DOI QR코드

DOI QR Code

Identification and Characterization of the Acid Phosphatase HppA in Helicobacter pylori

  • Ki, Mi-Ran (Department of Pathology, College of Veterinary Medicine, Kyungbook National University) ;
  • Yun, Soon-Kyu (Department of Bioinformatics, College of Science and Technology, Korea University) ;
  • Choi, Kyung-Min (Institute of Jinan Red Ginseng) ;
  • Hwang, Se-Young (Department of Bioinformatics, College of Science and Technology, Korea University)
  • 투고 : 2011.01.13
  • 심사 : 2011.02.19
  • 발행 : 2011.05.28

초록

An acid phosphatase (HppA) activated by $NH_4Cl$ was purified 192- and 34-fold from the periplasmic and membrane fractions of Helicobacter pylori, respectively. SDS-polyacrylamide gel electrophoresis revealed that HppA from the latter appears to be several kilodaltons larger in molecular mass than from the former by about 24 kDa. Under acidic conditions (pH${\leq}$4.5), the enzyme activity was entirely dependent on the presence of certain mono- and/or divalent metal cations (e.g., $K^+$,$ NH_4{^+}$, and/or $Ni^{2+}$). In particular, $Ni^{2+}$ appeared to lower the enzyme's $K_m$ for the substrates, without changing $V_{max}$. The purified enzyme showed differential specificity against nucleotide substrates with pH; for example, the enzyme hydrolyzed adenosine nucleotides more rapidly at pH 5.5 than at pH 6.0, and vice versa for CTP or TTP. Analyses of the enzyme's N-terminal sequence and of an $HppA^-$ H. pylori mutant revealed that the purified enzyme is identical to rHppA, a cloned H. pylori class C acid phosphatase, and shown to be the sole bacterial 5'-nucleotidase uniquely activated by $NH_4Cl$. In contrast to wild type, $HppA^-$ H. pylori cells grew more slowly. Strikingly, they imported $Mg^{2+}$ at a markedly lowered rate, but assimilated urea rapidly, with a subsequent increase in extracellular pH. Moreover, mutant cells were much more sensitive to extracellular potassium ions, as well as to metronidazole, omeprazole, or thiophenol, with considerably lowered MIC values, than wild-type cells. From these data, we suggest that the role of the acid phosphatase HppA in H. pylori may extend beyond 5'-nucleotidase function to include cation-flux as well as pH regulation on the cell envelope.

