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Characterization of the respiratory chain of Helicobacter pylori
FEMS Immunology and Medical Microbiology 24 (1999) 169^174
Characterization of the respiratory chain of Helicobacter pylori Ming Chen a
a;b;
*, Leif P. Andersen a , Lin Zhai
a;b
, Arsalan Kharazmi
a
Department of Clinical Microbiology, 7806, University Hospital (Rigshospitalet), Tagensvej 20, DK-2200 Copenhagen, Denmark b Statens Serum Institut, Copenhagen, Denmark Received 25 November 1998 ; accepted 18 February 1999
Abstract The respiratory chain of Helicobacter pylori has been investigated. The total insensitivity of activities of NADH dehydrogenase to rotenone and of NADH-cytochrome c reductase to antimycin is indicative of the absence of the classical complex I of the electron transfer chain in this bacterium. NADPH-dependent respiration was significantly stronger than NADH-dependent respiration, indicating that this is a major respiratory electron donor in H. pylori. Fumarate and malonate exhibited a concentration-dependent inhibitory effect on the activity of succinate dehydrogenase. The activity of succinatecytochrome c reductase was inhibited by antimycin, implying the presence of a classical pathway from complex II to complex III in this bacterium. The presence of NADH-fumarate reductase (FRD) was demonstrated in H. pylori and fumarate could reduce H2 O2 production from NADH, indicating fumarate to be an endogenous substrate for accepting electrons from NADH. The activity of NADH-FRD was inhibited by 2-thenoyltrifluoroacetone. A tentative scheme for the electron transfer pathway in H. pylori is proposed, which may be helpful in clarifying the pathogenesis of H. pylori and in opening new lines for chemotherapy against this bacterium. ß 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Helicobacter pylori; Respiratory chain; Succinate reductase; Fumarate reductase; NADH dehydrogenase; NADPH dehydrogenase
1. Introduction Helicobacter pylori is a Gram-negative, oxidasepositive, microaerophilic, £agellate, curved or spiral bacterium that colonizes the mucous layer of the human gastric epithelium [1^3]. The organism is now accepted as the etiological agent for type B gastritis and the majority of duodenal and gastric ulcers, and is linked to the development of gastric cancer [4^ 6]. Treatment of H. pylori infection has undergone signi¢cant development; from initial monotherapy to
* Corresponding author. Tel.: +45 35 45 77 38; Fax: +45 35 45 68 31; E-mail: [email protected]
dual, triple and, in some cases, quadruple therapy [7^11]. However, drug resistance has caused problems in the treatment of H. pylori infection [12^16]. The ability of H. pylori to develop resistance to 5nitroimidazoles, one of the most important drugs of the therapy, is a major contributing factor to the failure of therapy. So, beside the new combination treatment and the development of a vaccine, there is certainly a need for the development of new therapeutic agents speci¢cally targeted against H. pylori, which will represent a signi¢cant advance in the treatment of the infection. The physiology and metabolism of H. pylori are not fully understood, even though some e¡orts have been devoted to this ¢eld during the last several years [17^
0928-8244 / 99 / $20.00 ß 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 9 2 8 - 8 2 4 4 ( 9 9 ) 0 0 0 2 2 - X
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27]. Understanding these fundamental factors could help to control the infection, for example, by identifying potential drug targets in Helicobacter spp. The aim of this study was to characterize the respiratory chain of H. pylori. We have investigated the activities of the enzymes which are involved in the electron transport chain in the bacterium. A tentative scheme for the electron transfer pathway in H. pylori is proposed.
2. Materials and methods 2.1. Biochemicals All biochemicals and reagents were from Sigma Chemicals, USA, unless otherwise stated. 2.2. Bacterial cultivation and preparation Six clinical isolates of H. pylori (FH1, FH3, FH5, FH8, FH29 and FH47) were used for this study. However, we found little strain variation. Therefore, most of the experiments were carried out on strain FH5. H. pylori was grown microaerobically on 7% lysed horse blood agar plates at 37³C for 48 h. Bacteria were harvested in phosphate bu¡er saline (PBS), pH 7.4, and washed twice. The bacteria were disrupted by sonication at 20 000 Hz for 45 s ¢ve times with a Rapids 300 (19-mm probe with a 9.5-mm tip). During the sonication, preparations were cooled by immersion in iced water. The crude sonicates were centrifuged at 18 000Ug for 30 min at 4³C, the supernatant was collected and then frozen at 380³C until use. 2.3. Exposure of H. pylori preparations to inhibitors Protein concentrations of H. pylori preparations were determined by a Bio-Rad protein assay (BioRad Laboratories, Hercules, CA, USA). H. pylori preparations were adjusted to 1 mg ml31 and incubated with each inhibitor in 50 mM potassium phosphate bu¡er, pH 7.4 or bu¡er alone for 3 min at 37³C, before the enzyme determinations. 2.4. Enzymatic assays All enzymatic activities were determined using a
¢nal protein concentration of 1 mg ml31 in 1-ml cuvettes in 50 mM potassium phosphate bu¡er, pH 7.4 at 37³C. All spectrophotometric determinations were carried out in a Perkin-Elmer Lambda 40 UV/ Vis spectrophotometer. Succinate dehydrogenase activity was measured spectrophotometrically at 600 nm (O = 20.5 mM31 cm31 ) using 3 mM succinate, 0.5 mM 2,6-dichlorophenolindophenol and 0.1 phenazine methosulfate [28]. NADH dehydrogenase activity was determined spectrophotometrically at 420 nm by measuring the rate of potassium ferricyanide (0.5 mM) reduction in the presence of NADH (O = 1 mM31 cm31 ) [29]. Succinate- and NADH-cytochrome c reductase activities were measured spectrophotometrically at 550 nm (O = 18.9 mM31 cm31 ) in the presence of 20 WM cytochrome c and either 5 mM succinate or 0.2 mM NADH [30,31]. NADH-fumarate dehydrogenase (FRD) activity was determined as the rate of NADH oxidation on addition of 1 mM fumarate to the H. pylori sonication. The reaction was monitored by spectrophotometer at 340 nm (O = 6.2 mM31 cm31 ) using 100 WM NADH [32]. 2.5. NADH and NADPH oxidation The NADH and NADPH oxidation were determined by measuring changes in the dissolved oxygen concentrations in a Clarke-type oxygen electrode calibrated with air-saturated 20 mM potassium phosphate bu¡er, pH 7.2 at 37³C [17]. 2.6. Statistical analysis A paired two-tailed t-test was used for analysis of the data. P values of 6 0.05 were considered signi¢cant.
