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. 1996 Mar;64(3):905–912. doi: 10.1128/iai.64.3.905-912.1996

Surface localization of Helicobacter pylori urease and a heat shock protein homolog requires bacterial autolysis.

S H Phadnis 1, M H Parlow 1, M Levy 1, D Ilver 1, C M Caulkins 1, J B Connors 1, B E Dunn 1
PMCID: PMC173855  PMID: 8641799

Abstract

Helicobacter pylori is a gram-negative bacterium which causes chronic gastritis and is associated with peptic ulcer disease, gastric carcinoma, and gastric lymphoma. The bacterium is characterized by potent urease activity, thought to be located on the outer membrane, which is essential for survival at low pH. The purpose of the present study was to investigate mechanisms whereby urease and HspB, a GroEL homolog, become surface associated in vitro. Urease, HspB, and catalase were located almost exclusively within the cytoplasm in fresh log-phase cultures assessed by cryo- immunoelectron microscopy. In contrast, significant amounts of surface-associated antigen were observed in older or subcultured preparations concomitantly with the appearance of significant amounts of extracellular antigen, amorphous debris, and membrane fragments. By use of a variety of biochemical methods, a significant fraction of urease and HspB was associated with the outer membrane in subcultured preparations of H. pylori. Taken together, these results strongly suggest that H. pylori cells undergo spontaneous autolysis during culture and that urease and HspB become surface associated only concomitant with bacterial autolysis. By comparing enzyme sensitivity to flurofamide (a potent, poorly diffusible urease inhibitor) in whole cells with that in deliberately lysed cells, we show that both extracellular and intracellular urease molecules are active enzymatically. Autolysis of H. pylori is an important phenomenon to recognize since it likely exerts significant effects on the behavior of H. pylori. Furthermore, the surface properties of H. pylori must be unique in promoting adsorption of cytoplasmic proteins.