키워드

참고문헌

  1. Agranoff, D. D. and S. Krishna. 1998. Metal ion homeostasis and intracellular parasitism. Mol. Microbiol. 28: 403-412. https://doi.org/10.1046/j.1365-2958.1998.00790.x
  2. Aravind, L. and E. V. Koonin. 1998. The HD domain defines a new superfamily of metal-dependent phosphohydrolases. Trends Biochem. Sci. 23: 469-472. https://doi.org/10.1016/S0968-0004(98)01293-6
  3. Bayle, D., S. Wangler, T. Weitzenegger, W. Steinhilber, J. Volz, M. Przybylski, K. P. Schafer, G. Sachs, and K. Melchers. 1998. Properties of the P-type ATPases encoded by the copAP operons of Helicobacter pylori and Helicobacter felis. J. Bacteriol. 180: 317-329.
  4. Bury-Mone, S., J. M. Thiberge, M. Contreras, A. Maitournam, A. Labigne, and H. De Reuse. 2004. Responsiveness to acidity via metal ion regulators mediates virulence in the gastric pathogen Helicobacter pylori. Mol. Microbiol. 53: 623-638. https://doi.org/10.1111/j.1365-2958.2004.04137.x
  5. Chevalier, C., J. M. Thiberge, R. L. Ferrero, and A. Labigne. 1999. Essential role of Helicobacter pylori gamma-glutamyltranspeptidase for the colonization of the gastric mucosa of mice. Mol. Microbiol. 31: 1359-1372. https://doi.org/10.1046/j.1365-2958.1999.01271.x
  6. Doig, P., B. L. de Jonge, R. A. Alm, E. D. Brown, M. Uria- Nickelsen, B. Noonan, et al. 1999. Helicobacter pylori physiology predicted from genomic comparison of two strains. Microbiol. Mol. Biol. Rev. 63: 675-707.
  7. Furriel, R. P., D. C. Masui, J. C. McNamara, and F. A. Leone. 2004. Modulation of gill $Na^+$,$K^+$-ATPase activity by ammonium ions: Putative coupling of nitrogen excretion and ion uptake in the freshwater shrimp Macrobrachium olfersii. J. Exp. Zool. A Comp. Exp. Biol. 301: 63-74.
  8. Gang, J. G., S. K. Yun, K. M. Choi, W. J. Lim, J. K. Park, and S. Y. Hwang. 2001. Significance of urease distribution across Helicobacter pylori membrane. J. Microbiol. Biotechnol. 11: 315-325.
  9. Godlewska, R., J. M. Bujnicki, J. Ostrowski, and E. K. Jagusztyn- Krynicka. 2002. The hppA gene of Helicobacter pylori encodes the class C acid phosphatase precursor. FEBS Lett. 525: 39-42. https://doi.org/10.1016/S0014-5793(02)03064-8
  10. Gunshin, H., B. Mackenzie, U. V. Berger, Y. Gunshin, M. F. Romero, W. F. Boron, S. Nussberger, J. L. Gollan, and M. A. Hediger. 1997. Cloning and characterization of a mammalian proton-coupled metal-ion transporter. Nature 388: 482-488. https://doi.org/10.1038/41343
  11. Hughes, N. J., P. A. Chalk, C. L. Clayton, and D. J. Kelly. 1995. Identification of carboxylation enzymes and characterization of a novel four-subunit pyruvate:flavodoxin oxidoreductase from Helicobacter pylori. J. Bacteriol. 177: 3953-3959.
  12. Ki, M. R., S. K. Yun, W. J. Lim, B. S. Hong, and S. Y. Hwang. 1999. Synergistic inhibition of membrane ATPase and cell growth of Helicobacter pylori by ATPase inhibitors. J. Microbiol. Biotechnol. 9: 414-421.
  13. Ki, M. R., S. K. Yun, K. M. Choi, and S. Y. Hwang. 2003. Potential and significance of ammonium production from Helicobacter pylori. J. Microbiol. Biotechnol. 13: 673-679.
  14. Ki, M. R., S. K. Yun, and S. Y. Hwang. 1999. Effect of omeprazole on membrane P-type ATPase and peptide transport in Helicobacter pylori. J. Microbiol. Biotechnol. 9: 235-242.
  15. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680- 685. https://doi.org/10.1038/227680a0
  16. Macaskie, L. E. and A. C. Dean. 1984. Cadmium accumulation by a Citrobacter sp. J. Gen. Microbiol. 130: 53-62.
