Factors of metronidazole resistance in Trichomonas vaginalis

Trichomonas vaginalis is a worldwide occurring single cell parasite which infects the urogenital tract of humans, mainly in women. Those affected often develop vaginitis and cervicitis, symptoms usually referred to as trichomoniasis. In pregnant women, T. vaginalis infections can lead to preterm deliveries or even the death of the foetus. In addition, trichomoniasis does also predispose for HIV infection making it a considerable health problem in Southern Africa where both pathogens are highly prevalent. Since there is no vaccine available against T. vaginalis, management of the disease exclusively rests on chemotherapy. Of the chemotherapeutics available, the 5-nitromidazole drug metronidazole is by far the most frequently used. Metronidazole was developed more than 60 years ago and is still widely effective. However, in some parts of the world, treatment failures with metronidazole occur with up to 20% of the people treated. At least in a proportion of these cases treatment failure is caused by metronidazole resistance in the trichomonads. The mechanisms behind metronidazole resistance are still badly understood, mainly due to insufficient data. Based on previous results it is assumed that enzymes involved in the antioxidant defence have a role in metronidazole resistance. Furthermore, it is known that metronidazole resistance only becomes manifest in the presence of oxygen. It is important to note that T. vaginalis is an anaerobe, i.e. a microorganism which is harmed by oxygen and therefore quickly removes it, a process termed oxygen scavenging. An impaired capacity to scavenge intracellular oxygen had been described before in metronidazole-resistant T. vaginalis. It is the main goal of this project to considerably deepen our understanding of metronidazole resistance. To this end, candidate enzymes, possibly involved in resistance, will be expressed and characterized. Moreover, their expression levels will be measured in resistant and susceptible strains and compared, mainly by application of methods that identify and quantify the total protein content in cells (proteomics). It is hypothesized that resistant and susceptible cells display discrete and relevant differences with regard to their protein expression profiles. We expect that after completion of this project the understanding of molecular mechanisms casing metronidazole resistance in T. vaginalis will have improved greatly.

Trichomonas vaginalis is a single-celled parasite that infects humans, primarily affecting the urogenital tract in women, where it can cause inflammation. Although trichomoniasis is not life-threatening, it can cause long-lasting and painful symptoms and may also lead to premature birth. In addition, an underlying T. vaginalis infection can increase susceptibility to HIV infection. Trichomoniasis is treated almost exclusively with metronidazole, a drug that is toxic only to microaerophiles and anaerobes (which include T. vaginalis). The reason for this is that only in these organisms the nitro group of the compound is reduced and thus activated. Metronidazole has been used to treat trichomoniasis for more than 60 years, but the development of resistance is a growing problem. In this project, we investigated metronidazole resistance in T. vaginalis by analyzing protein expression in a number of resistant strains. We identified a single enzyme that, without exception, is expressed at lower levels or is completely silenced in all resistant strains: flavin reductase 1. This enzyme directly reduces the flavin FMN, but thereby also indirectly reduces intracellular iron (Fe to Fe). Reduced iron, in turn, is highly reactive and, in combination with other compounds (i.e., thiols such as cysteine), can reduce the nitro group of metronidazole. Resistance to metronidazole is thus attributable to lower levels of reduced iron in the cell, caused, in turn, by reduced activity of flavin reductase 1. In the final third of the project period, we applied a similar approach to elucidate metronidazole resistance in the bacterium Gardnerella vaginalis. G. vaginalis is the primary cause of bacterial vaginosis and is also treated with metronidazole. Again, metronidazole resistance is a common problem in treatment. We found that in metronidazole-resistant G. vaginalis strains a nitroreductase is expressed very weakly or not at all. Using the respective genetically engineered enzyme, we were able to demonstrate that the G. vaginalis nitroreductase very efficiently reduces metronidazole and related compounds, thereby converting them into their active forms. We suspect that this mechanism is responsible for metronidazole resistance in G. vaginalis.