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JAC Advance Access originally published online on August 8, 2006
Journal of Antimicrobial Chemotherapy 2006 58(4):768-772; doi:10.1093/jac/dkl332
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© The Author 2006. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

Selective intracellular accumulation of the major metabolite issued from the activation of the prodrug ethionamide in mycobacteria

Xavier Hanoulle1,{dagger}, Jean-Michel Wieruszeski1, Pierre Rousselot-Pailley1, Isabelle Landrieu1, Camille Locht2, Guy Lippens1,{ddagger} and Alain R. Baulard2,*,{ddagger}

1 UMR 8525 CNRS-Universíté de Lille 2 Lille, F-59021, France 2 U629 INSERM-Institut Pasteur de Lille F-59021, France

*Corresponding author. Tel: +33-320-87-11-55; Fax: +33-320-87-11-58; E-mail: Alain.Baulard{at}pasteur-lille.fr

Received 16 May 2006; returned 14 June 2006; revised 4 July 2006; accepted 18 July 2006


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Background: Ethionamide is one of the most widely used drugs for the treatment of multidrug-resistant tuberculosis (MDR-TB). Like isoniazid, and pyrazinamide, ethionamide is a prodrug that needs to be activated by a mycobacterial enzyme. Activation pathways of prodrugs are generally problematic to uncover as they produce intermediates potentially difficult to characterize, to purify and that might prove unstable outside of their cellular context.

Objectives and methods: We have used high resolution magic angle spinning-NMR (HRMAS-NMR) to follow ethionamide activation directly within living mycobacterial cells.

Results: Data indicated that the intracellular metabolization of ethionamide strictly depends on the presence of the monooxygenase EthA and that EthA-dependent activation of ethionamide is coupled to a precise molecular sorting mechanism of the ethionamide metabolites. We found that the previously identified ethionamide metabolite 2-ethyl-4-hydroxymethylpyridine is produced in substantial amounts by the ethionamide-treated mycobacteria and that it is present exclusively outside of the bacteria. In contrast, the still unidentified ethionamide metabolite ETH* is the only ethionamide derivative detected within the bacterial cell. Moreover, ETH* appears to be unable to cross the bacterial envelope and consequently accumulates within the cytoplasm of the ethionamide-treated mycobacteria.

Conclusions: These results strongly suggest that ETH* is the active antimycobacterial ethionamide derivative and open new perspectives for the understanding of the mode of action of prodrugs.

Keywords: HRMAS-NMR , thioamide , tuberculosis


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Several antituberculosis compounds are prodrugs that require activation by mycobacterial enzymes to acquire bacteriotoxicity. These include pyrazinamide, isoniazid and ethionamide. Pyrazinamide is activated by the mycobacterial pyrazinamidase PncA, which presumably catalyses the generation of pyrazinoic acid. Acidic pH appears to enhance the intracellular accumulation of pyrazinoic acid, which is unable to diffuse across the mycobacterial cell wall.1 Because no pyrazinoic acid efflux mechanism exists, this accumulation process causes a remarkable susceptibility of Mycobacterium tuberculosis to pyrazinamide.