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Selected References

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  1. Andrutis K. A., Fox J. G., Schauer D. B., Marini R. P., Murphy J. C., Yan L., Solnick J. V. Inability of an isogenic urease-negative mutant stain of Helicobacter mustelae to colonize the ferret stomach. Infect Immun. 1995 Sep;63(9):3722–3725. doi: 10.1128/iai.63.9.3722-3725.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Barer M. R., Elliott T. S., Berkeley D., Thomas J. E., Eastham E. J. Cytopathic effects of Campylobacter pylori urease. J Clin Pathol. 1988 May;41(5):597–597. doi: 10.1136/jcp.41.5.597-a. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Berry A. M., Paton J. C., Hansman D. Effect of insertional inactivation of the genes encoding pneumolysin and autolysin on the virulence of Streptococcus pneumoniae type 3. Microb Pathog. 1992 Feb;12(2):87–93. doi: 10.1016/0882-4010(92)90111-z. [DOI] [PubMed] [Google Scholar]
  4. Blaser M. J. Hypotheses on the pathogenesis and natural history of Helicobacter pylori-induced inflammation. Gastroenterology. 1992 Feb;102(2):720–727. doi: 10.1016/0016-5085(92)90126-j. [DOI] [PubMed] [Google Scholar]
  5. Bode G., Malfertheiner P., Lehnhardt G., Nilius M., Ditschuneit H. Ultrastructural localization of urease of Helicobacter pylori. Med Microbiol Immunol. 1993 Nov;182(5):233–242. doi: 10.1007/BF00579622. [DOI] [PubMed] [Google Scholar]
  6. Corthésy-Theulaz I., Porta N., Glauser M., Saraga E., Vaney A. C., Haas R., Kraehenbuhl J. P., Blum A. L., Michetti P. Oral immunization with Helicobacter pylori urease B subunit as a treatment against Helicobacter infection in mice. Gastroenterology. 1995 Jul;109(1):115–121. doi: 10.1016/0016-5085(95)90275-9. [DOI] [PubMed] [Google Scholar]
  7. Cover T. L., Tummuru M. K., Cao P., Thompson S. A., Blaser M. J. Divergence of genetic sequences for the vacuolating cytotoxin among Helicobacter pylori strains. J Biol Chem. 1994 Apr 8;269(14):10566–10573. [PubMed] [Google Scholar]
  8. Craig P. M., Territo M. C., Karnes W. E., Walsh J. H. Helicobacter pylori secretes a chemotactic factor for monocytes and neutrophils. Gut. 1992 Aug;33(8):1020–1023. doi: 10.1136/gut.33.8.1020. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Cussac V., Ferrero R. L., Labigne A. Expression of Helicobacter pylori urease genes in Escherichia coli grown under nitrogen-limiting conditions. J Bacteriol. 1992 Apr;174(8):2466–2473. doi: 10.1128/jb.174.8.2466-2473.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Doig P., Trust T. J. Identification of surface-exposed outer membrane antigens of Helicobacter pylori. Infect Immun. 1994 Oct;62(10):4526–4533. doi: 10.1128/iai.62.10.4526-4533.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Dunn B. E., Perez-Perez G. I., Blaser M. J. Two-dimensional gel electrophoresis and immunoblotting of Campylobacter pylori proteins. Infect Immun. 1989 Jun;57(6):1825–1833. doi: 10.1128/iai.57.6.1825-1833.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Dunn B. E., Roop R. M., 2nd, Sung C. C., Sharma S. A., Perez-Perez G. I., Blaser M. J. Identification and purification of a cpn60 heat shock protein homolog from Helicobacter pylori. Infect Immun. 1992 May;60(5):1946–1951. doi: 10.1128/iai.60.5.1946-1951.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Dunn B. E., Sung C. C., Taylor N. S., Fox J. G. Purification and characterization of Helicobacter mustelae urease. Infect Immun. 1991 Sep;59(9):3343–3345. doi: 10.1128/iai.59.9.3343-3345.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Eaton K. A., Brooks C. L., Morgan D. R., Krakowka S. Essential role of urease in pathogenesis of gastritis induced by Helicobacter pylori in gnotobiotic piglets. Infect Immun. 1991 Jul;59(7):2470–2475. doi: 10.1128/iai.59.7.2470-2475.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Eschweiler B., Bohrmann B., Gerstenecker B., Schiltz E., Kist M. In situ localization of the 60 k protein of Helicobacter pylori, which belongs to the family of heat shock proteins, by immuno-electron microscopy. Zentralbl Bakteriol. 1993 Sep;280(1-2):73–85. doi: 10.1016/s0934-8840(11)80942-4. [DOI] [PubMed] [Google Scholar]
  16. Evans D. J., Jr, Evans D. G., Kirkpatrick S. S., Graham D. Y. Characterization of the Helicobacter pylori urease and purification of its subunits. Microb Pathog. 1991 Jan;10(1):15–26. doi: 10.1016/0882-4010(91)90062-f. [DOI] [PubMed] [Google Scholar]
