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1 k v r 52 r 3 225~231 (2008) Yakhak Hoeji Vol. 52, No. 3 Helicobacter pylori Fur Metronidazole w Douglas E. BergÁ *,# School of Medicine, Washinghton University in St. Louis, MO , U.S.A. * w w w (Received April 14, 2008; Revised May 8, 2008) Transcriptional Regulatory Protein Fur of Helicobacter pylori and its Role in Metronidazole Resistance Douglas E. Berg and Sung-Sook Choi*,# School of Medicine, Washinghton University in St. Louis, MO 63043, U.S.A. *School of Pharmacy, Sahmyook University, Gongneung-dong, Nowon-gu, Seoul , Korea Abstract To clarify effects of the structural changes of Fur protein on the resistance to metronidazole (Mtz), the mutational analysis of structure and function of the protein in Helicobacter pylori (Hp) was undertaken. It was identified that some changes in Hp Fur protein resulted in increase of resistance to Mtz, and other changes resulted in decrease of resistance. Increase of Mtz resistance came from the enzyme's decreased ability of reducing prodrug Mtz to the form of bactericidal agent. Some sites that affects Mtz resistance (i) in Fur's N terminal extension, and (ii) in its central region, which links DNA binding and Fe-binding modules were identified. It was also found that the addition of FLAG tag to Fur's C terminus also significantly impairs Fur function. Keywords H. pylori, Fur protein, metronidazole resistance Helicobacter pylori(hp) p p or rp n k o p. rp r p p 50% m l o k, omp ppˆ okp op p t p. Hp p p 1-3) l v v 2% p p sq lk sp rp qp o 5~8%p rp. Hp tp p or l r rl t l qp mv pp ppˆ t t e p pp l p rl p p s(reactive oxygen species, ROS)p. 4,5) Hp t l v p o o l r rl o p ROS Hp tl rp r d d qn. p ROSp l Hp tp or l m rp r l v p o # l p rql (r ) (Žd) ( ) sschoi@syu.ac.kr p p Hp p r d dl l rp p l tl iron-cofactored superoxide dismutase(sodb), catalase(kata), hydroperoxide reductase(ahpc) p k r p. ~ p p 6) sl rp mk rqr l l l v p cofactor p p k rp ~ p mp p sq l n p p sp. p rp p ~ p 7) p ov o l r pp sr mrna p p pn p n ferric uptake regulator(fur)p pn p ov p k r. Hp p 8,9) pe l k p ~ l l lp Hpp n ~ p p ov l r d d dd p p tp sl n tn. p rl E. colim v Fur vp t n l p. 10,11) Fur vp p tn r sr pq(transcriptional regulator) t ~, r d dl l r l l orqp srp. 12) 225

226 Douglas E. BergË Fig. 1 Unique and conserved sequence of Helicobacterpylori Fur protein. 13) Hpp Fur vp 150 aa v q p d dp Fur v k 53% r p o p pp srp, rp l r p p p p k r p (Fig. 1).

2 226 Douglas E. BergË Fig. 1 Unique and conserved sequence of Helicobacterpylori Fur protein. 13) Hpp Fur vp 150 aa v q p d dp Fur v k 53% r p o p pp srp, rp l r p p p p k r p (Fig. 1). Hp Fur vp N-terminal mlp DNA bindingl, C-terminal mlp dimerizationl l p k r pp q p d dm vrp prp Hpp Fur vp N- p q k 9~10 r p k dp eq l p p. 13) Albert p Hp rp metronidazole (Mtz)l p Hp t Fur vp N p 3 w k p Argininep Isoleucinep p t p mp k e Mtzl p v p m. p p k 14) Fur vp pp pro-drug p Mtz oe bactericidal hydroxylaminep eˆ rl l p qnp lr. 15) v Hpp Fur vp redox potential srl l q np srl l p l l Fur vp N- p 3 w k p p Mtzl p v eˆ l k l k N- l p p Mtzl p p p l Fur vp N- p p kk q m. x Hp ³ e l n t Hp t o ~ Table Il ˆ lp tp kl rp brain heart infusion agar(difco) vl horse blood(invitrogen) 7%, Isovitalex(BBL) 0.4%, vancomycin(6 µg/ml), amphothericn B (8 µg/ml), Trimethoprim(5 µg/ml)p ~ l n m (p BHI agar ). l p~ p o nl BHI agar vl chloramphenicolp 