Both isoniazid and the structurally analogous thioamide prodrug ethionamide act as inhibitors of InhA. However, the large majority of isoniazid-resistant strains remain fully susceptible to ethionamide.2 This paradox is due to the fact that isoniazid and ethionamide are activated by two different mechanisms. Whereas isoniazid is activated by the catalase/peroxidase KatG,3,4 ethionamide is activated by the monooxygenase EthA.5–7 KatG has no effect on the activation of ethionamide, and reciprocally EthA is unable to activate isoniazid. A higher frequency of mutations in the katG gene than in inhA is the reason for the rarity of cross-resistance to the two drugs. Therefore, ethionamide may be a useful alternative to isoniazid for the treatment of isoniazid-resistant tuberculosis. The activation of isoniazid produces a hypothetical isonicotinic acyl radical that reacts with NADH,8 forming a competitive inhibitor of InhA. In contrast to the mechanism of action of isoniazid, the molecular mechanisms leading to the inhibition of InhA by activated ethionamide are less well understood. A recent in vitro study revealed that EthA is a bi-functional enzyme that transforms with a low efficiency ethionamide into the S-oxide derivative (ETH-SO) and subsequently into the 2-ethyl-4-amidopyridine derivative (ETH-amide).7 Because of the absence of toxicity of the amide derivative against mycobacteria, it has been proposed that a sulphinic acid intermediate is the putative active compound. In an independent study, Debarber et al.6 followed the metabolic process of [14C]-ETH within living mycobacteria by thin layer chromatography (TLC) and found several ethionamide metabolites, including ETH-SO, 2-ethyl-4-cyanopyridine (ETH-nitrile), ETH-amide, 2-ethyl-4-carboxypyridine (ETH-acid) and 2-ethyl-4-hydroxymethylpyridine (ETH-alcohol). In both studies, the ethionamide metabolites had been purified by TLC and/or HPLC, and their identity had been established by mass spectroscopy. It can therefore not be ruled out that the extraction/purification steps may have resulted in spontaneous modification/oxidation of the metabolites.

To avoid any spontaneous modification by the extraction/purification of the metabolites, we have recently used high resolution magic angle spinning-NMR (HRMAS-NMR) to follow ethionamide activation in vivo upon entering the mycobacterial cells.9 Surprisingly, we have found no traces of the ethionamide-derived compounds previously identified by classical biochemical methods. Instead, we have demonstrated the presence of a novel aromatic compound, named ETH*, derived from ethionamide.

In the present study, we used HRMAS-NMR to demonstrate the enzymatic implication of EthA in this process, to observe the distribution of ethionamide-derived metabolites inside and outside the bacteria and to monitor the kinetics of the transformation of this drug by living mycobacteria. The results revealed that activation of ethionamide is coupled to a complex molecular sorting of the metabolites and to a selective intracellular accumulation of one of them.


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Bacterial strains and growth conditions

Mycobacterium smegmatis mc2155 and the two derivatives Ms-ethA and Ms-ethR harbouring respectively pMV261-ethA and pMV261-ethR were described previously.5 Cells were grown under standard conditions in Sauton medium10 supplemented with 0.001% ZnSO4 and 0.25% Triton WR1339, in 400 mL Erlenmeyer flasks, under constant shaking at 200 rpm and at 37°C. For Ms-ethA and Ms-ethR cultures, kanamycin (Sigma) was added at a final concentration of 100 mg/L.

NMR analyses

Bacterial samples were introduced into the 4 mm Zr rotor by centrifugation through a plastic tip, as described previously.9 The rotor was introduced in the HRMAS probe on a 600 MHz Bruker DMX spectrometer, keeping the temperature at 293 K. Spinning speed was 6 kHz, and TOCSY spectra were acquired with a rotor synchronized pulse sequence,11 using a MLEV-17 spin-lock train with a spin-lock field of 12 kHz and a total duration of 54 ms. Spectra were recorded as a 1024 x 200 complex matrix. Double Fourier transformation was applied after zero-filling and squared sine bell multiplication. The integral of the aromatic cross peak at (7.59, 8.68) ppm corresponding to ETH* was normalized to the integral of the two aromatic cross peaks at (6.95, 7.61) and (6.89, 7.57) ppm. When normalized to the number of scans the absolute value of their integral varied only slightly over the different spectra recorded and was therefore used for the normalization.

For the time series with strain Ms-ethA, we recorded the spectra with 16 scans per increment, whereas this number was increased to 480 for both the Ms-ethR and the M. smegmatis mc2155 samples.


