Iron-sulfur flavoproteins (Isf) are flavin mononucleotide (FMN)- and FeS cluster-containing proteins commonly encountered in anaerobic prokaryotes. However, with the exception of Isf from Methanosarcina thermophila, which participates in oxidative stress management by removing oxygen and hydrogen peroxide, none of these proteins has been characterized in terms of function. Trichomonas vaginalis, a sexually transmitted eukaryotic parasite of humans, was found to express several iron-sulfur flavoprotein (TvIsf) homologs in its hydrogenosomes. We show here that in addition to having oxygen-reducing activity, the recombinant TvIsf also functions as a detoxifying reductase of metronidazole and chloramphenicol, both of which are antibiotics effective against a variety of anaerobic microbes. TvIsf can utilize both NADH and reduced ferredoxin as electron donors. Given the prevalence of Isf in anaerobic prokaryotes, we propose that these proteins are central to a novel defense mechanism against xenobiotics.

Department of Parasitology Charles University Prague Prague Czech Republic

NMR Spectroscopy Unit Institute of Organic Chemistry and Biochemistry Czech Academy of Sciences Prague Czech Republic

Zobrazit více v PubMed

Zhao T, Cruz F, Ferry JG. 2001. Iron-sulfur flavoprotein (Isf) from Methanosarcina thermophila is the prototype of a widely distributed family. J. Bacteriol. 183:6225–6233. 10.1128/JB.183.21.6225-6233.2001 PubMed DOI PMC

Andrade SLA, Cruz F, Drennan CL, Ramakrishnan V, Rees DC, Ferry JG, Einsle O. 2005. Structures of the iron-sulfur flavoproteins from Methanosarcina thermophila and Archaeoglobus fulgidus. J. Bacteriol. 187:3848–3854. 10.1128/JB.187.11.3848-3854.2005 PubMed DOI PMC

Leartsakulpanich U, Antonkine ML, Ferry JG. 2000. Site-specific mutational analysis of a novel cysteine motif proposed to ligate the 4Fe-4S cluster in the iron-sulfur flavoprotein of the thermophilic methanoarchaeon Methanosarcina thermophila. J. Bacteriol. 182:5309–5316. 10.1128/JB.182.19.5309-5316.2000 PubMed DOI PMC

Becker DF, Leartsakulpanich U, Surerus KK, Ferry JG, Ragsdale SW. 1998. Electrochemical and spectroscopic properties of the iron-sulfur flavoprotein from Methanosarcina thermophila. J. Biol. Chem. 273:26462–26469. 10.1074/jbc.273.41.26462 PubMed DOI

Latimer MT, Painter MH, Ferry JG. 1996. Characterization of an iron-sulfur flavoprotein from Methanosarcina thermophila. J. Biol. Chem. 271:24023–24028. 10.1074/jbc.271.39.24023 PubMed DOI

Cruz F, Ferry JG. 2006. Interaction of iron-sulfur flavoprotein with oxygen and hydrogen peroxide. Biochim. Biophys. Acta 1760:858–864. 10.1016/j.bbagen.2006.02.016 PubMed DOI

Johnston VJ, Mabey DC. 2008. Global epidemiology and control of Trichomonas vaginalis. Curr. Opin. Infect. Dis. 21:56–64. 10.1097/QCO.0b013e3282f3d999 PubMed DOI

World Health Organization. 2001. Global prevalence and incidence of selected curable sexually transmitted infections. World Health Organization, Geneva, Switzerland

Petrin D, Delgaty K, Bhatt R, Garber G. 1998. Clinical and microbiological aspects of Trichomonas vaginalis. Clin. Microbiol. Rev. 11:300–317 PubMed PMC

Hrdý I, Tachezy J, Muller M. 2008. Metabolism of trichomonad hydrogenosomes. Microbiol. Monogr. 9:113–145

Lloyd D, Kristensen B. 1985. Metronidazole inhibition of hydrogen production in vivo in drug-sensitive and resistant strains of Trichomonas vaginalis. J. Gen. Microbiol. 131:849–853. 10.1099/00221287-131-4-849 PubMed DOI

