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ANCOVA combines features of ANOVA (analysis of variance) and regression. It augments the ANOVA model with more additional quantitative variables (covari- ates), which are related to the response variable. ANCOVA was used as a way to assess the effect of the different nutrient concentrations in either growth or metabo- lite consumption and excretion. ANCOVA can be used to compare two or more regression lines by testing the effect of a factor (in this case the three media CM, BL and LG) on a dependent variable (parasite size/metabolite consumption/excretion) while controlling for the effect of a continuous co-variable (in this case time). AN- COVA allows us to find out if intercepts and slopes are different between factors.

Chapter 3

A tailored method to identify

and quantify metabolites of

Plasmodium falciparum

in cell

and media samples using

Nuclear Magnetic Resonance

(NMR)

3.1

Introduction:

P. falciparum

intra-erythrocyte meta-

bolic network revealed by metabolomics

Understanding of the parasite’s metabolism is paramount, not only because of its role in malaria pathogenesis but also because it is a target of antimalarial drugs [221] and for many of them the mode of action is not well characterised [244]. Attempts to unveil the metabolic network of the parasite has been investigated by

in vivobiochemistry or indirectly by inference from genomic data and bioinformatic studies [71, 72]. The development of metabolomic technologies enabled the study at the systems level of the parasite and consequently the first metabolomic studies to understandP. falciparumblood stages metabolism were published. Shortly after the publication of Nuclear Magnetic Resonance (NMR) analyses of metabolic responses of mice infected withPlasmodium bergheiusing biofluids [218], two metabolic studies in cellular extractsP. falciparum were published in 2009 by independent groups.

Tenget al. [219] used 1H NMR spectroscopy to analyse the metabolome of late trophozoite stages ofP. falciparum that were isolated from the host red blood cell (RBC) by a treatment with saponin. Sample sizes of 1-4 ×108 _{cells (equiva-}

lent to 105 µL of cell pellet) were extracted using 4 different extraction solutions: perchloric acid, methanol/water, methanol/chloroform/water or methanol and they were compared. Around 40 metabolites were identified and quantified followed by estimation of intracellular concentrations. Partial least squares was used to com- pare the extraction methods. The authors concluded that perchloric acid was the most advantageous solution although results were broadly similar among extrac- tions. However perchloric acid poses a problem for metabolomic studies by NMR spectroscopy. As an acidic solution, perchloric acid would vary the sample’s pH, which can severely affect spectra acquisition. Thus a protocol that involved the use of an acid as extraction solution would require an obligatory step to adjust pH prior to NMR acquisition, increasing the complexity of the sample processing. Super- natants were also collected and used to assess metabolite loss during the separation of parasites and RBCs. Overall the work by Teng et al. set out the methodology and precedence for metabolomics by NMR spectroscopy of P. falciparum. More- over, the same group has recently use this method to profile chloroquine sensitive and resistant strains ofP. falciparum [217].

The Llin´as group [195] used synchronised cultures of P. falciparum 3D7 to take samples at seven time points during its 48-hour blood stage, which were analysed using a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method developed by Lu et al. [212]. Samples consisted of 50 µL (equivalent to 9.5×107 cells) of cell pellet of either infected (at 10% parasitaemia) or non-infected RBCs and the supernatants that were extracted as described in [239]. Overall 90 metabolites were detected and quantified over the time course. These span a wide range of metabolic pathways such as amino acids, nucleotides and central carbon metabolism intermediates. This study revealed a modulation of metabo- lite levels by the parasite (in contrast with the low metabolism of the RBC) with numerous metabolites varying in phase with intra-erythrocytic development, gen- erally increasing concentration from ring stages to trophozoite stages and slightly decreasing towards the end of the incubation, coinciding with very mature schizonts and formation of merozoites. The authors considered noteworthy the high parasite consumption of arginine, consistent with the typical hypoargininemea observed in humans infected with malaria, which is associated with the cerebral pathogenesis of the disease.