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Production of ETH* depends on ethA expression

To determine whether the appearance of ETH* in the mycobacteria depends on the presence of the monooxygenase EthA, we used recombinant bacteria expressing various levels of ethA. The EthA-overproducing M. smegmatis strain Ms-ethA was constructed previously.5 Decreased production of EthA was achieved in the M. smegmatis Ms-ethR, a strain that overproduces the transcriptional repressor EthR, known to strongly repress ethA.5 Cultures of Ms-ethA, Ms-ethR and M. smegmatis mc2155 were treated with 100 mg/L of ethionamide. The cells were then extensively washed before being compacted in a HRMAS-NMR rotor, and NMR Total Correlated SpectroscopY (TOCSY) spectra were recorded [Figure 1S; available as Supplementary data at JAC Online (http://jac.oxfordjournals.org/)]. Whereas significant amounts of ETH* were detected by HRMAS-NMR in Ms-ethA, no trace of this ethionamide metabolite was observed in the Ms-ethR cells, even after prolonged treatment (up to 16 h). Compared with Ms-ethA, non-recombinant M. smegmatis mc2155 accumulated 34-fold less ETH*. These observations indicate that ETH* production correlates with the level of ethA expression. When the Ms-ethA culture was saturated with argon before being treated with ethionamide, no trace of ETH* was detected in the mycobacteria (data not shown), indicating that molecular oxygen is necessary for ETH* production, which is consistent with the monooxygenase function of EthA.

Interestingly, the TOCSY spectra of Ms-ethA treated with high concentrations of ethionamide revealed no traces of unmetabolized ethionamide inside the cells, even after prolonged contact (up to 24 h) with the drug. In addition, no trace of ethionamide was found within treated Ms-ethR cells, suggesting that, in the absence of EthA, ethionamide is quickly expelled or is unable to penetrate the mycobacterial cell.

Analysis of the metabolites in the growth medium of ethionamide-treated cultures

As the metabolization of ethionamide appears to be correlated with ethA expression, we investigated the fate of ethionamide in the culture medium of Ms-ethR and Ms-ethA treated with ethionamide. The growth medium of both cultures treated with 100 mg/L of ethionamide for 0.5, 3 and 6 h was analysed by NMR TOCSY. By integrating the aromatic cross-correlation peak of ethionamide, we found that the ethionamide concentration in the growth medium of the Ms-ethA cell culture dropped to 60% after 3 h and to 44% after another 3 h (Figure 1). In contrast, the ethionamide concentration in the growth medium of the Ms-ethR culture did not change over a period of 6 h, indicating that no drug metabolization, incorporation into the cells or significant spontaneous oxidation had occurred during this period. These observations indicate that, at minute levels of ethA expression, ethionamide is not metabolized into an alternative compound but remains intact in the culture supernatant of the mycobacteria. In addition, they support the hypothesis that ethionamide either is expelled very rapidly if not activated or that its penetration into the bacterial cell is coupled to its activation by EthA.


Figure 1
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  		Figure 1. Kinetics of ethionamide metabolism. Decrease of ethionamide concentration in the growth medium of Ms-ethA treated with 100 mg/L ethionamide for indicated times. Decrease of ethionamide concentration was determined by integration of correlation peaks at the different time points and is expressed in arbitrary units relatively to the ethionamide NMR signal recorded after 30 min of culture.

 
Interestingly, in addition to the peaks of unmetabolized ethionamide at {delta} = 7.56 and {delta} = 8.52 ppm, signals corresponding to two other aromatic molecules, at {delta} = 7.46 and {delta} = 8.58 ppm and {delta} = 7.30 and {delta} = 8.40 ppm, respectively, were monitored in the growth medium of ethionamide-treated Ms-ethA. These signals were absent in the growth medium of ethionamide-treated Ms-ethR. NMR TOCSY spectra of synthetic standards corresponding to various previously proposed ethionamide-derived metabolites, including ETH S-oxide (ETH-SO), 2-ethyl-4-cyanopyridine (ETH-nitrile), 2-ethyl-4-amidopyridine (ETH-amide), 2-ethyl-4-carboxypyridine (ETH-acid), 2-ethyl-4-aldehydopyridine (ETH-aldehyde) and 2-ethyl-4-hydroxymethylpyridine (ETH-alcohol; ETH-OH), were compared with the signals observed in the culture supernatant. The absence of a proton signal around 10 ppm in the NMR spectra of the two compounds present in the growth medium excludes the ETH-aldehyde intermediate as one of them. Comparison of the standard with the extracellular metabolites identifies the cross-peak at {delta} = 7.46 and {delta} = 8.58 ppm as ETH-SO, and the second, less intense cross-peak at {delta} = 7.38 and {delta} = 8.39 ppm as ETH-OH (Figure 2a).