3. Results Table 1 summarizes the activities of several respiratory chain segments and the e¡ect of inhibitors, as determined spectrophotometrically in the sonicate of H. pylori. Antimycin did not inhibit the succinate dehydrogenase of H. pylori. Fumarate and malonate
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Table 1 Mitochondrial redox reduction catalyzed by H. pylori sonicate Enzymes+inhibitors
Succinate dehydrogenase +fumarate (0.1 mM) +malonate (1 mM) +antimycin (4 WM) NADH-dehydrogenase +rotenone (50 WM) Succinate-cytochrome c reductase +antimycin (2 WM) NADH-cytochrome c reductase +antimycin (2 WM) Fumarate reductase +succinate (25 mM) +malonate (10 mM) +antimycin (200 WM) +3-MPA (20 Wg)
Speci¢c activity (nmol min31 mg protein31 ) FH5a
FH1b
FH3b
FH8b
FH29b
FH47b
18.7 þ 0.45 10.5 þ 0.42* 5.9 þ 0.62* 17.7 þ 0.62 175.5 þ 18.2 178.1 þ 6.7 14.7 þ 1.77 9.2 þ 0.59* 21.3 þ 0.79 20.7 þ 1.0 18.2 þ 1.5 14.8 þ 1.4* 14.2 þ 0.38* 16.3 þ 0.54 16.9 þ 0.65
18.0 9.9 5.3 18 170 172 14.2 8.8 20.5 20 17.6 14.6 14 16.5 17.1
19.4 10.2 5.9 18.2 188 186 15.6 9.2 22.4 22.2 20 14.6 14.5 16 16.8
17.3 9.7 5.2 17.4 165 166 15.2 9.1 20.1 20 16.4 14.5 14 16.1 16.6
18.4 10.1 5.3 17.5 171 170 14.5 8.8 21 20.8 18 14.3 13.8 15.8 16.1
17.5 9.2 5 16.8 161 160 13.8 8.2 19.5 20 11.6 14.1 13.5 15.4 15.9
a
Data are from four experiments and are given as mean þ S.E.M. Data are means from two replicate experiments. *P 6 0.05. b
exhibited a concentration-dependent inhibitory e¡ect on the activity of succinate dehydrogenase (Fig. 1). In comparison with the control, fumarate showed a signi¢cant inhibitory e¡ect on the enzyme activity at concentrations of 0.1 mM and above (P 6 0.05), malonate also showed the same e¡ect at concentrations of 1 mM and above (P 6 0.05). NADH dehydrogenase activity was completely insensitive to rotenone (Table 1), indicating the absence of a classical complex I in the bacterium. Furthermore, antimycin could not inhibit NADH-
Fig. 1. E¡ect of fumarate on the succinate dehydrogenase activity of H. pylori sonicate (FH5). The results are from ¢ve experiments and are given as mean þ S.E.M. Similar results have also been observed with other strains of H. pylori.
cytochrome c reductase (complex ICIII), which also suggests that a classical complex I does not exist in H. pylori. Oxygen uptake by H. pylori in the presence of NADPH and NADH was examined. Table 2 shows that the sonicate of H. pylori exhibited both NADH-dependent and NADPH-dependent respiration. However, NADPH-dependent respiration was signi¢cantly stronger than NADH-dependent respiration (P 6 0.05), indicating that this is a major respiratory electron donor in H. pylori. Antimycin signi¢cantly inhibited the activity of succinate-cytochrome c reductase (Table 1, P 6 0.05), implying the presence of a classical pathway from complex II to complex III (from succinate dehydrogenase to quinone pool) in the bacterium. Fig. 2 shows that 2-thenoyltri£uoroacetone (TTFA) exhibited a concentration-dependent inhibitory e¡ect on the activity of NADH-FRD and at a concentration of 0.2 mM almost completely inhibited the activity of NADH-FRD (P 6 0.05). At a high concentration (25 mM) succinate also exhibited signi¢cant inhibition on the activity of FRD (P 6 0.05). However, there was no signi¢cant di¡erence in the e¡ect on FRD at the concentrations between 25 mM and 100 mM of succinate. At a concentration of 10 mM malonate also inhibited the activity of FRD (P 6 0.05), while antimycin and 3-MPA did not inhibit it (Table 1).