15 µg/ml, kanamycinp 20 µg/ml l k mp e p o Mtz p nl k ~ l n m. tp k p 37 o C k l 5% O 2, 10% CO 2 85% N 2 d l ˆ ov t k m. e l l pp o l Mtzl r / p spectrump k o l p Mtzl Table I The strains that used in this study Strains Genotypes Phenoypes ref. Hp #1.4 wt fur rdxa-, frxa-, mdab-, ribf- 190R/220S 14,17 Hp #1.4(delata-fur) Delta fur::kan was introduced into # R/190S this study Hp # 1.4 wt fur-cat cat was introduced into # R/220S this study Hp #1.4 wt fur R3I Hp 1027(R3I) 220R/250S 14,17 J. Pharm. Soc. Korea

3 s r Fur v 227 Table II Primers used for H. pylori gene amplification. Italicized means complementary sequences that served as a tail for 3 fragment assembly. Complementary to the Pylori Gene coordinates listed Target site fur flanking region fur-cat fur-kan Arg3 random Flag tag apha gene cat gene Primer name Pylori gene coordinates (5' 3') x5k-f cct taa ttt agc cgc ttc ttg ttt g x4-r ctg tag agt tgc attg gaa ttt gtc a Sequences (5' 3') C1x atc cac ttt tca atc tat atc cca tta aag ata gcc cta tct aag c C2x ccc agt ttg tcg cac tga taa gct tag ata ggg cta tct tta atg g kanf x atg gtt cgc tgg gtt tat cct gat atc ttc ctt atc cgt aaa atg a aphar x tta ctg gat gaa ttg ttt tag tac cgc tta gat agg gct atc ttt aat gg R3n-F gga taa gga aga tat cag cat gaa ann Btt aga aac ttt gga atc ca R3n-R tgg att cca aag ttt cta avn Ntt tca tgc tga tat ctt cct tat cc R2A ttt atc atc gta atc ttt ata atc aca ttc act ctc ttg gca ttc t F3A gat tatt aaa gat gac gat gat aaa taa aag att tta aaa aag aag ctt aga tag ggc tat kanf kanr camf camr gat aaa ccc agc gaa cca ttt ga ggt act aaa aca att cat cca gta a gat ata gat tga aaa gtg gat aga ttt a tta tca gtg cga gaa act ggg orq p orr p p Hp #1.4 (rdxa -, frxa -, mdab -, ribf -, 190R/220S) n m. 16,17) Hp Hpp tn r srpqp Fur vp p k o k fur orq knockout eˆ l p~ l vr e p recipient cell n mp Fur v N- l random mutation p C- l FLAG tag pp o k orqp n vr ~p p d o rp chloramphenicol orqp cat genep marker or q wild type fur orqp down stream o l p l n m. e l n primer Invitrogen(CA, U.S.A.) l l n mp Table IIl n primer ˆ l. Hp fur::kan Tan p 18,19) ure AB :: cat rsm o p pn l r n v k PCR p pn l rs m. fur orq (HP1027) mr r kanamycin resistance cassette (apha, plasmid pip 1433)p p o l fur orq flanking regionl primer x5k-fm x4-r, apha orq p o primer kanfx6m aphar x3 kanfm aphar(1402 bp of kanamycin resitance cssette) n l PCR A(upstream region of fur), B(aphA) C(downstream region of fur) v 3 p PCR p l primer x5k-fm x4-rp pn l q v l s m. Hp wild type fur-cat k fur orql l p o l p~p prp p o l marker orq chloramphenicol resistance cassettep fur orq downstream l p q m. n primer fur orq flanking regionl primer x5k-fm x4-rm cat orq v o l camfm camr (Campylobacter coli plasmid C589) c1x2m c2x3 n m. PCR A(from 500 bp upstream of fur to entire fur region), B(cat region) C(downstream region of fur) v 3 p PCR p primer x5k-fm x4-rp pn l l q v l s m. Error prone PCR fur orql ro k r~orql qo l p o p~p Mtz p p o l error prone PCRp p p ee m. 20) PCR pp 20 f mol wild type genomic DNA(wt fur-cat), 30 pmolep primer(x5k-f, x4-r), 5 unit Taq polymerase, 0.2 mm dgtp, 0.2 mm datp, 1 mm dctp, 1 mm dttp, 0.5 mm MnCl 2, 10 ËPCR Buffer [1 buffer 7mM MgCl 2, 50 mm KCl, 10 mm Tris-Cl(pH 8.3)]p o mp 94 o C 10 denaturaiotn, 94 o C 40, 55 o C 40, 72 o C 2 min rp 30 ee 72 Cl 10 p o s m. PCR» w Fur N- C- Flag tag Random mutation k fur orqp r o vr l l p p q mp r o N l 3 w k p argininep ˆ m. p 3 w k p Argp Ile p n k t Mtz p v p p l, v k v 14,17) R3I Vol. 52, No. 3, 2008