Figure 2
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  		Figure 2. (a) TOCSY spectra of the culture medium of Ms-ethA treated with 100 mg/L ethionamide, after 30 min (left-hand panel), 3 h 30 min (middle panel) or 6 h (right-hand panel). Ethionamide-derived products were assigned by comparison with spectra of reference compounds.9 (b) Relative concentrations of the different ethionamide metabolites in the supernatant of an Ms-ethA culture treated with 100 mg/L ethionamide. The values correspond to the peak integrals of the indicated aromatic compounds expressed in arbitrary units relatively to the ‘combined’ signals recorded at 30 min.

 
The progressive reduction of the ethionamide concentration in the growth medium of Ms-ethA cells correlated with the appearance of, first, the S-oxide derivative, followed by the alcohol derivative (Figure 2). In the Ms-ethA culture supernatant, the S-oxide represented 13% of the remaining ethionamide concentration after 30 min, and this proportion increased to 56% and 85% after 3 and 6 h, respectively. However, the latter increase was not due to a further accumulation of S-oxide but rather due to the decrease of the ethionamide concentration. Indeed, the absolute quantity of S-oxide reached a threshold after 3 h, whereas between 3 and 6 h the ETH-alcohol concentration roughly doubled. This observation suggests that the ETH-alcohol derives from the SO metabolite in a secondary reaction. Even if the appearance of some minor resonances at the longest incubation time of 6 h is taken into account, the sum of the integrals of all aromatic correlation peaks recorded at 6 h (i.e. the remaining ethionamide, ETH-SO and ETH-alcohol) points to a global loss of ethionamide-derived compounds in the supernatant. This loss correlates with the appearance of ETH* within the bacterial cells, and from the combined integrals (Figure 2b), we estimate that after 6 h 20% of the ethionamide had accumulated in the form of ETH* within the mycobacteria.

Intrabacterial accumulation of ETH*

NMR TOCSY analysis of the growth medium of ethionamide-treated Ms-ethA cells revealed no trace of ETH*, strongly suggesting that ETH* is unable to cross the bacterial envelope, but accumulates within the cells during ethionamide treatment. To test this hypothesis, the kinetics of the ETH* appearance within the bacterial cells was recorded by NMR after treatment with ethionamide. Cells were treated with the drug for 0.5, 3 and 6 h, extensively washed with PBS to eliminate growth medium, compacted by centrifugation in an NMR rotor and analysed. A substantial amount of ETH* was already detected at 30 min. The amount of ETH* increased 4-fold after 3 h and ~5-fold after 6 h. [Supplementary Figure 2S and Figure 3; Figure 2S is available as Supplementary data at JAC Online (http://jac.oxfordjournals.org/)].


Figure 3
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  		Figure 3. Kinetics of intracellular ETH* appearance. Relative concentrations (in arbitrary units) of the intracellular ETH* metabolite at the indicated time points, as derived from the integral of the cross-peak at {delta} = 7.59 ppm and {delta} = 8.68 ppm.

 
To confirm that the generated ETH* was cell associated, we used an NMR diffusion filter methodology.12 This method suppresses NMR signals of molecules that diffuse freely along the gradient axis in the HRMAS rotor, which might have corresponded to ethionamide metabolites present in the extracellular milieu. When the diffusion filter was applied after TOCSY analyses performed at 0.5, 3 and 6 h of ethionamide treatment, the ETH* signal consistently survived the filter. These results confirm that even after prolonged period of treatment, ETH* remains located exclusively within the cells.