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Fig. 2. E¡ect of succinate and 2-thenoyltri£uoroacetone (TTFA) on the FRD activity of H. pylori sonicate (FH5). The results are from ¢ve experiments and are given as mean þ S.E.M. Similar results have also been observed with other strains of H. pylori.
4. Discussion The results presented in this report show little strain variation and suggest that the respiratory chain of H. pylori is substantially di¡erent from that of other bacteria, such as Escherichia coli. Although NADH dehydrogenase is a classical complex I in the respiratory chain, it appears that this enzyme is absent in H. pylori. Our results show that NADH dehydrogenase was completely insensitive to rotenone, a NADH ubiquinone reductase (complex I) inhibitor, and NADH-cytochrome c reductase was also completely insensitive to antimycin, the reduced ubiquinone cytochrome c reductase (complex III) inhibitor. Furthermore, NADPH-dependent respiration in H. pylori was signi¢cantly stronger than NADH-dependent respiration (Table 2), indicating that NADPH is a major respiratory electron donor in H. pylori. Similar results were reported by other laboratories. Chang et al. [18] reported that NADPH oxidation was six times more rapid than that of
NADH. Kelly and his colleagues [17,19] also reported that NADPH-dependent respiration in the sonicate of H. pylori was signi¢cantly stronger than NADH-dependent respiration and NADH-dependent respiration was not detectable in the membrane fraction. They suggested that NADPH was the most e¡ective electron donor to the respiratory chain and NADH-dependent respiration in the sonicate was presumably due to the presence of additional (soluble) dehydrogenases. Chang et al. [18] reported that there were both succinate oxidation and D-lactate oxidation in the whole cells and cell membranes of H. pylori. The data presented in this study show that the activity of succinate dehydrogenase was inhibited by malonate and fumarate, two competitive inhibitors of the enzyme, indicating that there is a succinate dehydrogenase in H. pylori. Moreover, succinate-cytochrome c reductase was inhibited by antimycin, suggesting the presence of a classical pathway from complex II to complex III in H. pylori. Marcelli et al. [20] reported that H. pylori cells and membranes contained b- and c-type cytochromes, but not terminal oxidases of the a or d types. The major isoprenoid quinone was menaquinone-6. Nagata et al. [21] also reported that the cbb3 -type cytochrome c oxidase functioned as a terminal oxidase in the respiratory chain of H. pylori. Our previous study [22] showed that the activity of a cytochrome c-like water-soluble oxidant of H. pylori seemed to be primarily important for the destruction of ascorbic acid in the gastric juice of infected patients. Furthermore, Alderson et al. [23] reported that apart from cytochrome c oxidase, there was also a cytochrome c peroxidase in H. pylori and it might play a role in energy conservation. The presence of NADH-FRD was demonstrated in the sonicate of H. pylori (Table 1 and Fig. 2) and
Table 2 NADH and NADPH oxidation of the sonicate of H. pylori Substrate
0.5 mM NADPH 0.5 mM NADH
Respiration rate (nmol of O2 min31 mg of protein31 )a FH5a
FH1b
FH3b
FH8b
FH29b
FH47b
4.5 þ 0.09 1.4 þ 0.09*
4.2 1.35
4.6 1.6
4.2 1.3
4.4 1.4
4.1 1.2
a
Data are from four experiments and are given as mean þ S.E.M. Data are means from two replicate experiments. *P 6 0.05. b
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fumarate could reduce H2 O2 production from NADH indicating fumarate to be an endogenous substrate for accepting electrons from NADH. The oxidation of succinate to fumarate could be reversed by increasing the concentration of fumarate (Table 1 and Fig. 1), whereas the reduction of fumarate to succinate was not easily reversed by increasing the concentration of succinate, requiring a very high concentration of succinate (Table 1 and Fig. 2). This phenomenon implies that the fumarate to succinate pathway is predominant in H. pylori. Mendz et al. [24,25] investigated the metabolism of fumarate by H. pylori with one- and two-dimensional 1 H and 13 C NMR spectroscopy, and reported the presence of FRD activity and active fumarate catabolism in the bacterium. The presence of FRD activity suggested the possibility of ATP generation via anaerobic respiration in H. pylori. They suggested that FRD is an essential component of the metabolism of H. pylori and is a possible target for therapeutic intervention in the treatment of the bacterium. Recently, Tomb et al. have reported a complete genome sequence of H. pylori [26] and Ge et al. have cloned and sequenced the genes encoding FRD of H. pylori, and reported that FRD of H. pylori had three subunits, frdA, frdB and frdC [27]. They found that inactivation of frdA led to the loss of the enzyme activity and the mutant H. pylori cells were delayed in entering the mid-exponential phase, suggesting that FRD-driven metabolism plays an active but non-essential role for growth of H. pylori cells in vitro. Further studies in this ¢eld are warranted. Based on our data and previous ¢ndings, a tentative scheme has been proposed for electron transfer in H. pylori, which is depicted in Fig. 3. Several dehydrogenases may be present, depending on the [H] donor. It seems that NADH dehydrogenase, a classical complex I, is absent in the respiratory chain of H. pylori. NADPH might be a major respiratory donor in the bacterium. There are a succinate dehydrogenase, a classical complex II, and a succinatecytochrome c reductase, a classical pathway from complex II to complex III, in the respiratory chain of H. pylori. The complex II could be inhibited by malonate and the pathway from complex II to complex III could be inhibited by antimycin. There are a cytochrome bc oxidase [20,21] and a cytochrome c peroxidase [23] in the respiratory chain of the bacte-
173
Fig. 3. Proposed electron transfer chain in Helicobacter pylori. Malonate inhibits succinate dehydrogenase. Antimycin inhibits the pathway from succinate dehydrogenase to the quinone pool. Malonate and TTFA inhibit FRD.