  17. Macaskie, L. E., B. C. Jeong, and M. R. Tolley. 1994. Enzymically accelerated biomineralization of heavy metals: Application to the removal of americium and plutonium from aqueous flows. FEMS Microbiol. Rev. 14: 351-367. https://doi.org/10.1111/j.1574-6976.1994.tb00109.x
  18. Mallery, C. H. 1983. A carrier enzyme basis for ammonium excretion in teleost gill. $NH_4^^+$-stimulated Na-dependent ATPase activity in Opsanus beta. Comp. Biochem. Physiol. A Comp. Physiol. 74: 889-897. https://doi.org/10.1016/0300-9629(83)90364-X
  19. Marshall, B. J. and J. R. Warren. 1984. Unidentified curved bacilli in the stomach of patients with gastritis and peptic ulceration. Lancet 1: 1311-1315.
  20. Mattar, R., S. B. Marques, S. Monteiro Mdo, A. F. Dos Santos, K. Iriya, and F. J. Carrilho. 2007. Helicobacter pylori cag pathogenicity island genes: Clinical relevance for peptic ulcer disease development in Brazil. J. Med. Microbiol. 56: 9-14. https://doi.org/10.1099/jmm.0.46824-0
  21. McGowan, C. C., T. L. Cover, and M. J. Blaser. 1997. Analysis of $F_1F_0$-ATPase from Helicobacter pylori. Infect. Immun. 65: 2640-2647.
  22. Melchers, K., T. Weitzenegger, A. Buhmann, W. Steinhilber, G. Sachs, and K. P. Schafer. 1996. Cloning and membrane topology of a P-type ATPase from Helicobacter pylori. J. Biol. Chem. 271: 446-457. https://doi.org/10.1074/jbc.271.1.446
  23. Michel, L. J., L. E. Macaskie, and A. C. Dean. 1986. Cadmium accumulation by immobilized cells of a Citrobacter sp. using various phosphate donors. Biotechnol. Bioeng. 28: 1358-1365. https://doi.org/10.1002/bit.260280910
  24. Mobley, H. L. T., G. L. Mendz, and S. L. Hazell. 2001. Helicobacter pylori: Physiology and Genetics. American Society for Microbiology Press, Washington, DC.
  25. Naseri, J. I., N. T. Truonga, J. Horentrup, P. Kuballa, A. Vogel, A. Rompel, F. Spenera, and B. Krebs. 2004. Porcine purple acid phosphatase: Heterologous expression, characterization, and proteolytic analysis. Arch. Biochem. Biophys. 432: 25-36. https://doi.org/10.1016/j.abb.2004.08.008
  26. Nielsen, H., J. Engelbrecht, S. Brunak, and G. von Heijne. 1997. Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Protein Eng. 10: 1-6. https://doi.org/10.1093/protein/10.1.1
  27. Reilly, T. J. and M. J. Calcutt. 2004. The class C acid phosphatase of Helicobacter pylori is a 5' nucleotidase. Protein Expr. Purif. 33: 48-56. https://doi.org/10.1016/j.pep.2003.08.020
  28. Reilly, T. J., B. A. Green, G. W. Zlotnick, and A. L. Smith. 2001. Contribution of the DDDD motif of H. influenzae e (P4) to phosphomonoesterase activity and heme transport. FEBS Lett. 494: 19-23. https://doi.org/10.1016/S0014-5793(01)02294-3
  29. Robinson, J. D. 1970. Interactions between monovalent cations and the ($Na^+ + K^+$)-dependent adenosine triphosphatase. Arch. Biochem. Biophys. 139: 17-27. https://doi.org/10.1016/0003-9861(70)90040-8
  30. Rokkas, T., D. Pistiolas, P. Sechopoulos, I. Robotis, and G. Margantinis. 2007. The long-term impact of Helicobacter pylori eradication on gastric histology: A systemic review and metaanalysis. Helicobacter 12(Suppl. 2): 32-38.
  31. Sachs, G., D. L. Weeks, Y. Wen, E. A. Marcus, D. R. Scott, and K. Melchers. 2005. Acid acclimation by Helicobacter pylori. Physiology 20: 429-438. https://doi.org/10.1152/physiol.00032.2005
