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1.  Unexpected properties of NADP-dependent secondary alcohol dehydrogenase (ADH-1) in Trichomonas vaginalis and other microaerophilic parasites 
Experimental Parasitology  2013;134(3):374-380.
Graphical abstract
CoA inhibits the oxidation of 2-propanol and the reduction of acetaldehyde, acetone and a yet unidentified “background” substrate by ADH-1.
•Trichomonas vaginalis NADPH-dependent alcohol dehydrogenase-1 (ADH-1) reduces acetaldehyde and acetone, and oxidizes 2-propanol.•In addition to its canonical function, a strong reducing background activity was observed.•All reactions catalyzed by ADH-1 are strongly inhibited by CoA.•These observations also apply for the parasites Entamoeba histolytica and Tritrichomonas foetus, but not for Giardia lamblia which lacks ADH-1.
Our previous observation that NADP-dependent secondary alcohol dehydrogenase (ADH-1) is down-regulated in metronidazole-resistant Trichomonas vaginalis isolates prompted us to further characterise the enzyme. In addition to its canonical enzyme activity as a secondary alcohol dehydrogenase, a pronounced, so far unknown, background NADPH-oxidising activity in absence of any added substrate was observed when the recombinant enzyme or T. vaginalis extract were used. This activity was strongly enhanced at low oxygen concentrations. Unexpectedly, all functions of ADH-1 were efficiently inhibited by coenzyme A which is a cofactor of a number of key enzymes in T. vaginalis metabolism, i.e. pyruvate:ferredoxin oxidoreductase (PFOR). These observations could be extended to Entamoeba histolytica and Tritrichomonas foetus, both of which have a homologue of ADH-1, but not to Giardia lamblia which lacks an NADP-dependent secondary alcohol dehydrogenase.
Although we could not identify the substrate of the observed background activity, we propose that ADH-1 functions as a major sink for NADPH in microaerophilic parasites at low oxygen tension.
PMCID: PMC3682184  PMID: 23578856
Trichomonas vaginalis; NADP-dependent alcohol dehydrogenase-1; Background activity; CoA
2.  Pyruvate:ferredoxin oxidoreductase and thioredoxin reductase are involved in 5-nitroimidazole activation while flavin metabolism is linked to 5-nitroimidazole resistance in Giardia lamblia 
The mechanism of action of, and resistance to, metronidazole in the anaerobic (or micro-aerotolerant) protozoan parasite Giardia lamblia has long been associated with the reduction of ferredoxin (Fd) by the enzyme pyruvate:ferredoxin oxidoreductase (PFOR) and the subsequent activation of metronidazole by Fd to toxic radical species. Resistance to metronidazole has been associated with down-regulation of PFOR and Fd. The aim of this study was to determine whether the PFOR/Fd couple is the only pathway involved in metronidazole activation in Giardia.
PFOR and Fd activities were measured in extracts of highly metronidazole-resistant (MTRr) lines and activities of recombinant G. lamblia thioredoxin reductase (GlTrxR) and NADPH oxidase were assessed for their involvement in metronidazole activation and resistance.
We demonstrated that several lines of highly MTRr G. lamblia have fully functional PFOR and Fd indicating that PFOR/Fd-independent mechanisms are involved in metronidazole activation and resistance in these cells. Flavin-dependent GlTrxR, like TrxR of other anaerobic protozoa, reduces 5-nitroimidazole compounds including metronidazole, although expression of TrxR is not decreased in MTRr Giardia. However, reduction of flavins is suppressed in highly MTRr cells, as evidenced by as much as an 80% decrease in NADPH oxidase flavin mononucleotide reduction activity. This suppression is consistent with generalized impaired flavin metabolism in highly MTRr Trichomonas vaginalis.
These data add to the mounting evidence against the dogma that PFOR/Fd is the only couple with a low enough redox potential to reduce metronidazole in anaerobes and point to the multi-factorial nature of metronidazole resistance.
PMCID: PMC3133484  PMID: 21602576
metronidazole; ronidazole; tinidazole; Blastocystis; NADPH oxidase
3.  Down-regulation of flavin reductase and alcohol dehydrogenase-1 (ADH1) in metronidazole-resistant isolates of Trichomonas vaginalis 
Molecular and Biochemical Parasitology  2012;183-222(2):177-183.
