The Amitochondriate Eukaryote Trichomonas vaginalis Contains a Divergent Thioredoxin-linked Peroxiredoxin Antioxidant System

Trichomonas is an amitochondriate parasitic protozoon specialized for an anaerobic lifestyle. Nevertheless, it is exposed to oxygen and is able to cope with the resultant oxidative stress. In the absence of glutathione, cysteine has been thought to be the major antioxidant. We now report that the parasite contains thioredoxin reductase, which functions together with thioredoxin and thioredoxin peroxidase to detoxify potentially damaging oxidants. Thioredoxin reductase and thioredoxin also reduce cystine and so may play a role in maintaining the cellular cysteine levels. The importance of the thioredoxin system as one of the major antioxidant defense mechanisms in Trichomonas was confirmed by showing that the parasite responds to environmental changes resulting in increased oxidative stress by up-regulating thioredoxin and thioredoxin peroxidases levels. Sequence data indicate that the thioredoxin reductase of Trichomonas differs fundamentally in structure from that of its human host and thus may represent a useful drug target. The protein is generally similar to thioredoxin reductases present in other lower eukaryotes, all of which probably originated through horizontal gene transfer from a prokaryote. The phylogenetic signal in thioredoxin peroxidase is weak, but evidence from trees suggests that this gene has been subject to repeated horizontal gene transfers from different prokaryotes to different eukaryotes. The data are thus consistent with the complexity hypothesis that predicts that the evolution of simple pathways such as the thioredoxin cascade are likely to be affected by horizontal gene transfer between species. Trichomonas is an amitochondriate parasitic protozoon specialized for an anaerobic lifestyle. Nevertheless, it is exposed to oxygen and is able to cope with the resultant oxidative stress. In the absence of glutathione, cysteine has been thought to be the major antioxidant. We now report that the parasite contains thioredoxin reductase, which functions together with thioredoxin and thioredoxin peroxidase to detoxify potentially damaging oxidants. Thioredoxin reductase and thioredoxin also reduce cystine and so may play a role in maintaining the cellular cysteine levels. The importance of the thioredoxin system as one of the major antioxidant defense mechanisms in Trichomonas was confirmed by showing that the parasite responds to environmental changes resulting in increased oxidative stress by up-regulating thioredoxin and thioredoxin peroxidases levels. Sequence data indicate that the thioredoxin reductase of Trichomonas differs fundamentally in structure from that of its human host and thus may represent a useful drug target. The protein is generally similar to thioredoxin reductases present in other lower eukaryotes, all of which probably originated through horizontal gene transfer from a prokaryote. The phylogenetic signal in thioredoxin peroxidase is weak, but evidence from trees suggests that this gene has been subject to repeated horizontal gene transfers from different prokaryotes to different eukaryotes. The data are thus consistent with the complexity hypothesis that predicts that the evolution of simple pathways such as the thioredoxin cascade are likely to be affected by horizontal gene transfer between species. Trichomonas vaginalis is the protozoan parasite responsible for trichomoniasis in humans (1Petrin D. Delgaty K. Bhatt R. Garber G. Clin. Microbiol. Rev. 1998; 11: 300-317Crossref PubMed Google Scholar). This is the most common non-viral sexually transmitted infection, with an estimated >170 million cases occurring each year (2World Health Organization Global Prevalence and Incidence of Selected Curable Sexually Transmitted Diseases: Overview and Estimates. 