rium, whereas E. coli contains no cytochrome a or c [33]. Moreover, H. pylori has a NADH-FRD, which could be inhibited by TTFA and malonate. This preliminary study may help to understand the respiratory chain of H. pylori and to clarify its pathogenesis. It is likely that detailed characterization of the special features of the electron transport pathway or target enzyme complexes may be helpful in opening new lines for chemotherapy against H. pylori. Work in this direction and an investigation of the e¡ect of metronidazole on the respiratory chain of H. pylori are now in progress at our laboratory.
Acknowledgements The technical assistance of Bent Jensen is greatly acknowledged. We thank Dr. David J. Kelly for useful discussions and comments. References [1] Goodwin, C.S. and Armstrong, J.A. (1990) Microbiological aspects of Helicobacter pylori (Campylobacter pylori). Eur. J. Clin. Microbiol. Infect. Dis. 9, 1^13. [2] Blaser, M.J. and Parsonnet, J. (1994) Parasitism by the `slow' bacterium Helicobacter pylori leads to altered gastric homeostasis and neoplasia. J. Clin. Invest. 94, 4^8. [3] Josenhans, C. and Suerbaum, S. (1997) Flagella and motility of Helicobacter pylori. In: Pathogenesis and Host Response in Helicobacter pylori Infections (Moron, A.P. and O'Morain, C.A., Eds.), pp. 6^15. Normed Verlag, Bad Homburg. [4] Graham, D.Y. (1989) Campylobacter pylori and peptic ulcer diseases. Gastroenterology 96, 615^625.
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[5] Dixon, M.F. (1991) Helicobacter pylori and peptic ulceration: hisopathological aspects. J. Gastroenterol. Hepatol. 6, 125^130. [6] Tytgat, G.N.J., Graham, D.Y., Dixon, M.F. and Rokkas, T. (1993) The role of infectious agents in peptic ulcer disease. Gastroenterol. Int. 6, 76^89. [7] Unge, P. and Ekstrom, P. (1993) E¡ects of combination therapy with omeprazole and an antibiotic on Helicobacter pylori and duodenal ulcer disease. Scand. J. Gastroenterol. 28, ((Suppl. 196)) 17^18. [8] Bazzoli, F., Zagari, R.M., Fossi, S., Pozzato, P., Roda, A. and Roda, E. (1993) Short-term low-dose triple therapy for the eradication of Helicobacter pylori. Gastroenterology 104, A40. [9] Bell, G.D., Bate, C.M., Axon, A.T.R., Tildesley Kerr, G.D., Gree, J.R.B., Emmas, C.E. and Taylor, M.D. (1995) Addition of metronidazole to omeprazole/amoxycillin dual therapy increases the rate of Helicobacter pylori eradication: a doubleblind, randomized trial. Aliment. Pharmacol. Ther. 9, 513^ 520. [10] Borody, T.J., Cole, P., Noonan, S., Morgan, A., Lenne, J., Hyland, L., Brandl, S., Borody, E.G. and Georage, L.L. (1989) Recurrence of duodenal ulcer and Campylobacter pylori infection after eradication. Med. J. Aust. 151, 431^435. [11] NIH Consensus Conference (1994) Helicobacter pylori in peptic ulcer disease. NIH Consensus Development Panel on Helicobacter pylori in Peptic Ulcer Disease. J. Am. Med. Assoc. 272, 65^69. [12] Glupczynski, Y., Burette, A., De Koster, E., Nyst, J.F., Deltenre, M., Cadranel, S., Bourdeaux, L. and De Vos, D. (1990) Metronidazole resistance in Helicobacter pylori. Lancet 335, 976^977. [13] Graham, D.Y., de Boer, W.A. and Tytgat, G.N.J. (1996) Choosing the best anti-Helicobacter pylori therapy : e¡ect of antimicrobial resistance. Am. J. Gastroenterol. 91, 1072^1076. [14] Logan, R.P.H., Gummett, P.A., Misiewicz, J.J., Karim, Q.N. and Waker, M.M. (1991) One week eradication regimen for Helicobacter pylori. Lancet 338, 1249^1252. [15] Rautelin, H., Swppala, K., Renkonen, O.V., Vaino, U. and Kosunen, T.U. (1992) Role of metronidazole resistance in therapy of Helicobacter pylori infection. Antimicrob. Agents Chemother. 