material. With the aim of studying parasite development in physiological conditions a culture medium mimicking human blood conditions was developed and tested on P. falciparum infected RBCs finding both phenotypic and metabolic differences. Further studies consisted of the development of tightly synchronised parasite cultures that were followed during 54 h using NMR-based metabolomics to assess consumption and excretion of metabolites in media, and high content imaging and bright field microscopy to assess parasite size and progeny. The measurements were taken under three different nutritional conditions: usual in vitro, physiological-like and hypoglycaemic. In usual culturing conditions P. falciparum 3D7 life cycle lasted around 45 h. During the early stages there was moderate consumption of glucose and glutamine and excretion of lactate, alanine and glycerol. During the mature trophozoite stages and schizonts, glucose uptake dramatically increased with a consequent augmentation of the lactate, alanine and glycerol production. These were excreted but their function was not clear. It was observed that these “wasteful" products were proportionally lower in the early developmental stages than in the later ones, suggesting a higher demand of raw materials (glucose) for biomass production during the early stages. During the late trophozoite stage the most abundant amino acids in the haemoglobin chain (leucine, valine and glycine) were excreted, a likely consequence of the need for space to nish maturation. Myoinositol, which is essential for creation of membranes was also avidly consumed. When comparing these findings with parasites growing in more physiological conditions there was a noticeable delay in the life cycle of at least 9 h. Consequently haemoglobin digestive products were excreted later in the time course. A decrease in the progeny resulting from schizonts containing significantly fewer merozoites was also observed. Parasites growing in physiological conditions but challenged with lower glucose availability also presented a further delay of the life-cycle and a decreased number of merozoites with respect to usual laboratory conditions. Haemoglobin degradation products were also excreted later in the life cycle and at lower rates compared to the parasites grown in complete media. These results suggest that there are significant differences between in vivo and in vitro life-cycles of P. falciparum. Such effects as the reduction in growth rates and elongation of the life cycle, if not accounted for, could severely compromise the in vivo results of in vitro drug killing rates assays.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:707422 |
| Date | January 2016 |
| Creators | Gutiérrez, Eva Caamaño |
| Publisher | University of Warwick |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://wrap.warwick.ac.uk/87354/ |
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