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Journal of Antimicrobial Chemotherapy 2004 54(1):56-59; doi:10.1093/jac/dkh280
JAC vol.54 no.1 © The British Society for Antimicrobial Chemotherapy 2004; all rights reserved.

Entamoeba histolytica alcohol dehydrogenase 2 (EhADH2) as a target for anti-amoebic agents

Avelina Espinosa1,*, David Clark2 and Samuel L. Stanley, Jr1,3

Departments of 1 Molecular Microbiology, and 3 Medicine, Washington University School of Medicine, St Louis, MO 63110; 2 Department of Microbiology, Southern Illinois University, Carbondale, IL 62901, USA

Received 6 July 2003; returned 1 December 2003; revised 5 January 2004; accepted 13 April 2004


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Objectives: The current use of metronidazole as an anti-amoebic agent causes significant side-effects. The purpose of this study was to identify alternative compounds with which to treat amoebiasis.

Methods: We tested the effects of cyclopropyl (CPC) and cyclobutyl (CBC) carbinols on the survival of Entamoeba histolytica trophozoites and on the enzymatic activities of E. histolytica alcohol dehydrogenase 2 (EhADH2), a crucial enzyme in the amoebic fermentation pathway.

Results: At 72 h, the estimated 50% inhibitory concentrations of CPC and CBC were 38.9 and 11.2 µM, respectively. The EhADH2 alcohol and aldehyde dehydrogenase activities were inhibited by 1.82 µM CPC and 0.89 µM CBC in vitro.

Conclusions: CPC and CBC are expected to be non-toxic to humans at the concentrations required to eliminate E. histolytica trophozoites. Similarities between EhADH2 and the Giardia lamblia AdhE enzyme indicate that CPC and CBC could be effective drugs for treatment of both amoebiasis and giardiasis.

Keywords: bifunctional proteins , glycolytic pathways , eukaryotic parasites , cycloalkanols , alcohol dehydrogenase E


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The intestinal protozoan Entamoeba histolytica is a major cause of morbidity and mortality worldwide, causing 50 million cases of diarrhoea and 100 000 deaths per year.1,2 Amoebiasis is primarily treated with the drug metronidazole, even though significant side-effects, such as neurological complications, and the possible selection of a resistant E. histolytica strain have been reported.3–6 These concerns have prompted the search for alternative therapeutic agents. E. histolytica lacks mitochondria and obtains its energy from the fermentation of glucose, producing carbon dioxide, acetate and ethanol as end products.7,8 E. histolytica alcohol dehydrogenase 2 (EhADH2), a bifunctional enzyme with both aldehyde dehydrogenase (ALDH) and alcohol dehydrogenase (ADH) activities, constitutes a key enzyme in this pathway.7–10 We have previously shown that EhADH2 expression is required for the growth and survival of E. histolytica trophozoites,11 suggesting this enzyme could be a target for new anti-amoebic drugs.

Successful complementation of a mutant Escherichia coli strain (SHH31{Delta}adhE) with recombinant EhADH212,13 provides an ideal system for studying potential inhibitors of EhADH2. In this study, we show that the cycloalkanol compounds cyclopropyl (CPC) and cyclobutyl (CBC) carbinols inhibit the growth and survival of E. histolytica trophozoites. We also demonstrate that these agents specifically affect the ALDH and ADH activities of the recombinant EhADH2.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 Acknowledgements
 References
 
Trophozoites of E. histolytica strain HM1:1MSS were cultured under axenic conditions in Diamond's TYI-S-33 medium.11–13 Trophozoites in the log phase of growth were used in all experiments. To determine inhibition of E. histolytica growth, standard culture tubes containing an initial inoculum of 5 x 103 trophozoites per tube were grown and counted at 48 and 72 h. Viable trophozoites were counted using a haemocytometer. Growth counts were averaged from three replicate tubes and three separate experiments. Amoebic cultures were closely examined to verify absence of bacterial contamination in tubes. E. coli strain SHH31 ({Delta}adh zch::Tn10 fadRmet tyrT)10,12,13 was used for transformation and expression of the recombinant EhADH2. For anaerobic growth, the transformed strain was grown on solid (1.5% Bacto Agar; Difco, Sparks, MD, USA) or liquid M9 media11,13 in anaerobic jars (BBL GasPak system; BBL, Cockeysville, MD, USA) with anaerobic system envelopes (BBL GasPak Plus). Indicator strips were used to confirm anaerobic conditions. A single colony of strain SHH31 ({Delta}adhE) transformed with pEhADH211 was grown overnight under aerobic conditions. Bacterial cells were processed as described elsewhere.10,11,13 Lysate samples were analysed by SDS–PAGE and western blot analyses. Bacterial lysates were used to measure NAD+-dependent ALDH and ADH activities. ALDH activity was assayed spectrophotometrically by measuring decrease in absorbance at 340 nm, following the oxidation of NADH to NAD using acetyl-CoA as the substrate.14,15 ADH activity was measured by the same assay, with the substitution of acetaldehyde for acetyl-CoA as the substrate.14,15 One unit of enzyme activity was defined as that which consumed 1 µmol of NADH or NAD+/min. Activity values were averaged from three independent samples. Substrate concentrations were based on standard protocols.13–15


