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Biotechnology and Applied Biochemistry (2003) 37, (195–204) (Printed in Great Britain)

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Low-molecular-mass thiol compounds from a free-living highly pathogenic amoeba, Naegleria fowleri

Raúl N. Ondarza*†‡¹, Angélica Iturbe*, Eva Hernández* and Gerardo Hurtado*

*Center of Research on Infectious Diseases, National Institute of Public Health, Cuernavaca, Morelos, Mexico, 62508, †Faculty of Medicine, Department of Biochemistry, National Autonomous University of Mexico, University City, Mexico, 04510, and ‡Center for Marine Biotechnology and Biomedicine, Scripps Institution of Oceanography, University of California San Diego, La Jolla, CA 92093–0204, U.S.A.

Key words: cysteine, glutathione, Naegleria thiol compound, ovothiol, potential drug target, trypanothione.

Abbreviations: Gspd, glutathionyl-spermidine; R1 and R2, reagent-derived peaks; DTT, dithiothreitol; NEM, N-ethylmaleimide; RSSR, oxidized thiol; RSH, reduced thiol; PCR, perchloric acid; MALDI-TOF, matrix-assisted laser-desorption ionization–time-of-flight; MALDI-FTMS, MALDI–Fourier-transform MS; mBBr, monobromobimane.

¹To whom correspondence should be addressed, at Olivar de Los Padres 941, Mexico, D.F. 01780 (e-mail ondarza@servidor.unam.mx).

Acid extracts labelled with the fluorescent reagent monobromobimane and separated by HPLC have enabled the detection of low-molecular-mass thiol compounds in Naegleria fowleri for the first time. The amounts detected are expressed in nmol/1×10⁶ trophozoites cultivated at various stages of growth in the appropriate culture medium. N. fowleri is a highly pathogenic free-living amoeba, in which we found important thiol compounds, some of them in their reduced and oxidized forms. Unlike cysteine and glutathione, a number of these are not represented in normal human lymphocytes. Some of these thiol compounds from Naegleria must have their respective disulphide reductases, although the presence of thiol-disulphide exchange reactions must be considered. Ovothiol A, with antioxidant properties, is an example of a compound that is kept reduced by trypanothione in trypanosomatids, although no disulphide reductase for ovothiol A has yet been discovered. In our case we were unable to detect this biothiol in Naegleria. The presence of thiol compounds that seem to be particular to this pathogen and which are not present in human lymphocytes opens the possibility of searching for disulphide-reducing enzymes that can serve as drug targets.

Received 24 September 2002/10 December 2002; accepted 11 December 2002

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