2001; (WHO/CDS/CSR/EDC/2001.10)Google Scholar), and has been implicated as a major risk factor in predisposition to human immunodeficiency virus/AIDS (3Sorvillo F. Smith L. Kerndt P. Ash L Emerg. Infect. Dis. 2001; 7: 927-932Crossref PubMed Scopus (202) Google Scholar). Indeed it has been suggested that successful treatment of trichomoniasis may be the most cost-effective means by which to reduce human immunodeficiency virus incidence (3Sorvillo F. Smith L. Kerndt P. Ash L Emerg. Infect. Dis. 2001; 7: 927-932Crossref PubMed Scopus (202) Google Scholar). Current chemotherapy relies upon a single group of drugs, the 5-nitroimidazoles, and there are worrying signs that drug resistance may be emerging as a significant problem (4Upcroft P. Upcroft J.A. Clin. Microbiol. Rev. 2001; 14: 150-164Crossref PubMed Scopus (433) Google Scholar). Thus there is a need for new chemotherapeutic tools. The parasite itself is an unusual eukaryote and has been considered to be one of the earliest branching organisms (5Sogin M.L. Curr. Opin. Genet. Dev. 1997; 7: 792-799Crossref PubMed Scopus (73) Google Scholar), although current evidence for this view is not compelling (6Baldauf S.L. Roger A.J. Wenk-Siefert I. Doolittle W.F. Science. 2000; 290: 972-977Crossref PubMed Scopus (975) Google Scholar, 7Embley T.M. Hirt R.P. Curr. Opin. Genet. Dev. 1998; 8: 624-629Crossref PubMed Scopus (231) Google Scholar, 8Roger A.J. Am. Nat. 1999; 154: S146-S163Crossref PubMed Google Scholar). It lacks conventional mitochondria but possesses organelles termed hydrogenosomes that share common ancestry with mitochondria (9Dyall S.D. Johnson P.J. Curr. Opin. Microbiol. 2000; 3: 404-411Crossref PubMed Scopus (109) Google Scholar, 10van der Giezen M. Slotboom D.J. Horner D.S. Dyal P.L. Harding M. Xue G.P. Embley T.M. Kunji E.R. EMBO J. 2002; 21: 572-579Crossref PubMed Scopus (80) Google Scholar) and appear to be adaptations for the parasite's existence in an environment containing only low oxygen concentrations. Trichomonads are fundamentally fermentative organisms, with oxygen apparently not making a significant contribution to energy metabolism (11Martin W. Muller M. Nature. 1998; 392: 37-41Crossref PubMed Scopus (905) Google Scholar). Nevertheless, the cells are exposed to oxygen in the natural environment. This was most elegantly demonstrated by the finding that T. vaginalis isolated from patients unresponsive to the standard chemotherapy treatment with metronidazole is drug-resistant in in vitro tests, but only if oxygen is present (4Upcroft P. Upcroft J.A. Clin. Microbiol. Rev. 2001; 14: 150-164Crossref PubMed Scopus (433) Google Scholar, 12Rasoloson D. Tomkova E. Cammack R. Kulda J. Tachezy J. Parasitology. 2001; 123: 45-56Crossref PubMed Scopus (48) Google Scholar, 13Rasoloson D. Vanacova S. Tomkova E. Razga J. Hrdy I. Tachezy J. Kulda J. Microbiology. 2002; 148: 2467-2477Crossref PubMed Scopus (69) Google Scholar, 14Land K.M. Johnson P.J. Drug Res. Updates. 1999; 2: 289-294Crossref PubMed Scopus (65) Google Scholar). The implications for T. vaginalis of exposure to oxygen have been pondered over many years. Despite a report of the beneficial effect of low oxygen concentrations (growth being significantly enhanced (15Paget T.A. Lloyd D. Mol. Biochem. Parasitol. 1990; 41: 65-72Crossref PubMed Scopus (49) Google Scholar)), it is generally considered that oxygen provides problems rather than benefits. Some of the parasite's enzymes are inactivated by oxygen itself, notably key proteins of the hydrogenosomes, and various metabolites likely to arise from the metabolism of oxygen (such as hydrogen peroxide and hydroxyl free radical) are generally harmful to cells and so need to be countered. Most eukaryotes have glutathione as a key redox buffer and antioxidant, but trichomonads lack this and similar thiols (16Ellis J.E. Yarlett N. Cole D. Humphreys M.J. Lloyd D. Microbiology. 