Ellis JE, Yarlett N, Cole D, Humphreys MJ, Lloyd D. 1994. Antioxidant defences in the microaerophilic protozoan Trichomonas vaginalis: comparison of metronidazole-resistant and sensitive strains. Microbiology 140(Part 9):2489–2494 PubMed

Linstead DJ, Bradley S. 1988. The purification and properties of two soluble reduced nicotinamide: acceptor oxidoreductases from Trichomonas vaginalis. Mol. Biochem. Parasitol. 27:125–133. 10.1016/0166-6851(88)90032-1 PubMed DOI

Lindmark DG, Muller M. 1974. Superoxide dismutase in the anaerobic flagellates, Tritrichomonas foetus and Monocercomonas sp. J. Biol. Chem. 249:4634–4637 PubMed

Pütz S, Gelius-Dietrich G, Piotrowski M, Henze K. 2005. Rubrerythrin and peroxiredoxin: two novel putative peroxidases in the hydrogenosomes of the microaerophilic protozoon Trichomonas vaginalis. Mol. Biochem. Parasitol. 142:212–223. 10.1016/j.molbiopara.2005.04.003 PubMed DOI

Mentel M, Zimorski V, Haferkamp P, Martin W, Henze K. 2008. Protein import into hydrogenosomes of Trichomonas vaginalis involves both N-terminal and internal targeting signals: a case study of thioredoxin reductases. Eukaryot. Cell 7:1750–1757. 10.1128/EC.00206-08 PubMed DOI PMC

Smutná T, Goncalves VL, Saraiva LM, Tachezy J, Teixeira M, Hrdy I. 2009. Flavodiiron protein from Trichomonas vaginalis hydrogenosomes: the terminal oxygen reductase. Eukaryot. Cell 8:47–55. 10.1128/EC.00276-08 PubMed DOI PMC

Carlton JM, Hirt RP, Silva JC, Delcher AL, Schatz M, Zhao Q, Wortman JR, Bidwell SL, Alsmark UCM, Besteiro S, Sicheritz-Ponten T, Noel CJ, Dacks JB, Foster PG, Simillion C, Van de Peer Y, Miranda-Saavedra D, Barton GJ, Westrop GD, Muller S, Dessi D, Fiori PL, Ren QH, Paulsen I, Zhang HB, Bastida-Corcuera FD, Simoes-Barbosa A, Brown MT, Hayes RD, Mukherjee M, Okumura CY, Schneider R, Smith AJ, Vanacova S, Villalvazo M, Haas BJ, Pertea M, Feldblyum TV, Utterback TR, Shu CL, Osoegawa K, de Jong PJ, Hrdy I, Horvathova L, Zubacova Z, Dolezal P, Malik SB, Logsdon JM, Henze K, Gupta A, Wang CC, Dunne RL, Upcroft JA, Upcroft P, White O, Salzberg SL, Tang P, Chiu CH, Lee YS, Embley TM, Coombs GH, Mottram JC, Tachezy J, Fraser-Liggett CM, Johnson PJ. 2007. Draft genome sequence of the sexually transmitted pathogen Trichomonas vaginalis. Science 315:207–212. 10.1126/science.1132894 PubMed DOI PMC

Schneider RE, Brown MT, Shiflett AM, Dyall SD, Hayes RD, Xie YM, Loo JA, Johnson PJ. 2011. The Trichomonas vaginalis hydrogenosome proteome is highly reduced relative to mitochondria, yet complex compared with mitosomes. Int. J. Parasitol. 41:1421–1434. 10.1016/j.ijpara.2011.10.001 PubMed DOI PMC

Beltrán NC, Horvathova L, Jedelsky PL, Sedinova M, Rada P, Marcincikova M, Hrdy I, Tachezy J. 2013. Iron-induced changes in the proteome of Trichomonas vaginalis hydrogenosomes. PLoS One 8:e65148. 10.1371/journal.pone.0065148 PubMed DOI PMC

Mertens E, Muller M. 1990. Glucokinase and fructokinase of Trichomonas vaginalis and Tritrichomonas foetus. J. Protozool. 37:384–388. 10.1111/j.1550-7408.1990.tb01161.x PubMed DOI