4 228 Douglas E. BergË Fig. 2 PCR-based site specific mutagenesis of fur. The following four primers were used; primer1 (x5k), 2 (R3n-R for codon 3 and R2A for flag tag) 3 (R3n-F for codon 3 and F3A for flag tag), 4 (x4). 3l k k p Mtzl p p q m. Random mutationp p q fur orqp flanking mll primer x5k-fm x4- Rm mutagenic primerp R3n-F R3n-Rp n m. p codon 3p nonsense mutationp p v o l 3 w codonp NNB(forward)/VNN(reverse) qp mp Fig. 2l p PCR p l p~ l. Hp Fur vp rr rp FLAG tagp fur orqp C- l p l p~ lp p p~ pn C- Mtz p l C- p kk q m. Flag tag pp o primer x5k-fm x4-r R2Am F3A n m. Hp x y Hpp vr p p p 21)p Israel p l q l vr p ee m. -70 Cl p o recipient t(hp fur::kan) BHI agarl ˆ l 3p k m. q q t n vl m r k m. p r k t loopp l p n BHI agar platep tkl 4~5 cm v 4~6e r k l exponential phase v k r genomic DNA PCR p mutated DNA ~1 µg p competent cell q over night k m. p t rr r o ˆ vl 2~3p r k vr ~ v p p m. Mtz w d Mtzl r p EOP l16) ee m. -70 o C l p Hp t Mtzp o pv kp BHI agarl 3p k k n drug-free vl e Fig. 3 Semi-quantitative tests of Mtz resistance: efficiency of colony formation by single cells. ~20-fold difference in efficiency colony formation (eop) on Mtz 220 agar. No such difference in eop on Mtz 190 agar (not shown). l k l l n m. r q Hp t PBS bufferl 10 l l lp p pr p Mtz o vl 10 µlj rs l k l k m. r p Žr p p lp spotting Mtz o platel q colony q 10 Mtz Žr m (Fig. 3). š Error prone PCR w fur Hp fur orql qorp l p o fur l J. Pharm. Soc. Korea

5 s r Fur v 229 p~m Mtz p o l error prone PCRp ee m. fur orq upstream ml 500 bp, downstream ml 500 bp r p m l o p p Error prone PCR p Hp #1.4 fur::kan(160r/190s) t l m o l ql vr chloramphenicolp 15 µg/ml o vl 3p k m. chloramphenicol vl q v 30 qo kanamycinp (20 µg/ml) o vm chloramphenicolp 15 µg/ml o v Mtzp 200 µg/ml o vl streaking kanamycin vl q v chloramphenicol vl q v t Mtz 200 µg/ml p l q t fur o rqp l pl p Mtz r p v p Ž p e l n m. qo v p 99% p p kans/camrp p ˆ l m l o l vr p p p n p k pl. Error prone PCR l p Mtzl r p v l p~ q mp k fur t ˆ Mtz p (190 µg/ml<)l q t l DNA m lp p m. Fig. 4m p l p~ l lp tp Mtzl phonotypep 220~250 µg/ml r p o ˆ p k pl. Fur vp r op l p Mtzl r p v eˆ p k pl. r target sitep N-terminal armmlp n s R3I mutantm p R3I l p~ error prone p l 1 t lp E8K(220R/250S) p p~ ll. e p Mtz p v l p~t Furp DNA binding domainp k v 57 (K57T), Fe-binding domainp k v 38(R38H), 40 (G40D), 41(T41A) 95(E95G) w k p l p~ llp p furp l p vr DNA l m p v Fe-binding l l DNA-binding pp l p l Mtz l l p p sr p. 13,22,23) Fur N codon 3 C Flag tag Mtz r l Fur vp N-terminal C-terminalp m p o l PCR p pn l fur o rq N C- r ol site-specific mutationp e e m. Hp Fur vp sp q p d d l l k 9 p k p p eq N- terminal armp p l l p N- terminal armp l l p v p o l l p o phenotypep Mtzl r p p q m. Error prone PCRp random mutation p N-terminal mll k l p~ lv mp v l p~p R3Ip n 3p Argp Ilep p Mtzl p ˆ l l(220r/250s vs 190R/220S) n 3 w k p Argp targetp 14,17) l p ee m. n codon 3p arginine o stop codonp rn k p n (AGAËNNB) mutagenic primer rq m (forward primerp R3 NNB, reverse primer VNNp design l stop codonp p r lr m ). wt fur-cat DNA template PCR pp ee fur orq upstream ml 500 bp, downstream ml 500 bp r p m l o p p PCR p Hp #1.4 fur::kan(160r/190s) tl m o l ql vr CamR/KanS v p qo l k tm Mtzl e p ee op p tp m lp p m. R3S(130R/160S)} p