1994; 140: 2489-2494Crossref PubMed Scopus (68) Google Scholar). Thus cysteine has been considered the major cellular reducing agent and antioxidant, although T. vaginalis is able to generate thiols (propanethiol, methanethiol, and hydrogen sulfide, from the action of the unusual bacterial-like enzyme methionine-γ-lyase (17McKie A.E. Edlind T. Walker J. Mottram J.C. Coombs G.H. J. Biol. Chem. 1998; 273: 5549-5556Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar)), which have been postulated to have antioxidant roles (16Ellis J.E. Yarlett N. Cole D. Humphreys M.J. Lloyd D. Microbiology. 1994; 140: 2489-2494Crossref PubMed Scopus (68) Google Scholar). Nevertheless, it was believed that the organism relies heavily upon cytosolic NADH oxidase (reducing oxygen to water) and NADPH oxidase (reducing oxygen to hydrogen peroxide) to prevent permeation of oxygen to the hydrogenosomes (18Linstead D.J. Bradley S. Mol. Biochem. Parasitol. 1988; 27: 125-133Crossref PubMed Scopus (45) Google Scholar). However, the generation of hydrogen peroxide by NADPH oxidase, and superoxide dismutase, poses the question as to how this and other reactive oxygen species (ROS) 1The abbreviations used are: ROSreactive oxygen speciesTRXRgene encoding thioredoxin reductaseTrxRprotein encoded by TRXRrTrxRrecombinant TrxR (equivalent nomenclature is used for thioredoxin (Trx) and thioredoxin peroxidase (TrxP) TrxP is also known as peroxiredoxin)DTNB5,5′-dithiobis(2-nitrobenzoic acid)E-64N-trans-epoxysuccinyl-l-leucine-4-guanidinobutylamideHGThorizontal gene transferESTexpressed sequence tagRACErapid amplification of cDNA endMLmaximum likelihood. are removed as T. vaginalis lacks catalase and glutathione-dependent peroxidase activities. An ascorbate peroxidase has been reported (19Page-Sharp M. Behm C.A. Smith G.D. Microbiology. 1996; 142: 207-211Crossref PubMed Scopus (21) Google Scholar), but it seemed likely that another system must also exist. reactive oxygen species gene encoding thioredoxin reductase protein encoded by TRXR TrxR (equivalent nomenclature is used for thioredoxin (Trx) and thioredoxin peroxidase (TrxP) TrxP is also known as horizontal gene transfer sequence amplification of cDNA likelihood. of peroxidases has been in that not glutathione as the but are by a protein known as thioredoxin which is itself by thioredoxin reductase J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar, E.R. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, E.R. 1999; PubMed Scopus Google Scholar). now known as are in eukaryotes with there being a of in different cellular E.R. 1999; PubMed Scopus Google Scholar, 1999; PubMed Scopus (202) Google Scholar, W. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus (202) Google Scholar, N. D. L. P. S. 1998; PubMed Scopus Google Scholar, L. Biol. Chem. 2002; Google Scholar). It has been that reduce hydrogen peroxide and and a major cellular system the of oxidative J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar, L. Biol. Chem. 2002; Google Scholar). may in all eukaryotes but has been to be of to and the parasite as a means of in the absence of enzymes such as catalase and glutathione peroxidases L. Biol. Chem. 2002; Google Scholar, Smith K. J. Parasitol. 1998; PubMed Scopus Google Scholar, Coombs G.H. Parasitol. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, R.P. S. Embley T.M. Coombs G.H. Parasitol. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar, S. E. Parasitol. Full Text Full Text PDF PubMed Scopus Google Scholar). metabolism may be of importance to amitochondriate eukaryotes, such as the and as as all lack glutathione S. E. Parasitol. Full Text Full Text PDF PubMed Scopus Google Scholar, L. P. 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M. was from T. vaginalis the The system was used to the of the and used for cDNA and amplification of and and and and as in I. was and the for of for for and for for and The sequence of the of each the was by the sequence of from each of in a new The was from and and the for the The to of the the The was with and and to and to generate In the and the the was from and and to The TRXR was from by cDNA was and as a to the The TRXR was and and the system for of for for and for for and The and containing the TRXR sequence was with the enzymes to The sequence of each was confirmed by the of Sequence was for protein proteins have a to of and T. vaginalis TrxR and TrxP proteins to proteins from the Res. 1994; PubMed Scopus Google Scholar). N. R. M.J. Res. 1996; PubMed Scopus Google Scholar). and than of an removed the to phylogenetic and removed for the for TrxR a of was for of was also used for a of for the The K. Mol. 