Delgadillo MG, Liston DR, Niazi K, Johnson PJ. 1997. Transient and selectable transformation of the parasitic protist Trichomonas vaginalis. Proc. Natl. Acad. Sci. U. S. A. 94:4716–4720. 10.1073/pnas.94.9.4716 PubMed DOI PMC

Sutak R, Dolezal P, Fiumera HL, Hrdy I, Dancis A, Delgadillo-Correa M, Johnson PJ, Muller M, Tachezy J. 2004. Mitochondrial-type assembly of FeS centers in the hydrogenosomes of the amitochondriate eukaryote Trichomonas vaginalis. Proc. Natl. Acad. Sci. U. S. A. 101:10368–10373. 10.1073/pnas.0401319101 PubMed DOI PMC

Gomes CM, Giuffre A, Forte E, Vicente JB, Saraiva LM, Brunori M, Teixeira M. 2002. A novel type of nitric-oxide reductase. Escherichia coli flavorubredoxin. J. Biol. Chem. 277:25273–25276. 10.1074/jbc.M203886200 PubMed DOI

Fischer DS, Price DC. 1964. A simple serum iron method using the new sensitive chromogen tripyridyl-s-triazine. Clin. Chem. 10:21–31. 10.1016/0009-8981(64)90210-4 PubMed DOI

Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265–275 PubMed

Vicente JB, Justino MC, Goncalves VL, Saraiva LM, Teixeira M. 2008. Biochemical, spectroscopic, and thermodynamic properties of flavodiiron proteins. Methods Enzymol. 437:21–45. 10.1016/S0076-6879(07)37002-X PubMed DOI

Wolff SP. 1994. Ferrous ion oxidation in presence of ferric ion indicator xylenol orange for measurement of hydroperoxides. Methods Enzymol. 233:182–189. 10.1016/S0076-6879(94)33021-2 DOI

Vidakovic MS, Fraczkiewicz G, Germanas JP. 1996. Expression and spectroscopic characterization of the hydrogenosomal [2Fe-2S] ferredoxin from the protozoan Trichomonas vaginalis. J. Biol. Chem. 271:14734–14739. 10.1074/jbc.271.25.14734 PubMed DOI

Hrdý I, Cammack R, Stopka P, Kulda J, Tachezy J. 2005. Alternative pathway of metronidazole activation in Trichomonas vaginalis hydrogenosomes. Antimicrob. Agents Chemother. 49:5033–5036. 10.1128/AAC.49.12.5033-5036.2005 PubMed DOI PMC

Bradley PJ, Lahti CJ, Plumper E, Johnson PJ. 1997. Targeting and translocation of proteins into the hydrogenosome of the protist Trichomonas: similarities with mitochondrial protein import. EMBO J. 16:3484–3493. 10.1093/emboj/16.12.3484 PubMed DOI PMC

Brown MT, Goldstone HM, Bastida-Corcuera F, Delgadillo-Correa MG, McArthur AG, Johnson PJ. 2007. A functionally divergent hydrogenosomal peptidase with protomitochondrial ancestry. Mol. Microbiol. 64:1154–1163. 10.1111/j.1365-2958.2007.05719.x PubMed DOI

Sarti P, Fiori PL, Forte E, Rappelli P, Teixeira M, Mastronicola D, Sanciu G, Giuffre A, Brunori M. 2004. Trichomonas vaginalis degrades nitric oxide and expresses a flavorubredoxin-like protein: a new pathogenic mechanism? Cell. Mol. Life Sci. 61:618–623. 10.1007/s00018-003-3413-8 PubMed DOI PMC

Chapman A, Cammack R, Linstead D, Lloyd D. 1985. The generation of metronidazole radicals in hydrogenosomes isolated from Trichomonas vaginalis. J. Gen. Microbiol. 131:2141–2144 PubMed

Yarlett N, Gorrell TE, Marczak R, Muller M. 1985. Reduction of nitroimidazole derivatives by hydrogenosomal extracts of Trichomonas vaginalis. Mol. Biochem. Parasitol. 14:29–40. 10.1016/0166-6851(85)90103-3 PubMed DOI

