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Human malaria is caused by protozoan parasites of the genus Plasmodium and is a major contributor to global morbidity and mortality. Plasmodium falciparum is the most virulent of five Plasmodium species that infect human. As in the other species, P. falciparum develops and proliferates in the nutrient-rich anucleated erythrocyte, consuming glucose for energy and metabolizing haemoglobin as a source of amino acids. In the erythrocyte, the parasite is also able to avoid some of the host’s immune responses during infection. To support the high growth rate of the parasite during infection, glucose consumption in the infected erythrocytes increases by up to 75-fold in comparison with uninfected erythrocytes. As a consequence, a high amount of the 2-oxoaldehyde methylglyoxal, a toxic by-product of glycolysis, is spontaneously formed in the parasite. Methylglyoxal reacts with and damages nucleic acids and proteins leading to the formation of the so-called advanced glycation end-products (AGE) in the cell. Glyoxalases 1 and 2 (Glo1 and Glo2) of the ubiquitous glyoxalase system catalyze the glutathione-dependent detoxification of methylglyoxal and other 2-oxoaldehydes to non-toxic 2-hydroxycarboxylic acids. In the P. falciparum infected erythrocyte, two functional glyoxalase systems operate; one in the cytosol of the erythrocyte (hGlo1 and hGlo2) and the other in the parasite cytosol (PfGlo1 and PfcGlo2). In addition to the cytosolic enzymes, P. falciparum also encodes a functional apicoplast-targeted Glo2 enzyme (PftGlo2), and an inactive Glo1-like protein (PfGILP) that also carries an apicoplast-targeting sequence. On account of the detoxification role the glyoxalase system plays during Plasmodium infection, there has been a long-standing hypothesis that inhibition or knockout of the Plasmodium glyoxalase-encoding genes would lead to an accumulation of methylglyoxal (2-oxoaldehydes) in the host-parasite unit and result in parasite death. And so, the glyoxalase system(s) of the host-parasite unit could be targeted for antimalarial drug development. This thesis investigated the relevance of the glyoxalase system to the blood-stage parasite survival by generating clonal PFGLO1 and PFcGLO2 knockout lines of P. falciparum 3D7 strain using the CRISPR-Cas9 system. SYBR green-based growth assays of the knockout clones showed that the 3D7Δglo1 knockout clones had an increased susceptibility to the external 2-oxoaldehyde glyoxal compared with the 3D7Δcglo2 knockout clone and the 3D7 wild-type strain. Western blot analyses also supported the accumulation of selected modified proteins in 3D7Δglo1 and 3D7Δcglo2 knockout strains in comparison with the 3D7 wild-type strain. The 3D7Δglo1 and 3D7Δcglo2 knockout lines were, however, viable and showed no significant morphological or growth phenotype under standard growth conditions. Furthermore, the lack of PfcGlo2, but not PfGlo1, resulted in increased gametocyte induction and gametocytogenesis in the knockout lines. PfGlo1 and PfcGlo2 are therefore dispensable during asexual blood-stage development and the loss of PfcGlo2 may actually contribute to the transmission of the malaria parasite. The results show that PfGlo1 and PfcGlo2 are non-essential and most likely not suitable for targeted antimalarial drug development. Several attempts to generate knockout clonal lines for the apicoplast targeted glyoxalase enzymes were unsuccessful. Transfectants resistant to both positive and negative selection were obtained in three knockout trials but were all confirmed to be false-positive transgenic parasites. The most probable explanation for this outcome is an off-target or unwanted integration of the positive selectable marker into the parasite genome. Methodological limitations, rather than the essentiality of the two enzymes, are therefore most likely the cause of the negative knockout results. The thesis also investigated the relevance of the hGlo1 enzyme to the survival of the parasite in the host-parasite unit using three tight-binding Glo1 inhibitors. Inhibitor treatments of uninfected erythrocytes showed an extremely slow inactivation of the host cell glyoxalase. Esterification did not confer improved pharmacokinetics nor increased the potency of the inhibitors. Inhibition of the erythrocyte Glo1 enzyme did not affect parasite development in the host cell, pointing to a potential dispensability of the host cell enzyme for parasite survival in the host-parasite unit. Finally, as a way of addressing the relevance of the so-called oxidative stress on parasite development, the thesis investigated the effects of the prooxidant H2O2 and the so-called antioxidants NAc, ascorbate, and DTT on the survival of P. falciparum 3D7 strain. IC50 values for the redox agents were determined for ring-stage synchronized parasites using a SYBR green-based growth assay. An IC50 value of 78 mM for H2O2 revealed the compound as a very poor prooxidant in parasite culture. The host-parasite unit appears to be very robust against challenges with H2O2 and parasite killing required extremely high concentrations with implications for host defence mechanisms. The reductants NAc, ascorbate and DTT also had antiproliferative instead of growth-promoting effects with IC50 values around 16, 4 and 0.3 mM, respectively. Taken together, the host-parasite unit appears more tolerant to high levels of H2O2, ascorbate and NAc, but is more susceptible to DTT. The inhibitory effect of the so-called antioxidants has implications for clinical trials and studies on oxidative stress. |