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Full Text PDF PubMed Google Scholar) the as as of NADPH of reductase of the was by the of NADPH in a of and concentrations of the The of upon the of and TrxP in T. in of in with for the which in with in a over was by with anaerobic The standard was by of ascorbate present of to of significantly affected over and was for the and as (17McKie A.E. Edlind T. Walker J. Mottram J.C. Coombs G.H. J. Biol. Chem. 1998; 273: 5549-5556Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). and of TRXR of T. of an from T. vaginalis with sequence to TRXR the of the The was confirmed by TRXR of T. vaginalis is to a protein of with a of Thus the enzyme is similar in to low of and many lower eukaryotes. proteins fundamentally from the of humans and other eukaryotes, P. R.P. S. Embley T.M. Coombs G.H. Parasitol. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar, S. M.L. K. J. 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E. and human and TrxP of T. vaginalis as a and TrxP of T. vaginalis and in in E. with a confirmed the of and and and that a of as a reductase, from NADPH to with as the T. vaginalis TrxR also the a low This is in to the E. which lacks such J. Biol. Chem. Full Text PDF PubMed Google Scholar). The of NADPH the was and the was In the of E. was the cystine also as the with the of cystine to The of and cystine by is and the of the to cystine that in this the cystine is Nevertheless, the finding that cystine is by the thioredoxin system suggests that it may be responsible for maintaining cysteine major redox buffer of in its cystine NADPH in the absence of The of hydrogen to a of and in a of the NADPH the was if was which that the T. vaginalis TrxR system is as as different from the of many prokaryotes 2000; PubMed Scopus Google Scholar, M. J. Biochem. 2000; PubMed Scopus Google Scholar) and the enzyme of E. I. S. E. Biochem. J. 1997; PubMed Scopus Google Scholar, I. S. E. Biochem. 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The TrxP a by of cysteine but not the other to the and this is in with the that cysteine as a major in T. of cysteine upon the of with the protein apparently being there was a increased of the The of and protein for was cysteine was but significantly increased by of and TrxR of T. vaginalis the and and TrxR as a which also the TrxR from the The of this is by the of which not of the used to the in all of The TrxR from the G. and S. which lack with the of this The of the T. vaginalis TrxR was not by although it also lacks the of the of and not with In most the T. vaginalis sequence the of the in However, M. Mol. 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TrxP to the of but a phylogenetic that the for eukaryotes is not of The is thus consistent with repeated horizontal gene transfers from prokaryotes to eukaryotes of for TrxP and T. vaginalis the gene for TrxP from the other eukaryotes gene transfer from prokaryotes to eukaryotes has J. M.L. J. 2002; PubMed Scopus Google Scholar, Embley T.M. Roger A.J. Curr. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar, T.A. Hirt R.P. Embley T.M. 154: PubMed Scopus (65) Google Scholar), with the thioredoxin system being suggested as an of a simple enzyme system for which from to eukaryote is likely to have R. J.A. S. 1999; PubMed Scopus Google Scholar). only are trees consistent with this but in the of T. vaginalis have also that with potentially different The of this that the system provides a means to detoxify in T. It to be not the system is for the of T. vaginalis in its but the finding that S. TrxR glutathione reductase for G. Rev. 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