Graphical abstract
In isolates of Trichomonas vaginalis with reduced susceptibility metronidazole, flavin reductase and alcohol dehydrogenase-1 (ADH1) activities are down-regulated.
► In clinical isolates of Trichomonas vaginalis with reduced metronidazole susceptibility flavin reductase is down-regulated. ► In clinical isolates of T. vaginalis with reduced metronidazole susceptibility alcohol dehydrogenase-1 (ADH1) is down-regulated. ► Thioredoxin reductase levels are not changed in metronidazole-resistant T. vaginalis clinical isolates.
The microaerophilic parasite Trichomonas vaginalis is a causative agent of painful vaginitis or urethritis, termed trichomoniasis, and can also cause preterm delivery or stillbirth. Treatment of trichomoniasis is almost exclusively based on the nitroimidazole drugs metronidazole and tinidazole. Metronidazole resistance in T. vaginalis does occur and is often associated with treatment failure. In most cases, metronidazole-resistant isolates remain susceptible to tinidazole, but cross resistance between the two closely related drugs can be a problem.
In this study we measured activities of thioredoxin reductase and flavin reductase in four metronidazole-susceptible and five metronidazole-resistant isolates. These enzyme activities had been previously found to be downregulated in T. vaginalis with high-level metronidazole resistance induced in the laboratory. Further, we aimed at identifying factors causing metronidazole resistance and compared the protein expression profiles of all nine isolates by application of two-dimensional gel electrophoresis (2DE).
Thioredoxin reductase activity was nearly equal in all strains assayed but flavin reductase activity was clearly down-regulated, or even absent, in metronidazole-resistant strains. Since flavin reductase has been shown to reduce oxygen to hydrogen peroxide, its down-regulation could significantly contribute to the impairment of oxygen scavenging as reported by others for metronidazole-resistant strains. Analysis by 2DE revealed down-regulation of alcohol dehydrogenase 1 (ADH1) in strains with reduced sensitivity to metronidazole, an enzyme that could be involved in detoxification of intracellular acetaldehyde.
PMCID: PMC3341570  PMID: 22449940
Trichomonosis; Metronidazole resistance; Thiordoxin reductase; Flavin reductase; Alcohol dehydrogenase 1
4.  Nitroimidazole drugs vary in their mode of action in the human parasite Giardia lamblia 
Graphical abstract
► Metronidazole and tinidazole bind Giardia proteins, including thioredoxin reductase. ► 5-Nitroimidazoles cause degradation of translation elongation factor-1γ (EF-1 γ). ► Nitroimidazoles deplete intracellular thiol levels. ► Nitroimidazoles affect Giardia differently than other parasites.
Giardia lamblia (syn. duodenalis, intestinalis) is a globally occurring micro-aerophilic human parasite that causes gastrointestinal disease. Standard treatment of G. lamblia infections is based on the 5-nitroimidazole drugs metronidazole and tinidazole. In two other micro-aerophilic parasites, Entamoeba histolytica and Trichomonas vaginalis, 5-nitroimidazole drugs bind to proteins involved in the thioredoxin-mediated redox network and disrupt the redox equilibrium by inhibiting thioredoxin reductase and depleting intracellular thiol pools. The major aim of this study was to assess whether nitroimidazoles exert a similar toxic effect on G. lamblia physiology.
The 5-nitroimidazoles metronidazole and tinidazole were found to bind to the same subset of proteins including thioredoxin reductase. However, in contrast to E. histolytica and T. vaginalis, none of the other proteins bound are candidates for being involved in the thioredoxin-mediated redox network. Translation elongation factor EF-1γ, an essential factor in protein synthesis, was widely degraded upon treatment with 5-nitroimidazoles. 2-Nitroimidazole (azomycin) and the 5-nitroimidazole ronidazole did not bind to any G. lamblia proteins, which is in contrast to previous findings in E. histolytica and T. vaginalis. All nitroimidazoles tested reduced intracellular thiol pools in G. lamblia, but metronidazole, also in contrast to the situation in the other two parasites, had the slightest effect. Taken together, our results suggest that nitroimidazole drugs affect G. lamblia in a fundamentally different way than E. histolytica and T. vaginalis.