36, 163^166. [16] Jorgensen, M., Daskalopoulos, G., Warburton, V., Mitchell, H.M. and Hazell, S.L. (1996) Multiple strain colonization and metronidazole resistance in Helicobacter pylori-infected patients: identi¢cation from sequential and multiple biopsy specimens. J. Infect. Dis. 174, 631^635. [17] Hughes, N.J., Clayton, C.L., Chalk, P.A. and Kelly, D.J. (1998) Helicobacter pylori porCDAB and oorDABC genes encode distinct pyruvate : Flavodoxin and 2-oxoglutarate : Acceptor oxidoreductases which mediate electron transport to NADP. J. Bacteriol. 180, 1119^1128. [18] Chang, H.T., Marcelli, S.W., Davison, A.A., Chalk, P.A., Poole, R.K. and Miles, R.J. (1995) Kinetics of substrate oxidation by whole cells and cells membranes of Helicobacter pylori. FEMS Microbiol. Lett. 129, 33^38. [19] Dennison, V.L., Hughes, N.J. and Kelly, D.J. (1998) 2-Oxoacid dependent electron transport pathways in Helicobacter pylori : A central role in metabolism and NADPH produc-
[20]
[21]
[22]
[23]
[24] [25]
[26]
[27]
[28]
[29]
[30]
[31]
[32]
[33]
tion? 3rd International Workshop on Pathogenesis and Host Response in Helicobacter Infections, HelsingÖr, Abstract H4. Marcelli, S.W., Chang, H.T., Chapman, T., Chalk, P.A., Miles, R.J. and Poole, R.K. (1996) The respiratory chain of Helicobacter pylori : identi¢cation of cytochromes and the effects of oxygen on cytochrome and menaquinone levels. FEMS Microbiol. Lett. 138, 59^64. Nagata, K., Tsukita, S., Tamura, T. and Sone, N. (1996) A cb-type cytochrome-c oxidase terminates the respiratory chain in Helicobacter pylori : Microbiology. Microbiology 142, 1757^1763. Òdum, L. and Andersen, L.P. (1995) Investigation of Helicobacter pylori ascorbic acid oxidating activity. FEMS Immunol. Med. Microbiol. 10, 289^294. Alderson, J., Clayton, C.L. and Kelly, D.J. (1998) Analysis of cytochrome c containing proteins in Helicobacter pylori: what is the physiological role of cytochrme c peroxidase ? 3rd International Workshop on Pathogenesis and Host Response in Helicobacter Infections, HelsingÖr, Abstract E10. Mendz, G.L. and Hazell, S.L. (1993) Fumarate catabolism in Helicobacter pylori. Biochem. Mol. Biol. Int. 31, 325^332. Mendz, G.L., Hazell, S.L. and Srinivasan, S. (1995) Fumarate reductase : A target for therapeutic intervention against Helicobacter pylori. Arch. Biochem. Biophys. 321, 153^159. Tomb, J.F., White, O., Kerlavage, A.R., Clayton, R.A., Sutton, G.G., Fleischmann, R.D., Ketchum, K.A., Klenk, H.P., Gill, S., Dougherty, B.A., Nelson, K., Quackenbush, J., Zhou, L., Kirkness, E.F., Peterson, S., Loftus, B., Richardson, D., Dodson, R., Khalak, H.G., Glodek, A., McKenney, K., Fitzegerald, L.M., Lee, N., Adams, M.D., Hickey, E.K., Berg, D.E., Gocayne, J.D., Utterback, T.R., Peterson, J.D., Kelley, J.M., Cotton, M.D., Weidman, J.M., Fujii, C., Bowman, C., Watthey, L., Wallin, E., Hayes, W.S., Borodovsky, M., Karp, P.D., Smith, H.O., Fraser, C.M. and Venter, J.C. (1997) The complete genome sequence of the gastric pathogen Helicobacter pylori. Nature 388, 539^547. Ge, Z., Jiang, Q., Kalisiak, M.S. and Taylor, D.E. (1997) Cloning and functional characterization of Helicobacter pylori fumarate reductase operon comprising three structural genes coding for subunits C, A and B. Gene 204, 227^234. King, T.E. (1967) Preparation of succinate dehydrogenase and reconstitution of succinate oxidase. Methods Enzymol. 10, 323^331. King, T.E. and Howard, R.L. (1967) Preparation and properties of soluble NADH dehydrogenase from cardiac muscle. Methods Enzymol. 10, 275^294. Tisdale, H.D. (1967) Preparation and properties of succinatecytochrome c reductase (complex II^III). Methods Enzymol. 10, 213^225. Hate¢, F. and Rieske, J.S. (1967) Preparation and properties of DPNH-cytochrome c reductase (complex I^III of the respiratory chain). Methods Enzymol., 225^231. Holwerda, A.D. and De Zwan, A. (1980) On the role of FRD in anaerobic carbohydrate metabolism of Mytilus edulis L. Comp. Biochem. Physiol. B 67, 447^453. Harris, D.A. (1995) Bioenergetics at a Glance, pp. 36^37. Blackwell Science, Oxford.