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Ethionamide, like many other powerful antimycobacterial drugs, is a prodrug, and its activation takes place within the targeted bacterium itself. We5 and others6 have recently shown that mycobacteria are to some degree resistant to ethionamide because of their limited capacity to activate the prodrug. Accordingly, genetic constructs leading to an artificial overproduction of EthA resulted in a drastically increased susceptibility of the bacteria to ethionamide. In the present study, we show that repression of the production of EthA leads not only to the absence of activation of ethionamide, but also to the incapacity for the prodrug to enter or remain within the cell. This unexpected result suggests that the penetration or retention of ethionamide in the bacterial cell is coupled to the active EthA monooxygenase enzyme.

As revealed here, the overproduction of EthA in mycobacteria leads to a better activation of the prodrug, and, as demonstrated before,5 to a drastic increase of the susceptibility to the drug. Three metabolites of ethionamide were identified using HRMAS-NMR whereby intra- and extracellular molecules could be distinguished. Since the HRMAS-NMR signal intensity requires a high degree of rotational freedom for the molecules, we could exclude a residual quantity of molecules stuck to the envelope as a source of the NMR signal, provided the cells were carefully washed before NMR analysis. The signal of molecules from the growth medium that may nevertheless have accidentally survived the washing procedure would have been efficiently eliminated by the NMR diffusion filter. In these experimental conditions, only one of the three molecules, named ETH*, was observed within the bacterial cells, whereas the two others, identified as ETH-SO and ETH-alcohol, were exclusively found in the extracellular milieu. This excludes an effective antibiotic action for the latter two and makes the remaining ETH* the prime active compound candidate. ETH* is an ethionamide-derived metabolite, different from all the metabolites identified previously. However, for the time being, we have not been able to purify and characterize the precise nature of ETH* by using classical HPLC/mass spectrometry procedures. This is most likely due to problems of product alteration during the purification steps.

DeBarber et al.6 used the very sensitive radioactive labelling as a detection method for the metabolites of [14C]-ETH and identified them as ETH-SO and ETH-alcohol. However, their use of TLC after cell lysis implies that the molecules are exposed to unspecific air or silica-catalysed oxidation. ETH*, however, might correspond to the very polar compound which stayed at the bottom of the TLC in the DeBarber et al.6 study. This polar compound remained unidentified by the authors, but was EthA and molecular oxygen dependent and was found to accumulate over time.

Vannelli et al.7 and more recently Fraaije et al.13 have shown that recombinant EthA is able to convert ethionamide to ETH-SO in vitro. Both groups have shown that EthA is membrane associated when produced in E. coli. The same behaviour was observed when EthA was overproduced in recombinant mycobacteria (data not shown), suggesting a functional association of the protein with the cellular membrane (Figure 4). Hence, a plausible hypothesis might be that the transformation of ethionamide into ETH-SO and ETH-OH occurs at the membrane, and that these two metabolites are then rapidly released into the extracellular milieu.


Figure 4
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  		Figure 4. Model of the compartmentalized activation of ethionamide. Ethionamide is metabolized by EthA into ETH-SO, which is subsequently transformed into ETH-OH. Both metabolites are exclusively present outside of the bacterial cell and accumulate over time (see the culture medium graph). In parallel, ethionamide is metabolized into ETH*, which accumulates exclusively in the cytoplasmic compartment (see the intracellular graph). The letter ‘a’ denotes concentrations of ETH metabolites are expressed in arbitrary units based on NMR signal intensities (see Figures 2 and 3 for details).

 
ETH-SO is toxic to the mycobacteria and its MIC varies according to the levels of EthA in the bacterial cell.6 This observation is in agreement with the capacity of EthA to transform in vitro ETH-SO into another metabolite (Figure 4).7 ETH*, observed exclusively within cells, and the final toxic compound are likely to be identical, although we cannot formally exclude at this stage that the true antibiotic species derives from ETH*.

In conclusion, similar to pyrazinamide, the accumulation of ETH* appears to be critical for the susceptibility of mycobacteria to ethionamide. This may turn out to be a common theme for antimicrobial prodrugs, and it may thus be interesting to study a possible accumulation of activated isoniazid or other prodrugs in mycobacteria. Particularly hydrophobic properties of the mycobacterial cell wall have hampered the development of new efficient antimycobacterial drugs. In that context, prodrugs may represent an interesting alternative of compounds with low toxicity that would be designed to efficiently cross the envelope before being activated and subsequently trapped in the bacteria.


