Thioredoxin reductase in Giardia lamblia

Giardia lamblia is a microaerophilic, unicellular parasite of the human gut which annually causes gastrointestinal disease in hundreds of million people worldwide. Giardiasis is not a life-threatening disease but causes severe and prolonged diarrhoea which can favour growth defects in children. Treatment of G. lamblia infections is mainly based on the nitroimidazole drug metronidazole which only exerts toxicity in organisms with a microaerohilic or anaerobic metabolism. This selectivity is due the necessity of the reduction of the nitro group for the activation of metronidazole which, however, only takes place quantitatively in the (near) absence of oxygen. The target molecules of metronidazole are probably manifold but are known to include several enzymes, including thioredoxin reductase (TrxR), a central factor in the antioxidative defense in most organisms. In the context of metronidazole toxicity, TrxR is especially interesting because it was not only shown to be a target of metronidazole but also to have enough reductive power in order to activate the drug. Thus, TrxR might have a key role in metronidazole toxicity. In previous studies it was shown that G. lamblia TrxR is bound by activated metronidazole, an event known in other parasites to reduce the enzyme`s activity. However, the consequences of this have remained unclear because virtually nothing is known about the TrxR-mediated redox system in G. lamblia and no interaction partners have been identified yet. Therefore, it will be the main objective of this project to identify and characterize the interaction partners of G. lamblia TrxR. In addition, an immunofluorescence microscopy study will be performed in order to identify TrxR`s localization in the parasite. Further, the role of TrxR in metronidazole toxicity in G. lamblia will be assessed by manipulating expression levels of the enzyme in transgenic G. lamblia cell lines. In the third year of the project, i.e. during the return phase to my home institute, newly acquired skills will be applied to study the influence of G. lamblia flavin reductase on metronidazole resistance. This enzyme activity was identified in an earlier study as being of central importance in the development of metronidazole resistance in Trichomonas vaginalis. Since metronidazole resistance has also been observed in G. lamblia, further research is warranted in order to develop counter-strategies in the future.

It was the major aim of this project to elucidate the physiological role of the enzyme thioredoxin reductase (TrxR) in the human unicellular parasite Giardia lamblia and to assess its potential as a drug target.Giardia lamblia colonizes the small intestine of humans or animals and is adapted to low oxygen levels in its surroundings, i.e. it is a microaerophilic microorganism. As high concentrations of oxygen are detrimental for such organisms, they dispose over efficient mechanisms to remove oxygen and to quickly repair oxidative damage inflicted directly or indirectly by oxygen. In this regard, TrxR is often of central importance through reducing other factors, e.g. thioredoxin and peroxiredoxin, that instrumental in the repair of damaged proteins and the removal of hydrogen peroxide, which is formed intracellularly after exposition to oxygen. Consequently, it is expected that inhibition of TrxR activity has a detrimental effect on G. lamblia, qualifying this enzyme as a drug target for chemotherapy. Interestingly, TrxR has another activity which, however, is disadvantageous for the parasite: in addition to its major substrate thioredoxin (Trx), TrxR also reduces nitro group containing compounds, thereby transforming them into toxic agents. One of these compounds is metronidazole, the gold standard for treating G. lamblia infections and infections with microaerophilic or anaerobic pathogens in general.In this project, this double role of TrxR in G. lamblia was studied. It was tested if TrxR has essential physiological importance in the parasite and if it has a relevant role in the reduction of metronidazole. To this end, the parasite was genetically manipulated and expression of TrxR was strongly augmented. The resulting mutant was assayed for physiological abnormalities and susceptibility to metronidazole and other nitro compounds. Indeed, the mutant proved to be more susceptible to metronidazole. However, the physiological consequences of TrxR overexpression were minimal and hardly relevant. A further aim was to identify binding partners of TrxR in G. lamblia in order to improve our understanding its role in G. lamblia. Our results showed that TrxR interacts with a large number of proteins, albeit in an unspecific manner.To summarize, the results of this project support the role of TrxR as activating enzyme of several drugs, including metronidazole. The physiological importance of TrxR, however, seems to be less significant as previously suggested. It is, therefore, questionable whether TrxR can be a relevant drug target for novel therapeutics.