FEMSIM 1013 6-5-99
Characterization of the respiratory chain of Helicobacter pylori Ming Chen a
a;b;
*, Leif P. Andersen a , Lin Zhai
a;b
, Arsalan Kharazmi
a
Department of Clinical Microbiology, 7806, University Hospital (Rigshospitalet), Tagensvej 20, DK-2200 Copenhagen, Denmark b Statens Serum Institut, Copenhagen, Denmark Received 25 November 1998 ; accepted 18 February 1999
Abstract The respiratory chain of Helicobacter pylori has been investigated. The total insensitivity of activities of NADH dehydrogenase to rotenone and of NADH-cytochrome c reductase to antimycin is indicative of the absence of the classical complex I of the electron transfer chain in this bacterium. NADPH-dependent respiration was significantly stronger than NADH-dependent respiration, indicating that this is a major respiratory electron donor in H. pylori. Fumarate and malonate exhibited a concentration-dependent inhibitory effect on the activity of succinate dehydrogenase. The activity of succinatecytochrome c reductase was inhibited by antimycin, implying the presence of a classical pathway from complex II to complex III in this bacterium. The presence of NADH-fumarate reductase (FRD) was demonstrated in H. pylori and fumarate could reduce H2 O2 production from NADH, indicating fumarate to be an endogenous substrate for accepting electrons from NADH. The activity of NADH-FRD was inhibited by 2-thenoyltrifluoroacetone. A tentative scheme for the electron transfer pathway in H. pylori is proposed, which may be helpful in clarifying the pathogenesis of H. pylori and in opening new lines for chemotherapy against this bacterium. ß 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Helicobacter pylori; Respiratory chain; Succinate reductase; Fumarate reductase; NADH dehydrogenase; NADPH dehydrogenase
1. Introduction Helicobacter pylori is a Gram-negative, oxidasepositive, microaerophilic, £agellate, curved or spiral bacterium that colonizes the mucous layer of the human gastric epithelium [1^3]. The organism is now accepted as the etiological agent for type B gastritis and the majority of duodenal and gastric ulcers, and is linked to the development of gastric cancer [4^ 6]. Treatment of H. pylori infection has undergone signi¢cant development; from initial monotherapy to
* Corresponding author. Tel.: +45 35 45 77 38; Fax: +45 35 45 68 31; E-mail: [email protected]
dual, triple and, in some cases, quadruple therapy [7^11]. However, drug resistance has caused problems in the treatment of H. pylori infection [12^16]. The ability of H. pylori to develop resistance to 5nitroimidazoles, one of the most important drugs of the therapy, is a major contributing factor to the failure of therapy. So, beside the new combination treatment and the development of a vaccine, there is certainly a need for the development of new therapeutic agents speci¢cally targeted against H. pylori, which will represent a signi¢cant advance in the treatment of the infection. The physiology and metabolism of H. pylori are not fully understood, even though some e¡orts have been devoted to this ¢eld during the last several years [17^
0928-8244 / 99 / $20.00 ß 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 9 2 8 - 8 2 4 4 ( 9 9 ) 0 0 0 2 2 - X
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27]. Understanding these fundamental factors could help to control the infection, for example, by identifying potential drug targets in Helicobacter spp. The aim of this study was to characterize the respiratory chain of H. pylori. We have investigated the activities of the enzymes which are involved in the electron transport chain in the bacterium. A tentative scheme for the electron transfer pathway in H. pylori is proposed.
2. Materials and methods 2.1. Biochemicals All biochemicals and reagents were from Sigma Chemicals, USA, unless otherwise stated. 2.2. Bacterial cultivation and preparation Six clinical isolates of H. pylori (FH1, FH3, FH5, FH8, FH29 and FH47) were used for this study. However, we found little strain variation. Therefore, most of the experiments were carried out on strain FH5. H. pylori was grown microaerobically on 7% lysed horse blood agar plates at 37³C for 48 h. Bacteria were harvested in phosphate bu¡er saline (PBS), pH 7.4, and washed twice. The bacteria were disrupted by sonication at 20 000 Hz for 45 s ¢ve times with a Rapids 300 (19-mm probe with a 9.5-mm tip). During the sonication, preparations were cooled by immersion in iced water. The crude sonicates were centrifuged at 18 000Ug for 30 min at 4³C, the supernatant was collected and then frozen at 380³C until use. 2.3. Exposure of H. pylori preparations to inhibitors Protein concentrations of H. pylori preparations were determined by a Bio-Rad protein assay (BioRad Laboratories, Hercules, CA, USA). H. pylori preparations were adjusted to 1 mg ml31 and incubated with each inhibitor in 50 mM potassium phosphate bu¡er, pH 7.4 or bu¡er alone for 3 min at 37³C, before the enzyme determinations. 2.4. Enzymatic assays All enzymatic activities were determined using a
¢nal protein concentration of 1 mg ml31 in 1-ml cuvettes in 50 mM potassium phosphate bu¡er, pH 7.4 at 37³C. All spectrophotometric determinations were carried out in a Perkin-Elmer Lambda 40 UV/ Vis spectrophotometer. Succinate dehydrogenase activity was measured spectrophotometrically at 600 nm (O = 20.5 mM31 cm31 ) using 3 mM succinate, 0.5 mM 2,6-dichlorophenolindophenol and 0.1 phenazine methosulfate [28]. NADH dehydrogenase activity was determined spectrophotometrically at 420 nm by measuring the rate of potassium ferricyanide (0.5 mM) reduction in the presence of NADH (O = 1 mM31 cm31 ) [29]. Succinate- and NADH-cytochrome c reductase activities were measured spectrophotometrically at 550 nm (O = 18.9 mM31 cm31 ) in the presence of 20 WM cytochrome c and either 5 mM succinate or 0.2 mM NADH [30,31]. NADH-fumarate dehydrogenase (FRD) activity was determined as the rate of NADH oxidation on addition of 1 mM fumarate to the H. pylori sonication. The reaction was monitored by spectrophotometer at 340 nm (O = 6.2 mM31 cm31 ) using 100 WM NADH [32]. 2.5. NADH and NADPH oxidation The NADH and NADPH oxidation were determined by measuring changes in the dissolved oxygen concentrations in a Clarke-type oxygen electrode calibrated with air-saturated 20 mM potassium phosphate bu¡er, pH 7.2 at 37³C [17]. 2.6. Statistical analysis A paired two-tailed t-test was used for analysis of the data. P values of 6 0.05 were considered signi¢cant.