r l p~m, R3G(160R/190S), R3D (160R/190S), R3T(160R/190S) null mutantm p p ˆ l p~ ll. R3P(190R/220S), R3L (190R/220S) wild type p p, R3K(220R250S), R3I(220R/250S) p wild type p v p p m. N-terminal l p~p genotype phenotypep Fig. 5l ˆ l. v N-terminal mlp 3 w k p Mtz l m p ˆ p k pl. Fur vp rr rp C-terminall Flag tagp p q m. p pn l C-terminal Mtz p p q m. Fur vp C-terminalp Fur Fig. 4 Error prone PCR mutagenesis of fur and selected for hyperresistance in many steps. Fur alleles have varied quantitative effects on Mtz resistance. Mutations located near: Fe-binding site (R38H, G40D, T41A, E95G), N- terminal arm (R3I, E8K), DNA-binding domain (K57T). Fig. 5 Spectrum of MtzR values for several R3 fur alleles. MtzR values (µg/ml) were determined as described in materials and methods. Vol. 52, No. 3, 2008

6 230 Douglas E. BergË vp dimerization l o p ˆ rrp l p 24) C-terminalp p l Furp l m p v Mtz r p m. Flag tag pp delta-fur p, fur k p p p plp (160R/190S, data not shown) p flag tag pl p Fur vp dimerizationl m p p p. kp l q p v vp ˆp PCR l k l p~ rs (DNA binding site, iron binding site ) s m p l l Hp Furp n sr (reductive potential ) p Hp t p or l r v p o rl Fur v p p q. Hpp r srpqp Fur vp sl s q p d d srp vp pp l l p vrp mlt N- p p q m. Random mutation site-specific mutationp l p~ rs Mtz p p p p l p mlp r k p Fur vp orq sr rl m p p p pl. v Fur vp p pro-drugp Mtz oe bactericidal hydroxylaminep eˆ rl redox potential l orqp srl l ppp p m. š x 1) Westblom, J. U., Czinn, S. J. and Nedrud, J. G. (ed.) : Current topics in microbiology and immunology, vol. 241, gastroduodenal disease and Helicobacter pylori: pathology, diagnosis and treatment. Springer Press, Berlin, Germany (1999). 2) Parsonnet, J. : Helicobacter and gastric adenocarcinoma, pp In J. Personnet (ed.). Microbes and malignancy: Infection as a cause of human cancer. Oxford University Press, New York, N.Y. (1999). 3) Blaser, M. J. and Berg, D. E. : Helicobacter pylori genetic diversity and risk of human disease. J. Clin. Investig. 107, 767 (2001). 4) Ramarao, N., Gray-Owen, S. D. and Meyer, T. F. : Helicobacter pylori induces but survives the extracellular release of oxygen radical from professional phagocytes using catalase activity. Mol. Microbiol. 38, 103 (2000). 5) Nardone, G., Rocco, A. and Malfertheiner, P. : Helicobacter pylori and molecular events in precancerous gastric lesions. Aliment. Pharmacol. Ther. 20, 261 (2004). 6) Hazell, S. L., Harris, A. G. and Trend, M. A. : Evasion of the toxic effects of oxygen. In Helicobacter pylori; physiology and genetics. Mobley, H. L. T., Mendz, G. L. and Hazell, S. L. (eds.). Washington DC; American Society for Microbiology Press, pp (2001). 7) Tourati, D. : Iron and oxidative stress in bacteria. Arch. Biochem. Biophys. 373, 1(2000). 8) Andrew, S. C., Robinson, A. K. and Rodriguez-Quinones, F. : Bacterial iron homeostasis. FEMS Microbial. Rev. 27, 215 (2003). 9) Bagg, A. and Neidland, J. B. : Ferric uptake regulation protein acts as a repressor, employing iron (II) as a cofactor to bind the operator of an iron transport operon in Escherichia coli. Biochemistry 26, 5471 (1987). 10) Hanke, K. : Iron and metal regulation in bacteria. Curr. Opion. Microbiol. 4, 172 (2001). 11) Florian, D. E., Stefan, B., Barbara, W., Jeroen, S., Ulrike, M., Johannes, G. K., Ernst, J. K., Manfred, K., Aronoud, H. M. van V. and Gerg, H. : Transcriptional profiles of Helicobacter pylori Fur- and iron regulated gene expression. Microbiology 151, 533 (2005). 