  17. Fath M. J., Kolter R. ABC transporters: bacterial exporters. Microbiol Rev. 1993 Dec;57(4):995–1017. doi: 10.1128/mr.57.4.995-1017.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Ferrero R. L., Cussac V., Courcoux P., Labigne A. Construction of isogenic urease-negative mutants of Helicobacter pylori by allelic exchange. J Bacteriol. 1992 Jul;174(13):4212–4217. doi: 10.1128/jb.174.13.4212-4217.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Ferrero R. L., Thiberge J. M., Huerre M., Labigne A. Recombinant antigens prepared from the urease subunits of Helicobacter spp.: evidence of protection in a mouse model of gastric infection. Infect Immun. 1994 Nov;62(11):4981–4989. doi: 10.1128/iai.62.11.4981-4989.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Griffiths G., McDowall A., Back R., Dubochet J. On the preparation of cryosections for immunocytochemistry. J Ultrastruct Res. 1984 Oct;89(1):65–78. doi: 10.1016/s0022-5320(84)80024-6. [DOI] [PubMed] [Google Scholar]
  21. Hawtin P. R., Stacey A. R., Newell D. G. Investigation of the structure and localization of the urease of Helicobacter pylori using monoclonal antibodies. J Gen Microbiol. 1990 Oct;136(10):1995–2000. doi: 10.1099/00221287-136-10-1995. [DOI] [PubMed] [Google Scholar]
  22. Hazell S. L., Evans D. J., Jr, Graham D. Y. Helicobacter pylori catalase. J Gen Microbiol. 1991 Jan;137(1):57–61. doi: 10.1099/00221287-137-1-57. [DOI] [PubMed] [Google Scholar]
  23. Hu L. T., Mobley H. L. Purification and N-terminal analysis of urease from Helicobacter pylori. Infect Immun. 1990 Apr;58(4):992–998. doi: 10.1128/iai.58.4.992-998.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Jarvis K. G., Girón J. A., Jerse A. E., McDaniel T. K., Donnenberg M. S., Kaper J. B. Enteropathogenic Escherichia coli contains a putative type III secretion system necessary for the export of proteins involved in attaching and effacing lesion formation. Proc Natl Acad Sci U S A. 1995 Aug 15;92(17):7996–8000. doi: 10.1073/pnas.92.17.7996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Jonsson A. B., Nyberg G., Normark S. Phase variation of gonococcal pili by frameshift mutation in pilC, a novel gene for pilus assembly. EMBO J. 1991 Feb;10(2):477–488. doi: 10.1002/j.1460-2075.1991.tb07970.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Loewen P. C., Switala J. Purification and characterization of catalase-1 from Bacillus subtilis. Biochem Cell Biol. 1987 Nov;65(11):939–947. doi: 10.1139/o87-122. [DOI] [PubMed] [Google Scholar]
  27. Macchia G., Massone A., Burroni D., Covacci A., Censini S., Rappuoli R. The Hsp60 protein of Helicobacter pylori: structure and immune response in patients with gastroduodenal diseases. Mol Microbiol. 1993 Aug;9(3):645–652. doi: 10.1111/j.1365-2958.1993.tb01724.x. [DOI] [PubMed] [Google Scholar]
  28. Mai U. E., Perez-Perez G. I., Allen J. B., Wahl S. M., Blaser M. J., Smith P. D. Surface proteins from Helicobacter pylori exhibit chemotactic activity for human leukocytes and are present in gastric mucosa. J Exp Med. 1992 Feb 1;175(2):517–525. doi: 10.1084/jem.175.2.517. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Marshall B. J., Barrett L. J., Prakash C., McCallum R. W., Guerrant R. L. Urea protects Helicobacter (Campylobacter) pylori from the bactericidal effect of acid. Gastroenterology. 1990 Sep;99(3):697–702. doi: 10.1016/0016-5085(90)90957-3. [DOI] [PubMed] [Google Scholar]
  30. McCord J. M., Fridovich I. Superoxide dismutase. An enzymic function for erythrocuprein (hemocuprein). J Biol Chem. 1969 Nov 25;244(22):6049–6055. [PubMed] [Google Scholar]
  31. Michetti P., Corthésy-Theulaz I., Davin C., Haas R., Vaney A. C., Heitz M., Bille J., Kraehenbuhl J. P., Saraga E., Blum A. L. Immunization of BALB/c mice against Helicobacter felis infection with Helicobacter pylori urease. Gastroenterology. 1994 Oct;107(4):1002–1011. doi: 10.1016/0016-5085(94)90224-0. [DOI] [PubMed] [Google Scholar]
  32. Mobley H. L., Cortesia M. J., Rosenthal L. E., Jones B. D. Characterization of urease from Campylobacter pylori. J Clin Microbiol. 1988 May;26(5):831–836. doi: 10.1128/jcm.26.5.831-836.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Mobley H. L., Garner R. M., Bauerfeind P. Helicobacter pylori nickel-transport gene nixA: synthesis of catalytically active urease in Escherichia coli independent of growth conditions. Mol Microbiol. 1995 Apr;16(1):97–109. doi: 10.1111/j.1365-2958.1995.tb02395.x. [DOI] [PubMed] [Google Scholar]
  34. Mobley H. L., Hausinger R. P. Microbial ureases: significance, regulation, and molecular characterization. Microbiol Rev. 1989 Mar;53(1):85–108. doi: 10.1128/mr.53.1.85-108.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Mobley H. L., Island M. D., Hausinger R. P. Molecular biology of microbial ureases. Microbiol Rev. 1995 Sep;59(3):451–480. doi: 10.1128/mr.59.3.451-480.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Nilius M., Ströhle A., Bode G., Malfertheiner P. Coccoid like forms (CLF) of Helicobacter pylori. Enzyme activity and antigenicity. Zentralbl Bakteriol. 1993 Sep;280(1-2):259–272. doi: 10.1016/s0934-8840(11)80964-3. [DOI] [PubMed] [Google Scholar]