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None to declare.


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Figures S1 and S2 are available as Supplementary data at JAC Online (http://jac.oxfordjournals.org/).


   Footnotes
 
{dagger}Present address. Department of Medical Protein Research (VIB09), Flanders Interuniversity Institute for Biotechnology, Faculty of Medicine and Health Sciences, Ghent University, A. Baertsoenkaai 3, B-9000 Gent, Belgium Back

{ddagger}These authors contributed equally to this work. Back


   Acknowledgements
 
This work was supported by INSERM, CNRS, Région Nord-Pas de Calais, and Institut Pasteur de Lille (IFR142). X. H. and P. R.-P. were funded by a doctoral fellowship of the Ministry of Research and Technology (MRT, France).


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1 Zhang Y, Scorpio A, Nikaido H, et al. (1999) Role of acid pH and deficient efflux of pyrazinoic acid in unique susceptibility of Mycobacterium tuberculosis to pyrazinamide. J Bacteriol 181:2044–9.[Abstract/Free Full Text]

2 Morlock GP, Metchock B, Sikes D, et al. (2003) ethA, inhA, and katG loci of ethionamide-resistant clinical Mycobacterium tuberculosis isolates. Antimicrob Agents Chemother 47:3799–805.[Abstract/Free Full Text]

3 Zhang Y, Heym B, Allen B, et al. (1992) The catalase peroxidase gene and isoniazid resistance of Mycobacterium tuberculosis. Nature (London) 358:591–3.[CrossRef][Medline]

4 Heym B, Zhang Y, Poulet S, et al. (1993) Characterization of the katG gene encoding a catalase-peroxidase required for the isoniazid susceptibility of Mycobacterium tuberculosis. J Bacteriol 175:4255–9.[Abstract/Free Full Text]

5 Baulard AR, Betts JC, Engohang-Ndong J, et al. (2000) Activation of the pro-drug ethionamide is regulated in mycobacteria. J Biol Chem 275:28326–31.[Abstract/Free Full Text]

6 DeBarber AE, Mdluli K, Bosman M, et al. (2000) Ethionamide activation and sensitivity in multidrug-resistant Mycobacterium tuberculosis. Proc Natl Acad Sci USA 97:9677–82.[Abstract/Free Full Text]

7 Vannelli TA, Dykman A, Ortiz De Montellano PR. (2002) The antituberculosis drug ethionamide is activated by a flavoprotein monooxygenase. J Biol Chem 277:12824–9.[Abstract/Free Full Text]

8 Rozwarski DA, Grant GA, Barton DHR, et al. (1998) Modification of the NADH of the isoniazid target (InhA) from Mycobacterium tuberculosis. Science 279:98–102.[Abstract/Free Full Text]

9 Hanoulle X, Wieruszeski JM, Rousselot-Pailley P, et al. (2005) Monitoring of the ethionamide pro-drug activation in mycobacteria by 1H high resolution magic angle spinning NMR. Biochem Biophys Res Commun 331:452–8.[CrossRef][ISI][Medline]

10 Sauton MB. (1912) Sur la nutrition minérale du bacille tuberculeux. C R Acad Sci 155:860–1.

11 Wieruszeski JM, Montagne G, Chessari G, et al. (2001) Rotor synchronization of radiofrequency and gradient pulses in high-resolution magic angle spinning NMR. J Magn Reson 152:95–102.[CrossRef][ISI][Medline]

12 Warrass R, Wieruszeski J-M, Lippens G. (1999) Efficient suppression of solvent resonances in HR-MAS of resin-supported molecules. J Am Chem Soc 121:3787–8.[CrossRef]

13 Fraaije MW, Kamerbeek NM, Heidekamp AJ, et al. (2004) The prodrug activator EtaA from Mycobacterium tuberculosis is a Baeyer-Villiger monooxygenase. J Biol Chem 279:3354–60.[Abstract/Free Full Text]


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