PMCID: PMC3862438  PMID: 24533278
Nitroimidazoles; Protein binding; Translation elongation factor-1γ; Thiol depletion
5.  In vitro activity of N-chlorotaurine (NCT) in combination with NH4Cl against Trichomonas vaginalis 
Trichomoniasis, caused by the protozoan Trichomonas vaginalis, is usually treated with metronidazole, however resistance is on the rise. In this study, N-chlorotaurine (NCT), a new endogenous mild active chlorine compound for topical use, killed T. vaginalis in vitro within 15 min of treatment at a concentration of 55 mM (1%), which is well tolerated by human tissue. The activity of NCT was further enhanced by addition of ammonium chloride (NH4Cl). A combination of 5.5 mM (0.1%) NCT plus 19 mM (0.1%) NH4Cl killed 100% of trichomonads within 5 min.
PMCID: PMC3030749  PMID: 21074373
Trichomonas vaginalis; Susceptible; N-Chlorotaurine; Oxidant; In vitro
6.  Major Role for Cysteine Proteases during the Early Phase of Acanthamoeba castellanii Encystment ▿ † 
Eukaryotic Cell  2010;9(4):611-618.
Acanthamoeba castellanii is a facultative pathogen that has a two-stage life cycle comprising the vegetatively growing trophozoite stage and the dormant cyst stage. Cysts are formed when the cell encounters unfavorable conditions, such as environmental stress or food deprivation. Due to their rigid double-layered wall, Acanthamoeba cysts are highly resistant to antiamoebic drugs. This is problematic as cysts can survive initially successful chemotherapeutic treatment and cause relapse of the disease. We studied the Acanthamoeba encystment process by using two-dimensional gel electrophoresis (2DE) and found that most changes in the protein content occur early in the process. Truncated actin isoforms were found to abound in the encysting cell, and the levels of translation elongation factor 2 (EF2) were sharply decreased, indicating that the rate of protein synthesis must be low at this stage. In the advanced stage of encystment, however, EF2 levels and the trophozoite proteome were partly restored. The protease inhibitors PMSF (phenylmethylsulfonyl fluoride) and E64d [(2S,3S)-trans-epoxysuccinyl-l-leucylamido-3-methylbutane ethyl ester] inhibited the onset of encystment, whereas the protein synthesis inhibitor cycloheximide was ineffective. Changes in the protein profile, similar to those of encysting cells, could be observed with trophozoite homogenates incubated at room temperature for several hours. Interestingly, these changes could be inhibited significantly by cysteine protease inhibitors but not by inhibitors against other proteases. Taken together, we conclude that the encystment process in A. castellanii is of a bipartite nature consisting of an initial phase of autolysis and protein degradation and an advanced stage of restoration accompanied by the expression of encystment-specific genes.
PMCID: PMC2863413  PMID: 20190073
7.  Nitroimidazole Action in Entamoeba histolytica: A Central Role for Thioredoxin Reductase 
PLoS Biology  2007;5(8):e211.
Metronidazole, a 5-nitroimidazole drug, has been the gold standard for several decades in the treatment of infections with microaerophilic protist parasites, including Entamoeba histolytica. For activation, the drug must be chemically reduced, but little is known about the targets of the active metabolites. Applying two-dimensional gel electrophoresis and mass spectrometry, we searched for protein targets in E. histolytica. Of all proteins visualized, only five were found to form adducts with metronidazole metabolites: thioredoxin, thioredoxin reductase, superoxide dismutase, purine nucleoside phosphorylase, and a previously unknown protein. Recombinant thioredoxin reductase carrying the modification displayed reduced enzymatic activity. In treated cells, essential non-protein thiols such as free cysteine were also affected by covalent adduct formation, their levels being drastically reduced. Accordingly, addition of cysteine allowed E. histolytica to survive in the presence of otherwise lethal metronidazole concentrations and reduced protein adduct formation. Finally, we discovered that thioredoxin reductase reduces metronidazole and other nitro compounds, suggesting a new model of metronidazole activation in E. histolytica with a central role for thioredoxin reductase. By reducing metronidazole, the enzyme renders itself and associated thiol-containing proteins vulnerable to adduct formation. Because thioredoxin reductase is a ubiquitous enzyme, similar processes could occur in other eukaryotic or prokaryotic organisms.