  37. Nomura A., Stemmermann G. N., Chyou P. H., Kato I., Perez-Perez G. I., Blaser M. J. Helicobacter pylori infection and gastric carcinoma among Japanese Americans in Hawaii. N Engl J Med. 1991 Oct 17;325(16):1132–1136. doi: 10.1056/NEJM199110173251604. [DOI] [PubMed] [Google Scholar]
  38. Osborn M. J., Munson R. Separation of the inner (cytoplasmic) and outer membranes of Gram-negative bacteria. Methods Enzymol. 1974;31:642–653. doi: 10.1016/0076-6879(74)31070-1. [DOI] [PubMed] [Google Scholar]
  40. Parsonnet J., Friedman G. D., Vandersteen D. P., Chang Y., Vogelman J. H., Orentreich N., Sibley R. K. Helicobacter pylori infection and the risk of gastric carcinoma. N Engl J Med. 1991 Oct 17;325(16):1127–1131. doi: 10.1056/NEJM199110173251603. [DOI] [PubMed] [Google Scholar]
  41. Parsonnet J., Hansen S., Rodriguez L., Gelb A. B., Warnke R. A., Jellum E., Orentreich N., Vogelman J. H., Friedman G. D. Helicobacter pylori infection and gastric lymphoma. N Engl J Med. 1994 May 5;330(18):1267–1271. doi: 10.1056/NEJM199405053301803. [DOI] [PubMed] [Google Scholar]
  42. Paton J. C., Andrew P. W., Boulnois G. J., Mitchell T. J. Molecular analysis of the pathogenicity of Streptococcus pneumoniae: the role of pneumococcal proteins. Annu Rev Microbiol. 1993;47:89–115. doi: 10.1146/annurev.mi.47.100193.000513. [DOI] [PubMed] [Google Scholar]
  43. Phadnis S. H., Ilver D., Janzon L., Normark S., Westblom T. U. Pathological significance and molecular characterization of the vacuolating toxin gene of Helicobacter pylori. Infect Immun. 1994 May;62(5):1557–1565. doi: 10.1128/iai.62.5.1557-1565.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Pugsley A. P. The complete general secretory pathway in gram-negative bacteria. Microbiol Rev. 1993 Mar;57(1):50–108. doi: 10.1128/mr.57.1.50-108.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Pérez-Pérez G. I., Gower C. B., Blaser M. J. Effects of cations on Helicobacter pylori urease activity, release, and stability. Infect Immun. 1994 Jan;62(1):299–302. doi: 10.1128/iai.62.1.299-302.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Rautelin H., Blomberg B., Fredlund H., Järnerot G., Danielsson D. Incidence of Helicobacter pylori strains activating neutrophils in patients with peptic ulcer disease. Gut. 1993 May;34(5):599–603. doi: 10.1136/gut.34.5.599. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Schmitt W., Haas R. Genetic analysis of the Helicobacter pylori vacuolating cytotoxin: structural similarities with the IgA protease type of exported protein. Mol Microbiol. 1994 Apr;12(2):307–319. doi: 10.1111/j.1365-2958.1994.tb01019.x. [DOI] [PubMed] [Google Scholar]
  48. Seifert H. S., Ajioka R. S., Marchal C., Sparling P. F., So M. DNA transformation leads to pilin antigenic variation in Neisseria gonorrhoeae. Nature. 1988 Nov 24;336(6197):392–395. doi: 10.1038/336392a0. [DOI] [PubMed] [Google Scholar]
  49. Slot J. W., Geuze H. J., Gigengack S., James D. E., Lienhard G. E. Translocation of the glucose transporter GLUT4 in cardiac myocytes of the rat. Proc Natl Acad Sci U S A. 1991 Sep 1;88(17):7815–7819. doi: 10.1073/pnas.88.17.7815. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Slot J. W., Geuze H. J., Gigengack S., Lienhard G. E., James D. E. Immuno-localization of the insulin regulatable glucose transporter in brown adipose tissue of the rat. J Cell Biol. 1991 Apr;113(1):123–135. doi: 10.1083/jcb.113.1.123. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Smoot D. T., Mobley H. L., Chippendale G. R., Lewison J. F., Resau J. H. Helicobacter pylori urease activity is toxic to human gastric epithelial cells. Infect Immun. 1990 Jun;58(6):1992–1994. doi: 10.1128/iai.58.6.1992-1994.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Spiegelhalder C., Gerstenecker B., Kersten A., Schiltz E., Kist M. Purification of Helicobacter pylori superoxide dismutase and cloning and sequencing of the gene. Infect Immun. 1993 Dec;61(12):5315–5325. doi: 10.1128/iai.61.12.5315-5325.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Suerbaum S., Thiberge J. M., Kansau I., Ferrero R. L., Labigne A. Helicobacter pylori hspA-hspB heat-shock gene cluster: nucleotide sequence, expression, putative function and immunogenicity. Mol Microbiol. 1994 Dec;14(5):959–974. doi: 10.1111/j.1365-2958.1994.tb01331.x. [DOI] [PubMed] [Google Scholar]
  54. Telford J. L., Ghiara P., Dell'Orco M., Comanducci M., Burroni D., Bugnoli M., Tecce M. F., Censini S., Covacci A., Xiang Z. Gene structure of the Helicobacter pylori cytotoxin and evidence of its key role in gastric disease. J Exp Med. 1994 May 1;179(5):1653–1658. doi: 10.1084/jem.179.5.1653. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Tokuyasu K. T. Immunochemistry on ultrathin frozen sections. Histochem J. 1980 Jul;12(4):381–403. doi: 10.1007/BF01011956. [DOI] [PubMed] [Google Scholar]

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