Investigating Toxoplasma gondii peroxisome and the discovery of two Bisabolane Sesquiterpenes as anti-leshmanials.

Abstract

The use of natural products for treating protozoan infections can be traced back to Rome in 1631, where cinchona tree bark was used to cure malaria. The discovery of novel naturally derived compounds for the treatment of cutaneous leishmaniasis and toxoplasmosis is vital for many vaccine deficient protozoan infections today. Here, the assessment of natural products against Leishmania mexicana (L. mexicana) was explored using a library of compounds screened against the mammalian stage of L. mexicana in in vitro assays. Two hit compounds from the screen were then used in metabolomic studies to determine mode of action. In addition, another natural compound, Aureobasidin A and its derivatives, and peroxisome inhibitors were screened against Toxoplasma gondii (T. gondii).
Peroxisomes are organelles involved in the metabolism of fatty acids and choles- terol. Other than lipid metabolism, peroxisomes contain many enzymes involved in several different metabolic processes. Catalase is involved in the neutralization of hydrogen peroxide thereby, preventing toxic build up within cells. This enzyme over time has become a key identifier of peroxisomes in many organisms. However, this is controversial when it comes to T. gondii. The use of catalase as a marker for peroxisomes in this parasite has been disputed, and in some cases led to the belief that the T. gondii does not possess these organelles. In this thesis, we take a dif- ferent approach in to establishing the existence of peroxisomes. Through evolution T. gondii has maintained, within its genome, genes encoding peroxisomal proteins, named peroxins (Pex). Here we investigated the presence of peroxisomes within T. gondii using Pex proteins. Our experimental approach involved characterization of putative TgPex5 and protein ligand TgSCP2. Using molecular biology, reverse genetics and protein characterization, we show that through pull-down assays, lo- calisation and complementation of TgPex5 in yeast expression systems, we are able to provide evidence to prove the presence of peroxisomes within T. gondii.