3. Results Table 1 summarizes the activities of several respiratory chain segments and the e¡ect of inhibitors, as determined spectrophotometrically in the sonicate of H. pylori. Antimycin did not inhibit the succinate dehydrogenase of H. pylori. Fumarate and malonate
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Table 1 Mitochondrial redox reduction catalyzed by H. pylori sonicate Enzymes+inhibitors
Succinate dehydrogenase +fumarate (0.1 mM) +malonate (1 mM) +antimycin (4 WM) NADH-dehydrogenase +rotenone (50 WM) Succinate-cytochrome c reductase +antimycin (2 WM) NADH-cytochrome c reductase +antimycin (2 WM) Fumarate reductase +succinate (25 mM) +malonate (10 mM) +antimycin (200 WM) +3-MPA (20 Wg)
Speci¢c activity (nmol min31 mg protein31 ) FH5a
FH1b
FH3b
FH8b
FH29b
FH47b
18.7 þ 0.45 10.5 þ 0.42* 5.9 þ 0.62* 17.7 þ 0.62 175.5 þ 18.2 178.1 þ 6.7 14.7 þ 1.77 9.2 þ 0.59* 21.3 þ 0.79 20.7 þ 1.0 18.2 þ 1.5 14.8 þ 1.4* 14.2 þ 0.38* 16.3 þ 0.54 16.9 þ 0.65
18.0 9.9 5.3 18 170 172 14.2 8.8 20.5 20 17.6 14.6 14 16.5 17.1
19.4 10.2 5.9 18.2 188 186 15.6 9.2 22.4 22.2 20 14.6 14.5 16 16.8
17.3 9.7 5.2 17.4 165 166 15.2 9.1 20.1 20 16.4 14.5 14 16.1 16.6
18.4 10.1 5.3 17.5 171 170 14.5 8.8 21 20.8 18 14.3 13.8 15.8 16.1
17.5 9.2 5 16.8 161 160 13.8 8.2 19.5 20 11.6 14.1 13.5 15.4 15.9
a
Data are from four experiments and are given as mean þ S.E.M. Data are means from two replicate experiments. *P 6 0.05. b
exhibited a concentration-dependent inhibitory e¡ect on the activity of succinate dehydrogenase (Fig. 1). In comparison with the control, fumarate showed a signi¢cant inhibitory e¡ect on the enzyme activity at concentrations of 0.1 mM and above (P 6 0.05), malonate also showed the same e¡ect at concentrations of 1 mM and above (P 6 0.05). NADH dehydrogenase activity was completely insensitive to rotenone (Table 1), indicating the absence of a classical complex I in the bacterium. Furthermore, antimycin could not inhibit NADH-
Fig. 1. E¡ect of fumarate on the succinate dehydrogenase activity of H. pylori sonicate (FH5). The results are from ¢ve experiments and are given as mean þ S.E.M. Similar results have also been observed with other strains of H. pylori.
cytochrome c reductase (complex ICIII), which also suggests that a classical complex I does not exist in H. pylori. Oxygen uptake by H. pylori in the presence of NADPH and NADH was examined. Table 2 shows that the sonicate of H. pylori exhibited both NADH-dependent and NADPH-dependent respiration. However, NADPH-dependent respiration was signi¢cantly stronger than NADH-dependent respiration (P 6 0.05), indicating that this is a major respiratory electron donor in H. pylori. Antimycin signi¢cantly inhibited the activity of succinate-cytochrome c reductase (Table 1, P 6 0.05), implying the presence of a classical pathway from complex II to complex III (from succinate dehydrogenase to quinone pool) in the bacterium. Fig. 2 shows that 2-thenoyltri£uoroacetone (TTFA) exhibited a concentration-dependent inhibitory e¡ect on the activity of NADH-FRD and at a concentration of 0.2 mM almost completely inhibited the activity of NADH-FRD (P 6 0.05). At a high concentration (25 mM) succinate also exhibited signi¢cant inhibition on the activity of FRD (P 6 0.05). However, there was no signi¢cant di¡erence in the e¡ect on FRD at the concentrations between 25 mM and 100 mM of succinate. At a concentration of 10 mM malonate also inhibited the activity of FRD (P 6 0.05), while antimycin and 3-MPA did not inhibit it (Table 1).
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Fig. 2. E¡ect of succinate and 2-thenoyltri£uoroacetone (TTFA) on the FRD activity of H. pylori sonicate (FH5). The results are from ¢ve experiments and are given as mean þ S.E.M. Similar results have also been observed with other strains of H. pylori.