Author Summary
The protist parasites Entamoeba histolytica, Trichomonas vaginalis, and Giardia intestinalis grow in environments with low oxygen concentration. Infections with these parasites are commonly treated with metronidazole, a nitroimidazole drug that must be reduced for activation, resulting in several toxic metabolites. We examined the soluble proteome of metronidazole-treated E. histolytica cells for target proteins of these metabolites, applying two-dimensional gel electrophoresis and mass spectrometry. Of about 1,500 proteins visualized, only five formed covalent adducts with metronidazole metabolites, including thioredoxin, thioredoxin reductase, and superoxide dismutase. Metronidazole-bound thioredoxin reductase displayed diminished activity. In addition to these proteins, small thiol molecules, including cysteine, formed adducts with metronidazole. Supplementation with cysteine allowed the cells to survive otherwise lethal metronidazole concentrations. Finally, we discovered that one of the modified proteins, thioredoxin reductase, reduces metronidazole, suggesting a central role for this enzyme with regard to metronidazole toxicity. Taken together, our work reveals a new area of molecular interactions of activated metronidazole with cellular components. Because thioredoxin reductase is a ubiquitous enzyme, similar processes could also occur in other eukaryotic or prokaryotic organisms.
Metronidazole is used for treatment of infections with microaerophilic protist parasites. Here, a new model of metronidazole activation is proposed, with a central role for thioredoxin reductase.
PMCID: PMC1933457  PMID: 17676992
8.  A Monoclonal Antibody to the Amebic Lipophosphoglycan-Proteophosphoglycan Antigens Can Prevent Disease in Human Intestinal Xenografts Infected with Entamoeba histolytica  
Infection and Immunity  2002;70(10):5873-5876.
Entamoeba histolytica trophozoites are covered by lipophosphoglycan-peptidoglycan molecules which may be key virulence factors. We found that pretreatment of severe combined immunodeficient mice bearing human intestinal xenografts with a monoclonal antibody to the amebic lipophosphoglycan-peptidoglycan molecules can prevent or significantly reduce the human intestinal inflammation and tissue damage that are normally seen with E. histolytica colonic infection.
PMCID: PMC128362  PMID: 12228321
9.  Cytotoxic Activities of Alkylphosphocholines against Clinical Isolates of Acanthamoeba spp. 
Free-living amoebae of the genus Acanthamoeba are causing serious chronic conditions such as destructive keratitis in contact lens wearers or granulomatous amoebic encephalitis in individuals with compromised immune systems. Both are characterized by the lack of availability of sufficiently effective and uncomplicated, manageable treatments. Hexadecylphosphocholine (miltefosine) is licensed for use as a topical antineoplastic agent, but it is also active in vitro against several protozoan parasites, and it was applied very successfully for the treatment of human visceral leishmaniasis. The aim of our study was to evaluate the efficacy of hexadecylphosphocholine and other alkylphosphocholines (APCs) against Acanthamoeba spp. The in vitro activities of eight different APCs against three Acanthamoeba strains of various pathogenicities were determined. All substances showed at least amoebostatic effects, and some of them disrupted the amoebae, as shown by the release of cytoplasmic enzyme activity. Hexadecylphosphocholine exhibited the highest degree of cytotoxicity against trophozoites, resulting in complete cell death at a concentration as low as 40 μM, and also displayed significant cysticidal activity. Hexadecylphosphocholine may be a promising new candidate for the topical treatment of Acanthamoeba keratitis and, conceivably, even for the oral treatment of granulomatous amoebic encephalitis.