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 Acknowledgements
 References
 
CPC and CBC inhibited the growth of E. histolytica trophozoites (Figures 1 and 2). A CPC concentration of at least 75 µM was required for complete trophozoite growth inhibition (Figure 1; Table 1). In contrast, a CBC concentration of only 30 µM (a 2.5-fold decrease) was required to completely block trophozoite growth (Figure 2; Table 1). The EIC50 value for CBC was much lower than that for CPC: 11.2 µM and 38.9 µM, respectively (Table 1). The two compounds also blocked anaerobic growth of wild-type E. coli (BL21 DE3) and E. coli strain SHH31{Delta}adhE transformed with EhADH2 in plasmid pET3a (data not shown).



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  		Figure 1.. Inhibition of trophozoite growth with CPC. A concentration of 75 µM inhibits trophozoite growth. An initial 5 x 103 HM1:1MSS E. histolytica trophozoites were inoculated per tube, and counted at 48 and 72 h.

 


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  		Figure 2.. Inhibition of trophozoite growth with CBC. A concentration of 30 µM completely blocks trophozoite growth. An initial 5 x 103 HM1:1MSS E. histolytica trophozoites were inoculated per tube, and counted at 48 and 72 h.

 

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  		Table 1.. Effect of cycloalkanols CPC and CBC on the growth of E. histolytica trophozoites (EIC50 at 48 and 72 h are shown)

 
CPC and CBC inhibited both enzymatic activities of the EhADH2 protein in vitro (Table 2). Assays of ALDH and ADH activities demonstrated a stronger effect of CBC than of CPC. The effects of CPC and CBC on the ALDH activity of EhADH2 were particularly dramatic. At 0.53 µM, CPC decreased the ALDH activity by 53%, while a similar concentration of CBC completely eliminated ALDH activity (Table 2). CBC completely eliminated the ADH activity of EhADH2 at 0.89 µM, while a two-fold increase in concentration (1.82 µM) of CPC was required to obtain similar results (Table 2).


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  		Table 2.. In vitro inhibition of EhADH2 enzymatic activities (ALDH and ADH) by CPC and CBC

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
Acknowledgements
 References
 
EhADH2 is a member of the microbial group III or ‘iron activated’ alcohol dehydrogenases, with higher homology to prokaryotic than to eukaryotic alcohol dehydrogenases.16,17 In an attempt to search for specific inhibitors for this enzyme, we tested two alkanols, CPC and CBC. This study shows that both of these carbinols have an inhibitory effect on in vivo trophozoite growth (Figures 1 and 2; Table 1) and on in vitro EhADH2 enzymatic activities (Table 1). CBC is a stronger inhibitor than CPC, but both compounds affect growth and enzymatic function at micromolar concentrations (Tables 1 and 2). These results suggest that CPC and CBC interfere with trophozoite growth by blocking EhADH2 and, therefore, fermentative metabolism. Our results show an important difference between the drug concentrations required to block parasite growth and to inhibit enzymatic activity in vitro. Since EhADH2 is mostly localized in the cytosol, these compounds probably cross the parasite's membrane to affect its metabolism. Although the mechanisms underlying the inhibitory effects of either compound on the anaerobic growth of E. histolytica are not known, a likely possibility is that EhADH2 converts both cyclic alcohols into corresponding aldehydes, which then attack the active site residues of the glycolytic enzyme, affecting its activity. This is the mechanism by which unsaturated alcohol analogues, such as allyl alcohol, inhibit ADH enzymes.18–20 Moreover, in preliminary fluorescence analysis of EhADH2, addition of CBC to a mix of EhADH2 and acetyl-CoA blocked quenching of tryptophans of EhADH2 by the substrate (data not shown). It is possible to speculate that CBC interferes irreversibly with substrate binding of acetyl-CoA.