  32. Scott, D. R., E. A. Marcus, D. L. Weeks, and G. Sachs. 2002. Mechanisms of acid resistance due to the urease system of Helicobacter pylori. Gastroenterology 123: 187-195. https://doi.org/10.1053/gast.2002.34218
  33. Shimomura, H., S. Hayashi, K. Yokota, K. Oguma, and Y. Hirai. 2007. Conversion of flavodoxin from holoenzyme to apoprotein during growth phase changes in Helicobacter pylori. J. Bacteriol. 189: 4960-4963. https://doi.org/10.1128/JB.00272-07
  34. Silva, P. and H. Geros. 2009. Regulation by salt of vacuolar H+- ATPase and $H^+$-pyrophosphatase activities and $Na^+/H^+$ exchange. Plant Signal Behav. 4: 718-726. https://doi.org/10.4161/psb.4.8.9236
  35. Skou, J. C. and M. Esmann. 1992. The Na,K-ATPase. J. Bioenerg. Biomembr. 24: 249-261.
  36. Skouloubris, S., J. M. Thiberge, A. Labigne, and H. De Reuse. 1998. The Helicobacter pylori UreI protein is not involved in urease activity but is essential for bacterial survival in vivo. Infect. Immun. 66: 4517-4521.
  37. Spiegelhalder, C., B. Gerstenecker, A. Kersten, E. Schiltz, and M. Kist. 1993. Purification of Helicobacter pylori superoxide dismutase and cloning and sequencing of the gene. Infect. Immun. 61: 5315-5325.
  38. Stingl, K., K. Altendorf, and E. P. Bakker. 2002. Acid survival of Helicobacter pylori: How does urease activity trigger cytoplasmic pH homeostasis? Trends Microbiol. 10: 70-74. https://doi.org/10.1016/S0966-842X(01)02287-9
  39. Tomb, J. F., O. White, A. R. Kerlavage, R. A. Clayton, G. G. Sutton, R. D. Fleischmann, et al. 1997. The complete genome sequence of the gastric pathogen Helicobacter pylori. Nature 388: 539-547. https://doi.org/10.1038/41483
  40. van Vliet, A. H., E. J. Kuipers, B. Waidner, B. J. Davies, N. de Vries, C. W. Penn, et al. 2001. Nickel-responsive induction of urease expression in Helicobacter pylori is mediated at the transcriptional level. Infect. Immun. 69: 4891-4897.
  41. Wall, S. M. 1996. Ammonium transport and the role of the Na,K-ATPase. Miner. Electrolyte Metab. 22: 311-317.
  42. Wall, S. M. 1996. $NH_4^^+$ augments net acid secretion by a ouabainsensitive mechanism in isolated perfused inner medullary collecting ducts. Am. J. Physiol. 270: F432-F439.
  43. Wall, S. M. 1997. Ouabain reduces net acid secretion and increases pHi by inhibiting $NH_4^^+$ uptake on rat tIMCD Na(+)- K(+)-ATPase. Am. J. Physiol. 273: F857-F868.
  44. Wall, S. M. and L. M. Koger. 1994.$NH_4^^+$ transport mediated by Na(+)-K(+)-ATPase in rat inner medullary collecting duct. Am. J. Physiol. 267: F660-F670.
  45. Weatherburn, M. W. 1967. Phenol-hypochlorite reaction for determination of ammonia. Anal. Biochem. 39: 971-974.
  46. Yoda, A. and L. E. Hokins. 1970. On the reversibility of binding of cardiotonic steroids to a partially purified (Na + K)-activated adenosinetriphosphatase from beef brain. Biochem. Biophys. Res. Commun. 40: 880-886. https://doi.org/10.1016/0006-291X(70)90985-X
  47. Yun, S. K. and S. Y. Hwang. 1997. Characteristics of ATPases present in everted membrane vesicles of Helicobacter pylori. J. Microbiol. Biotechnol. 7: 167-173.
  48. Yun, S. K., M. R. Ki, J. K. Park, W. J. Lim, and S. Y. Hwang. 2000. Cation flux-mediated activation of P-type ATPase in Helicobacter pylori. J. Microbiol. Biotechnol. 10: 441-449.

피인용 문헌

  1. Glutamine-Induced Production and Secretion of Helicobacter pylori ${\gamma}$-Glutamyltranspeptidase at Low pH and Its Putative Role in Glutathione Transport vol.23, pp.4, 2011, https://doi.org/10.4014/jmb.1210.10035