Gomes CM, Silva G, Oliveira S, LeGall J, Liu MY, Xavier AV, Rodrigues-Pousada C, Teixeira M. 1997. Studies on the redox centers of the terminal oxidase from Desulfovibrio gigas and evidence for its interaction with rubredoxin. J. Biol. Chem. 272:22502–22508. 10.1074/jbc.272.36.22502 PubMed DOI

Seedorf H, Dreisbach A, Hedderich R, Shima S, Thauer RK. 2004. F420H2 oxidase (FprA) from Methanobrevibacter arboriphilus, a coenzyme F420-dependent enzyme involved in O2 detoxification. Arch. Microbiol. 182:126–137. 10.1007/s00203-004-0675-3 PubMed DOI

Bogdan C. 2001. Nitric oxide and the immune response. Nat. Immunol. 2:907–916. 10.1038/ni1001-907 PubMed DOI

Park GC, Ryu JS, Min DY. 1997. The role of nitric oxide as an effector of macrophage-mediated cytotoxicity against Trichomonas vaginalis. Korean J. Parasitol. 35:189–195. 10.3347/kjp.1997.35.3.189 PubMed DOI

Schwebke JR, Burgess D. 2004. Trichomoniasis. Clin. Microbiol. Rev. 17:794–803. 10.1128/CMR.17.4.794-803.2004 PubMed DOI PMC

Di Matteo A, Scandurra FM, Testa F, Forte E, Sarti P, Brunori M, Giuffre A. 2008. The O2-scavenging flavodiiron protein in the human parasite Giardia intestinalis. J. Biol. Chem. 283:4061–4068. 10.1074/jbc.M705605200 PubMed DOI

Vicente JB, Tran V, Pinto L, Teixeira M, Singh U. 2012. A detoxifying oxygen reductase in the anaerobic protozoan Entamoeba histolytica. Eukaryot. Cell 11:1112–1118. 10.1128/EC.00149-12 PubMed DOI PMC

Moreno SN, Mason RP, Docampo R. 1984. Distinct reduction of nitrofurans and metronidazole to free radical metabolites by Tritrichomonas foetus hydrogenosomal and cytosolic enzymes. J. Biol. Chem. 259:8252–8259 PubMed

Carlier JP, Sellier N, Rager MN, Reysset G. 1997. Metabolism of a 5-nitroimidazole in susceptible and resistant isogenic strains of Bacteroides fragilis. Antimicrob. Agents Chemother. 41:1495–1499 PubMed PMC

Leiros HK, Kozielski-Stuhrmann S, Kapp U, Terradot L, Leonard GA, McSweeney SM. 2004. Structural basis of 5-nitroimidazole antibiotic resistance: the crystal structure of NimA from Deinococcus radiodurans. J. Biol. Chem. 279:55840–55849. 10.1074/jbc.M408044200 PubMed DOI

Mendz GL, Megraud F. 2002. Is the molecular basis of metronidazole resistance in microaerophilic organisms understood? Trends Microbiol. 10:370–375. 10.1016/S0966-842X(02)02405-8 PubMed DOI

Pal D, Banerjee S, Cui J, Schwartz A, Ghosh SK, Samuelson J. 2009. Giardia, Entamoeba, and Trichomonas enzymes activate metronidazole (nitroreductases) and inactivate metronidazole (nitroimidazole reductases). Antimicrob. Agents Chemother. 53:458–464. 10.1128/AAC.00909-08 PubMed DOI PMC

Vazquez D. 1966. Mode of action of chloramphenicol and related antibiotics p 169–191 In Newton PA, Reynolds PE. (ed), Symposium of the Society for General Microbiology, vol 16 Cambridge University Press, Cambridge, United Kingdom

Corbett MD, Chipko BR. 1978. Synthesis and antibiotic properties of chloramphenicol reduction products. Antimicrob. Agents Chemother. 13:193–198. 10.1128/AAC.13.2.193 PubMed DOI PMC

O'Brien RW, Morris JG. 1971. The ferredoxin-dependent reduction of chloramphenicol by Clostridium acetobutylicum. J. Gen. Microbiol. 67:265–271. 10.1099/00221287-67-3-265 PubMed DOI

Fe-S cluster assembly in the supergroup Excavata