The different phylogenetic origin and unique structural characteristics of EhADH2 with respect to the human dehydrogenases also suggest that CPC and CBC would bind specifically to this enzyme and probably not to the human ADH and ALDH homologues. We have hypothesized that EhADH2 originated from the fusion of ancestral ADH and ALDH microbial proteins, diverging functionally and structurally from classical unifunctional ADHs and ALDHs.11 The anaerobic EhADH2 uses iron as the catalytic ion and shares the co-factor binding site for the ADH and ALDH activities.11 CPC and CBC apparently have strong affinity for this shared binding site. In contrast, humans possess two structurally independent ADH and ALDH aerobic enzymes; ADH is zinc dependent and ALDH requires no catalytic ion. Based on these differences between the amoebic and the human dehydrogenases, we predict little affinity of cycloalkanols for the mammalian enzymes.

Although CPC and CBC can be toxic to humans at millimolar concentrations, our EIC50 data indicate that, if administered at micromolar concentrations, these or similar compounds could be used safely as anti-amoebic agents. As anaesthetics, cycloalkanols act on the cytochrome P450 enzyme family and on neuronal conduction mechanisms (nicotinic acetylcholine receptor)18,20–23 at much higher concentrations than the ones we used to inhibit trophozoite growth (Table 1). In a previous report,18 the anaesthetic EC50 value for cyclobutyl methanol (a CBC analogue) on vertebrates was 5.4 mM, 480 times higher than the CBC EIC50 value reported here (Table 1), and 6000-fold the concentration we needed to inhibit the ADH activity in vitro (Table 2). Mammalian toxicity data on the effects of n-alkanols on ion channels indicate that at least 140 mM cyclopropanemethanol and 14 mM cyclobutanemethanol were necessary to inhibit the nicotinic acetylcholine receptor in vitro.22 These values are 3600- and 1250-fold the concentrations used in our study to inhibit 50% of amoebic trophozoite growth by the analogues CPC and CBC, respectively (Table 1). A survey revealed that 1-aminocyclopropanol, an aldehyde dehydrogenase inhibitor, had no inhibitory effect on rat brain tryptophan hydrolase activity in vitro.21 A recent in vivo study showed that methyl isobutyl carbinol (MIBC) had low potential for toxicity in male rats, based on the plasma levels found after 12 h of a single 5 mmol/kg oral dose of MIBC. Neither mortality nor clinical signals of toxicity were observed in the animals.24 In summary, no study has reported any harmful effect of these or similar compounds on vertebrate dehydrogenases.

In conclusion, our results suggest that CPC and CBC can be used safely and effectively for the treatment of amoebiasis. These compounds are expected to be non-toxic to humans at the concentrations required to eliminate E. histolytica trophozoites. As anti-amoebic agents, CPC and CBC appear to specifically target EhADH2, while as anesthetics they act on P450 oxygenases and neuronal receptors. Similarities between EhADH2 and the Giardia lamblia AdhE enzyme17,25 indicate that CPC and CBC could be effective drugs for the treatment of both amoebiasis and giardiasis.


   Acknowledgements
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
References
 
We thank Guillermo Paz-y-Miño C. for comments on the manuscript and Ellen Li for discussion. Lynne Foster and Fairoz Radtke provided invaluable technical assistance. This work was funded by NIH grants AI51621 to S.L.S., and DK52574 to the Washington University Digestive Diseases Core Center. S.L.S. is a Burroughs Wellcome Scholar in Molecular Parasitology.


   Footnotes
 
* Correspondence address. University of Nebraska-Lincoln, Center for Biotechnology, N316 Beadle, PO Box 880660, Lincoln, NE 68588-0660, USA. Tel: +1-402-472-0500; Fax: +1-402-472-3139; Email: mespinosa2{at}unlnotes.unl.edu


   References
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 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
1 . Walsh, J. A. (1988). Prevalence of Entamoeba histolytica infection. In Amoebiasis: Human Infection by Entamoeba histolytica (Ravdin, J. I., Ed.), pp. 93–105. Wiley Medical, New York, NY, USA.

