The species Entamoeba histolytica is a microaerophilic protozoan parasite and causes amoebiasis in humans. Globally, amoebiasis is the third leading cause of death due to parasites especially in tropical and subtropical countries according to the World Health Organization (WHO). The infection can be treated orally with the low-cost nitroimidazole drug metronidazole. Although the drug has been used for more than 40 years, it is not fully understood why nitroimidazole kills amoebae, and two theories have been put forward. The common theory about the deadly effect of metronidazole on E. histolytica is that the pyruvate:ferredoxin oxidoreductase (PFOR) reaction is producing reduced ferredoxin which is responsible for the reduction of metronidazole into the active form, a nitroradical anion, which kills the amoeba. More recently it was demonstrated that there is an alternative reduction pathway of metronidazole. Thioredoxin reductase is also able to reduce metronidazole and turn it into an active form, even more efficiently than PFOR. It was also shown that a small group of amoebic proteins forms covalent adducts with activated metronidazole or other nitroimidazole drugs. So this could be a new link how the toxic impact of metronidazole works. The modified proteins are thioredoxin reductase (TrxR), thioredoxin (Trx), superoxide dismutase (Sod), purine nucleoside phosphorylase (Pnp), and an abundant small protein of unknown function named metronidazole target protein-1 (Mtp1).
In this thesis, our interest was concentrated on the investigation of the function Mtp1. The first aspired aim was to produce a recombinant Mtp1 for enzyme function studies and also for localisation in the amoebae by immunofluorescence staining. The vectors for expression in both expression systems, Escherichia coli and Pichia pastoris, were constructed and confirmed to be correct by sequencing, however, neither system produced any recombinant Mtp1, although all the other modified proteins could be expressed in E. coli. So it will be necessary to investigate more expression systems to study Mtp1 such as insect or mammalian cells or even in vitro cell free systems.
The second basic approach was to investigate if the toxic impact of metronidazole could be linked to the amount of Mtp1 in the amoebae. It could be possible that increased or lowered amounts of Mtp1 reduce or amplify the effect of metronidazole in amoebae or vice versa. For this purpose, the Mtp1 gene was inserted in two different vectors, an inducible expression vector pEhHYG-tetR-O and a constitutive expression vector pEhNEO/. In addition, antisense constructs and one construct with a herpes simplex virus (HSV) tag were generated, and all constructs were sequenced. Transfection of the E. histolytica laboratory strain HM-1:IMSS with the NEO (G418) resistance constructs did not give any resistant amoebae, but the amoebae transfected with the HYG construct became resistant up to 6 μg/ml hygromycin B, less than the published 10 μg/ml. The successful transfection was confirmed by RT-qPCR but no increased Mtp1 mRNA level could be detected. The protein investigation by two-dimensional gel electrophoresis showed no increased amount of Mtp1 protein as well. These results show that the control of transcription in E. histolytica is not understood well at all. During our work we found out that the group of Iris Bruchhaus at the Bernhard-Nocht-Institute for Tropical Medicine (Hamburg, Germany) has had similar problems with the same transfection vectors. The conclusion is that some E. histolytica genes may need their own upstream or downstream sequences for successful expression. Therefore more vector reconstruction work will be needed.