falciparum works, the Llin´as’ group traced 13C-labelled compounds and published a controversial paper in which they stated that the Krebs cycle was not only largely disconnected from glycolysis but also presented a branched structure, different from the canonical cyclical flux [104]. However this communication was retracted in 2013 [245]. Central carbon metabolism of the blood stages ofP. falciparum was finally elucidated by MacRaeet al. analysis13C-Glucose and13C-Glutamine flux through glycolysis and glutaminolysis using LC-MS/MS. This study confirmed that the par- asite uses a canonical Krebs cycle, despite the flux from glycolysis being very low during the asexual stages and that most of the carbon skeletons of the Krebs cycle are instead provided by glutaminolysis [103]. The lack of pyruvate dehydrogenase (PDH) in the mitochondrion that could convert pyruvate in to acetyl-CoA influ- enced the hypothesis of a dysfunctional Krebs cycle. However a branched chain ketoacid dehydrogenase (BCKDH) was found to functionally replace mitochondrial PDH [106]. These studies were done in mature trophozoite and schizont stages of the asexual life cycle of the parasite. Aliquots equivalent to 108cells were extracted with a chloroform:methanol (1:1) solution, yet another extraction solution, different from above-mentioned publications. Finally a study of the central carbon metabolism using knock-out (KO) parasites for the main enzymes involved in the Krebs cycle has been recently published by Llin´as group confirming above-mentioned results and proving that none of the enzymes ablated were essential for asexual development of the parasite [107]. For this study the authors were not very explicit with the nature of the samples used (cell suspension vs washed cellular extracts) where they only reported the use of a methanolic metabolite extraction on 800µL samples.

Metabolomic studies have unveiled the metabolic network of the asexual stages of P. falciparum. However the reason behind this metabolic rewiring is un- clear. During the asexual stages, P. falciparum exhibits a very high glycolytic flux (with up to 100× higher glucose intake than uninfected RBCs [223]) followed by a low flux into the Krebs cycle. During sexual stages, the Krebs cycle activity in- creases and parasites are more susceptible to mitochondrial inhibitors [103]. We have proposed that this metabolic rewiring, similar to the Warbug effect, which is well characterised in cancer cells [115], is a strategy to maximise biomass production by redirecting glycolytic intermediates into pathways that will lead to the construc- tion of nucleic acids, lipids and other key metabolites to meet the high demand for biomass production [111]. The limited flux into Krebs cycle might be explained by a need to keep lactic fermentation ongoing as it might serve as a regulatory mech- anism [117] (see Chapter 1, Section 1.3.1.3) or to reduce in general the otherwise high flux into the respiratory chain with the consequent ion leakage followed by the

production of reactive oxygen species (ROS) that would be harmful for the progeny. All the above-mentioned studies were performed in conditions far from phys- iological. Some of these studies used RBC-free parasites. This process would be greatly stressful for the parasite and it is likely that it would trigger stress responses atypical from healthy parasites. All the experiments were done in laboratory condi- tions, with rich media that has in general much higher concentrations of the metabo- lites compared to typical human blood [178]. As described in Chapter 1, one of the knowledge gaps to address is whether nutritional availability has an effect upon parasite development. We aim here to further pursue the validity of the proposed hypotheses regarding the role played by high glycolytic fluxes followed by high lactic fermentation.

Because of the limitations of previous methods, in this Chapter we focus on the development of a robust metabolomics method to identify and quantify metabo- lites of intra-erythrocytic stages of P. falciparum in a NMR spectroscopy-based platform. The Chapter will detail the progression of the assay development includ- ing the following sections:

1. Adequate signal detection on undisturbed parasites.

(a) Assess the signal difference between cell suspension and individual media and cell extractions for similar samples.

(b) Use of iRBC samples without parasite enrichment methods in order to keep conditions as natural as possible.

(c) Optimise NMR signal strength in order to identify a relevant number of metabolites by using 13C isotopic glucose and explore alternative NMR spectroscopy experiments.

2. Metabolite identification. 3. Metabolite quantification.

4. Validation and pipeline assembly 5. Pilot experiment to test the method.