2 . Walsh, J. A. (1986). Problems in recognition and diagnosis of amoebiasis: estimation of the global magnitude of morbidity and mortality. Reviews in Infectious Diseases 8, 228–38.[Web of Science][Medline]

3 . Goldman, P., Koch, R. L., Yeung, T. C. et al. (1986). Comparing the reduction of nitroimidazoles in bacteria and mammalian tissues and relating it to biological activity. Biochemical Pharmacology 35, 43–51.[CrossRef][Web of Science][Medline]

4 . Knight, R. C., Skolioowski, I. M. & Edwards, D. I. (1978). The interaction of reduced metronidazole with DNA. Biochemical Pharmacology 27, 2089–93.[CrossRef][Web of Science][Medline]

5 . Koch, C. J., Lord, E. M., Shapiro, I. M. et al. (1997). Imaging hypoxia and blood flow in normal tissues. Advances in Experimental Medicine and Biology 428, 585–93.[Web of Science][Medline]

6 . Kock, R. L., Beaulieu, B. B., Jr, Chrystal, E. J. T. et al. (1981). A metronidazole metabolite in human urine and its risk. Science 211, 398–400.[Abstract/Free Full Text]

7 . Reeves, R. E. (1984). Metabolism of Entamoeba histolytica Schaudinn, 1903. Advances in Parasitology 23, 105–42.[Web of Science][Medline]

8 . Lo, H. & Reeves, R. E. (1978). Pyruvate-to-ethanol pathway in Entamoeba histolytica. Biochemical Journal 171, 225–30.[Web of Science][Medline]

9 . Yang, W., Li, E., Kairong, T. et al. (1994). Entamoeba histolytica has an alcohol dehydrogenase homologous to the adhE gene product of Escherichia coli. Molecular and Biochemical Parasitology 64, 253–60.[CrossRef][Web of Science][Medline]

10 . Espinosa, A., Wang, L., Li, E. et al. (1997). Expression of the alcohol dehydrogenase (ADH) domain of Entamoeba histolytica EhADH2 enzyme. Archives of Medical Research 28, S78–81.

11 . Espinosa, A., Yang, L., Zhang, Z. et al. (2001). The bifunctional Entamoeba histolytica 2 (EhADH2) protein is necessary for amoebic growth and survival and requires an intact C-terminal domain for both alcohol dehydrogenase and aldehyde dehydrogenase activity. Journal of Biological Chemistry 276, 20136–43.[Abstract/Free Full Text]

12 . Goodlove, P. E., Cunningham, P. R., Parker, J. et al. (1989). Cloning and sequence analysis of the fermentative alcohol-dehydrogenase-encoding gene of Escherichia coli. Gene 85, 209–14.[CrossRef][Web of Science][Medline]

13 . Yong, T., Li, E., Clark, D. et al. (1996). Complementation of an Escherichia coli adhE mutant by the Entamoeba histolytica EhADH2 gene provides a method for the identification of new anti-amoebic drugs. Proceedings of the National Academy of Sciences, USA 93, 6464–9.[Abstract/Free Full Text]

14 . Kessler, D., Herth, W. & Knappe, J. (1992). Ultrastructure and pyruvate formate-lyase radical quenching property of the multienzymic AdhE protein of Escherichia coli. Journal of Biological Chemistry 267, 18073–9.[Abstract/Free Full Text]

15 . Kessler, D., Leibrecht, I. & Knappe, J. (1991). Pyruvate-formate-lyase-deactivase and acetyl-CoA reductase activities of Escherichia coli reside on a polymeric protein particle encoded by adhE. FEBS Letters 281, 59–63.[CrossRef][Web of Science][Medline]

16 . Reid, M. F. & Fewson, C. A. (1994). Molecular characterization of microbial alcohol dehydrogenases. Critical Reviews in Microbiology 20, 13–56.[Web of Science][Medline]

17 . Samuelson, J. (1999). Why metronidazole is active against both bacteria and parasites. Antimicrobial Agents and Chemotherapy 43, 1533–41.[Free Full Text]

18 . LaBella, F. S., Chen, Q. M., Stein, D. et al. (1997). The site of general anesthesia and cytochrome P450 oxygenases: similarities defined by straight chain and cyclic alcohols. British Journal of Pharmacology 120, 1158–64.[CrossRef][Web of Science]

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