4. Discussion The results presented in this report show little strain variation and suggest that the respiratory chain of H. pylori is substantially di¡erent from that of other bacteria, such as Escherichia coli. Although NADH dehydrogenase is a classical complex I in the respiratory chain, it appears that this enzyme is absent in H. pylori. Our results show that NADH dehydrogenase was completely insensitive to rotenone, a NADH ubiquinone reductase (complex I) inhibitor, and NADH-cytochrome c reductase was also completely insensitive to antimycin, the reduced ubiquinone cytochrome c reductase (complex III) inhibitor. Furthermore, NADPH-dependent respiration in H. pylori was signi¢cantly stronger than NADH-dependent respiration (Table 2), indicating that NADPH is a major respiratory electron donor in H. pylori. Similar results were reported by other laboratories. Chang et al. [18] reported that NADPH oxidation was six times more rapid than that of
NADH. Kelly and his colleagues [17,19] also reported that NADPH-dependent respiration in the sonicate of H. pylori was signi¢cantly stronger than NADH-dependent respiration and NADH-dependent respiration was not detectable in the membrane fraction. They suggested that NADPH was the most e¡ective electron donor to the respiratory chain and NADH-dependent respiration in the sonicate was presumably due to the presence of additional (soluble) dehydrogenases. Chang et al. [18] reported that there were both succinate oxidation and D-lactate oxidation in the whole cells and cell membranes of H. pylori. The data presented in this study show that the activity of succinate dehydrogenase was inhibited by malonate and fumarate, two competitive inhibitors of the enzyme, indicating that there is a succinate dehydrogenase in H. pylori. Moreover, succinate-cytochrome c reductase was inhibited by antimycin, suggesting the presence of a classical pathway from complex II to complex III in H. pylori. Marcelli et al. [20] reported that H. pylori cells and membranes contained b- and c-type cytochromes, but not terminal oxidases of the a or d types. The major isoprenoid quinone was menaquinone-6. Nagata et al. [21] also reported that the cbb3 -type cytochrome c oxidase functioned as a terminal oxidase in the respiratory chain of H. pylori. Our previous study [22] showed that the activity of a cytochrome c-like water-soluble oxidant of H. pylori seemed to be primarily important for the destruction of ascorbic acid in the gastric juice of infected patients. Furthermore, Alderson et al. [23] reported that apart from cytochrome c oxidase, there was also a cytochrome c peroxidase in H. pylori and it might play a role in energy conservation. The presence of NADH-FRD was demonstrated in the sonicate of H. pylori (Table 1 and Fig. 2) and
Table 2 NADH and NADPH oxidation of the sonicate of H. pylori Substrate
0.5 mM NADPH 0.5 mM NADH
Respiration rate (nmol of O2 min31 mg of protein31 )a FH5a
FH1b
FH3b
FH8b
FH29b
FH47b
4.5 þ 0.09 1.4 þ 0.09*
4.2 1.35
4.6 1.6
4.2 1.3
4.4 1.4
4.1 1.2
a
Data are from four experiments and are given as mean þ S.E.M. Data are means from two replicate experiments. *P 6 0.05. b
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fumarate could reduce H2 O2 production from NADH indicating fumarate to be an endogenous substrate for accepting electrons from NADH. The oxidation of succinate to fumarate could be reversed by increasing the concentration of fumarate (Table 1 and Fig. 1), whereas the reduction of fumarate to succinate was not easily reversed by increasing the concentration of succinate, requiring a very high concentration of succinate (Table 1 and Fig. 2). This phenomenon implies that the fumarate to succinate pathway is predominant in H. pylori. Mendz et al. [24,25] investigated the metabolism of fumarate by H. pylori with one- and two-dimensional 1 H and 13 C NMR spectroscopy, and reported the presence of FRD activity and active fumarate catabolism in the bacterium. The presence of FRD activity suggested the possibility of ATP generation via anaerobic respiration in H. pylori. They suggested that FRD is an essential component of the metabolism of H. pylori and is a possible target for therapeutic intervention in the treatment of the bacterium. Recently, Tomb et al. have reported a complete genome sequence of H. pylori [26] and Ge et al. have cloned and sequenced the genes encoding FRD of H. pylori, and reported that FRD of H. pylori had three subunits, frdA, frdB and frdC [27]. They found that inactivation of frdA led to the loss of the enzyme activity and the mutant H. pylori cells were delayed in entering the mid-exponential phase, suggesting that FRD-driven metabolism plays an active but non-essential role for growth of H. pylori cells in vitro. Further studies in this ¢eld are warranted. Based on our data and previous ¢ndings, a tentative scheme has been proposed for electron transfer in H. pylori, which is depicted in Fig. 3. Several dehydrogenases may be present, depending on the [H] donor. It seems that NADH dehydrogenase, a classical complex I, is absent in the respiratory chain of H. pylori. NADPH might be a major respiratory donor in the bacterium. There are a succinate dehydrogenase, a classical complex II, and a succinatecytochrome c reductase, a classical pathway from complex II to complex III, in the respiratory chain of H. pylori. The complex II could be inhibited by malonate and the pathway from complex II to complex III could be inhibited by antimycin. There are a cytochrome bc oxidase [20,21] and a cytochrome c peroxidase [23] in the respiratory chain of the bacte-
173
Fig. 3. Proposed electron transfer chain in Helicobacter pylori. Malonate inhibits succinate dehydrogenase. Antimycin inhibits the pathway from succinate dehydrogenase to the quinone pool. Malonate and TTFA inhibit FRD.
rium, whereas E. coli contains no cytochrome a or c [33]. Moreover, H. pylori has a NADH-FRD, which could be inhibited by TTFA and malonate. This preliminary study may help to understand the respiratory chain of H. pylori and to clarify its pathogenesis. It is likely that detailed characterization of the special features of the electron transport pathway or target enzyme complexes may be helpful in opening new lines for chemotherapy against H. pylori. Work in this direction and an investigation of the e¡ect of metronidazole on the respiratory chain of H. pylori are now in progress at our laboratory.
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