12) Hall, H. K. and Foster, J. W. : The role of Fur in the acid tolerance response of Salmonella typhimurium is physiologically separable from its role in iron acquisition. J. Bacteriol. 178, 5683 (1996). 13) Pohl, E., Haller, C. J., Mijovilovich, A., Meyer-Klaucke, W., Garman, E. and Vasil, L. M. : Architecture of a protein central to iron homeostasis: crystal structure and spectroscopic analysis of the ferric uptake regulator. Molecular Microbiology 47(4), 903 (2003). 14) Albert, D. T., Dailidien, D., Dalidien, G., Jason E. N., Awdhesh, K., Todd, A. R., Michale, M., Jaz, S., Roland, D. G. and Berg, D. E. : Mutational discovery in bacterial genomes: metronidazole resistance in Helicobacter pylori. Nature Meth. 2, 951 (2005). 15) Garry, S., Avery, G., Ausra, R., Nicky, J. H., Asish, K. M., Berg, D. E. and Hoffman, S. P. : Enzymes associated with reductive activation and action of nitrozoxamide, nitrofurans, and metronidazole in Helicobacter pylori. Antimicrob. Agents Chemother. 146(7), 2116 (2002). 16) Jeong, J. Y., Mukhopadhyay, A. K., Dailidien, D., Wang, Y., Velapatino, B., Gilman, R. H., Parkinson, A. J., Nair, G. B., Wong, B. Y., Lam, S. K., Mistry, R., Segal, I., Yuan, Y., Gao, H., Alarcon, T., Brea, M. L., Ito, Y., Kersulyte, D., Lee, H. K., Gong, Y., Goodwin, A., Hoffman, P. S. and Berg, D. E. : Sequential inactivation of rdxa (HP0954) and frxa (HP0642) nitroreductase genes cause moderate and high-level metronidazole resistance in Helicobacter pylori. J. Bacteriol. 182(18), 5082 (2000). J. Pharm. Soc. Korea

7 s r Fur v ) Dailidien, D., Chang, D., Dailidien, G. and Berg, E. D. : Finding mutations implicated in Mtz hyperrsistance. Unpublished data. 18) Tan, S. and Berg, E. D. : Motility of urease-deficient derivatives of Helicobacter pylori. J. Bacteriol. 186(3), 885 (2004). 19) Dailidien, D., Tan, S., Zhang, O. K., Lee, A. H., Severinor, K. and Berg, E. D. : Urea sensitization caused by separation of Helicobacter pylori RNA polymerase beta and beta' subunit. Helicobacter 12(2), 103 (2007). 20) Vartanian, J. P., Henrry, M. and Wain-Hobson, S. : Hypermutagenic PCR involving all four transition and a sizeable proportion of transversion. Nucleic Acids Resch. 24, 2627 (1996). 21) Israel, D. A., Lou, A. S. and Blaser, M. J. : Characteristics of Helicobacter pylori natural transformation. FEMS Microbial. Lett. 186, 275 (2000). 22) Vasil, L. M. and Ochsner, A. U. : The response of Pseudomonas aeruginosa to iron: genetics, biochemistry and virulence. Mol. Microbiol. 34(3), 399 (1999). 23) Peredo, A. G., Saint-Pierre, C., Latour, J. M., Michaud-Soret, I. and Forest, E. : Conformational changes of the ferric uptake regulation protein upon metal activation and DNA binding; first evidence of structural homologies with the diphtheria toxin repressor. J. Mol. Biol. 310, 83 (2001). 24) Delany, I., Sophon, G., Rappuoli, R. and Scarlato, V. : The Fur repressor controls transcription of iron-activated and -repressed genes in Helicobacter pylori. Mol. Microbiol. 42, 1297 (2001). Vol. 52, No. 3, 2008

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Æ¯ÇãÃ» '' - 1 - - 2 - - 3 - - 4 - - 5 - ' - 6 - - 7 - - 8 - - 9 - - 10 - - 11 - Super-Bio Co., Ltd. KWON, Suk-Tae Thermostable DNA Polymerase-encoding Gene from Thermus sp. X-1 an d Amino Acid Sequence