Molecular mechanisms of Entamoeba histolytica chemotherapy

The intestinal protozoan parasite Entamoeba histolytica is responsible for significant morbidity and mortality worldwide claiming at least 40 000 lives every year. The nitroimidazoles with metronidazole as the major representative are currently the only satisfying class of chemotherapeutic agents for the treatment of invasive amoebiasis. Alkylphosphocholines (alkyl-PCs) are antineoplastic agents which have also been successfully used for the treatment of human visceral leishmaniasis. In our laboratory we demonstrated that alkyl-PCs are also cytotoxic for E. histolytica. In the proposed project we plan to study the impact of these novel drug candidates and the established drug metronidazole on the amoebae. The aim of the proposed study is to contribute to our understanding of the cytotoxic action of alkyl-PCs and metronidazole in order to pave the way for further improvements in anti-amoebic chemotherapy. In the project we plan to approach this aim by studying the molecular reaction of the parasite to the two classes of substances, either the immediate reaction to treatment with lethal concentrations, or the changes observable after prolonged exposure to sublethal concentrations, leading to partial drug resistance. It is planned to assess the reaction of the parasite (1) by studying the changes on the protein level by performing two-dimensional electrophoresis and identifying up- or downregulated proteins, or (2) by isolating RNAs from the parasites and identifying changes on the mRNA level by molecular biological methods such as subtractive hybridization amplification or hybridization of fluorescently labeled cDNA to oligonucleotide arrays. Finally we plan to use biochemical or molecular biological methods to investigate the functional role of the up- or downregulated proteins during the parasite response to the anti-amoebic agents. We believe that this broad molecular perspective may lead to new ideas for improving anti-amoebic chemotherapy.

Every year, up to 100,000 patients lives are claimed by infections with the unicellular organism Entamoeba histolytica, the intestinal parasite that causes amoebic dysentery and liver abscess. Amoebiasis is treated almost exclusively with metronidazole, a drug used for various microorganisms in environments with low oxygen concentration. It is known that metronidazole must be chemically reduced to become active, but very little is known how the generated toxic products damage the microorganism and if the parasite tries to respond to the toxic action of the drug. Surprisingly, the amoebae did not show any significant stress response upon metronidazole treatment, neither new protein synthesis was observed, nor the induction of new mRNAs. When the total protein content of treated and untreated amoebae was separated by two-dimensional gel electrophoresis, careful inspection revealed that five proteins out of more than 1000 were chemically modified, and that they had been decorated by the activated drug. These proteins were thioredoxin reductase, thioredoxin, purine nucleoside phosphorylase, superoxide dismutase, and a protein of unknown function. We discovered that one of these enzymes, thioredoxin reductase, itself activates metronidazole and is the first to be modified. In addition to its action on proteins, the activated metronidazole diminished the level of free cysteine in the amoebae. The discovered processes result in a massive assault on the system protecting the amoebae from oxidative damage and define a novel mechanism of action for this pivotal drug. Finally, this mechanism could also be operative when other microorganisms are treated with metronidazole, in line with this assumption we found that thioredoxin reductase is also modified when Trichomonas vaginalis cells are treated with metronidazole. Taken together, the project defined for the first time the protein structures of E. histolytica that are hit when the amoebae are treated with metronidazole. This does not only provide a hypothesis on a new mechanism of drug action, but possibly, knowing specific targets provides opportunities to develop better anti-amoebic drug leads that could also be active against other pathogenic microorganisms. Another important experience was to be part of the E. histolytica genome project and to search for genes coding for enzymes and to establish metabolic pathways in this parasite. All our work started as basic science but the better understanding can certainly lead to better ideas for the treatment of amoebiasis, finally our work may be of general interest much beyond this single parasite, and it certainly opened the field for many new questions.