PMCID: PMC127497  PMID: 11850250
10.  Effects of Miltefosine and Other Alkylphosphocholines on Human Intestinal Parasite Entamoeba histolytica 
The protozoan parasite Entamoeba histolytica is the cause of amoebic dysentery and liver abscess. It is therefore responsible for significant morbidity and mortality in a number of countries. Infections with E. histolytica are treated with nitroimidazoles, primarily with metronidazole. At this time, there is a lack of useful alternative classes of substances for the treatment of invasive amoebiasis. Alkylphosphocholines (alkyl-PCs) such as hexadecyl-PC (miltefosine) were originally developed as antitumor agents, but recently they have been successfully used for the treatment of visceral leishmaniasis in humans. We examined hexadecyl-PC and several other alkyl-PCs with longer alkyl chains, with and without double bond(s), for their activity against two strains of E. histolytica. The compounds with the highest activity were oleyl-PC, octadecyl-PC, and nonadecenyl-PC, with 50% effective concentrations for 48 h of treatment between 15 and 21 μM for strain SFL-3 and between 73 and 98 μM for strain HM-1:IMSS. We also tested liposomal formulations of these alkyl-PCs and miltefosine. The alkyl-PC liposomes showed slightly lower activity, but are expected to be well tolerated. Liposomal formulations of oleyl-PC or closely related alkyl-PCs could be promising candidates for testing as broad-spectrum antiprotozoal and antitumor agents in humans.
PMCID: PMC90496  PMID: 11302818
11.  Protection against Invasive Amebiasis by a Single Monoclonal Antibody Directed against a Lipophosphoglycan Antigen Localized on the Surface of Entamoeba histolytica  
The Journal of Experimental Medicine  1997;186(9):1557-1565.
A panel of monoclonal antibodies was raised from mice immunized with a membrane preparation from Entamoeba histolytica, the pathogenic species causing invasive amebiasis in humans. Antibody EH5 gave a polydisperse band in immunoblots from membrane preparations from different E. histolytica strains, and a much weaker signal from two strains of the nonpathogenic species Entamoeba dispar. Although the exact chemical structure of the EH5 antigen is not yet known, the ability of the antigen to be metabolically radiolabeled with [32P]phosphate or [3H]glucose, its sensitivity to digestion by mild acid and phosphatidylinositol-specific phospholipase C, and its specific extraction from E. histolytica trophozoites by a method used to prepare lipophosphoglycans from Leishmania showed that it could be classified as an amebal lipophosphoglycan. Confocal immunofluorescence and immunogold labeling of trophozoites localized the antigen on the outer face of the plasma membrane and on the inner face of internal vesicle membranes. Antibody EH5 strongly agglutinated amebas in a similar way to concanavalin A (Con A), and Con A bound to immunoaffinity-purified EH5 antigen. Therefore, surface lipophosphoglycans may play an important role in the preferential agglutination of pathogenic amebas by Con A. The protective ability of antibody EH5 was tested in a passive immunization experiment in a severe combined immunodeficient (SCID) mouse model. Intrahepatic challenge of animals after administration of an isotype-matched control antibody or without treatment led to the development of a liver abscess in all cases, whereas 11 out of 12 animals immunized with the EH5 antibody developed no liver abscess. Our results demonstrate the importance and, for the first time, the protective capacity of glycan antigens on the surface of the amebas.
PMCID: PMC2199119  PMID: 9348313
12.  Acanthamoeba castellanii : growth on human cell layers reactivates attenuated properties after prolonged axenic culture 
Fems Microbiology Letters  2009;299(2):121-127.
The free-living, but potentially pathogenic, bacteriovorous amoebae of the genus Acanthamoeba can be easily grown axenically in a laboratory culture. This, however, often leads to considerable losses in virulence, and encystment capacity, and to changes in drug susceptibility. We evaluated potential options for a reactivation of a number of physiological properties, attenuated by prolonged axenic laboratory culture, including encystment potential, protease activity, heat resistance, growth rates and drug susceptibility against N-chlorotaurine (NCT). Toward this end, a strain that had been grown axenically for 10 years was repeatedly passaged on human HEp-2 cell monolayers or treated with 5′-azacytidine (AzaC), a methyltransferase inhibitor, and trichostatin A (TSA), a histone deacetylase inhibitor, in order to uplift epigenetic gene regulation. Culture on human cell monolayers resulted in significantly enhanced encystment potentials and protease activities, and higher susceptibility against NCT, whereas the resistance against heat shock was not altered. Treatment with AzaC/TSA resulted in increased encystment rates and protease activities, indicating the participation of epigenetic mechanisms. However, lowered resistances against heat shock indicate that possible stress responses to AzaC/TSA have to be taken into account. Repeated growth on human cell monolayers appears to be a potential method to reactivate attenuated characteristics in Acanthamoeba.
PMCID: PMC2810444  PMID: 19732153
Acanthamoeba; encystment; protease; axenic culture; N-chlorotaurine

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