E. histolytica: thioredoxin system as metronidazole target

Amoebic dysentery and liver abcess, caused by the intestinal protozoan parasite Entamoeba histolytica, are very common in many countries of this world. Up to 50 million cases of this disease per year have been estimated. Although amoebiasis is potentially lethal, only few of the patients die because of the wide-spread and affordable oral therapy with metronidazole. Metronidazole is a major antibiotic which has been used for as long as 50 years. Besides against E. histolytica, it is active against a broad spectrum of other microaerophilic and anaerobic microorganisms such as Trichomonas vaginalis, Giardia intestinalis, Bacteroides spp. and Helicobacter pylori. Although metronidazole has saved millions of human lives, we do not know how exactly on the molecular level it kills E. histolytica or other pathogens. It has clearly been shown that the drug needs to be chemically reduced for activity, and that there are several toxic metabolites. So far the primary reduction product, a nitroradical anion, was seen as the major damaging agent, and its action was thought to be indiscriminate, hitting a wide range of biomolecules such as proteins and in particular DNA. In contrast to this, we discovered in our previous FWF funded project, that in E. histolytica and T. vaginalis other activated metabolites of metronidazole covalently modify thiol groups in only a few defined proteins. In E. histolytica, these were thioredoxin reductase, thioredoxin, superoxide dismutase, purine nucleoside phosphorylase, and a small protein with a carbohydrate-binding domain named metronidazole target protein-1 (MTP1). Thioredoxin reductase itself was found to activate metronidazole, and so this enzyme and other protein components of the thiol-based redox network were the first targets of metronidazole action. This was accompanied by a strong decrease in small thiol molecules within the cells. While our past work has identified the thiol-based redox network of E. histolytica as a main target area of metronidazole action, the new proposed project aims to examine the following questions: What are the interactions of the primary targets, particularly thioredoxin reductase and thioredoxin? What is the localization of these components? Is there a link between the damage of the thioredoxin system and the damage to the cytoskeleton? What is the role of MTP1 in metronidazole toxicity? Which small thiol molecules are reacting with the activated metronidazole? Is the DNA damage cause or consequence of cell death? Taken together we want to gain a better understanding of the thiol- based redox system of E. histolytica and its role in metronidazole activity as well as to attempt to identify the relevant targets of the drug which could be exploited for new strategies of chemotherapy in the future.

Entamoeba histolytica is a single-cell infectious agent which is responsible for amoebic dysentery and liver abscess. Although invasive amoebiasis is a life-threatening disease, the infection can be treated with metronidazole, a very inexpensive and effective drug that has been in use for more than 40 years. The drug is also used against various other microorganisms which live in environments with little oxygen, but the amoebae are most sensitive and even after many years of drug use have never developed resistance. In spite of this great success, many questions remain open about the molecular mechanisms of metronidazole action, and some of them were addressed in our project. In a previous project we had identified the thioredoxin system as an important target. In this project we discovered that in E. histolytica thioredoxin interacts with a large number of other proteins. We could demonstrate for a prototype enzyme, serine acetyltransferase, how it can be damaged by oxidising agents and repaired by thioredoxin. Finally, we were able to examine the redox state of thioredoxin in the amoebae, and very unexpectedly, in the metronidazole-treated amoebae, thioredoxin was as much in the active reduced form as in the untreated ones. This shows that there must be further important and so far unknown targets of metronidazole in E. histolytica.In the late phase of metronidazole action, the parasite DNA is degraded as it is after treatment with several other toxic agents. As this process resembles programmed cell death, where cellular DNases cleave the chromosomal DNA, we searched the databases for E. histolytica enzymes which could be involved in this DNA destruction. Caspase-dependent DNases or endonuclease G are absent from E. histolytica, but we could identify and characterise a homologue of the TatD nuclease. The recombinant enzyme produced in E. coli exhibited magnesium-dependent DNase activity, so this is the first characterisation of an E. histolytica DNase which could play a role in the metronidazole-mediated DNA decay. In the world of antimicrobial agents the use of metronidazole against E. histolytica represents a lucky punch. As this is such a lasting success story, it is extremely interesting to understand exactly how metronidazole acts in the amoebae and why other microorganisms develop resistance much faster. Our approach is to investigate the relevant factors or possible targets one by one using biochemical and molecular biological methods. We believe that understanding precisely this successful example will make the development of new and very much needed antimicrobial agents easier in the future.