Comparative studies of Yersinia pestis outer membrane isolation techniques and their potential use in plague epidemiology

Abath, Frederico Guilherme Coutinho; Ferreira, Luís Carlos de Sousa

doi:10.1590/S0036-46651990000200002

Abstracts

In the present study three techniques for obtaining outer membrane enriched fractions from Yersinia pestis were evaluated. The techniques analysed were: differential solubilization of the cytoplasmic membrane with Sarkosyl or Triton X-100, and centrifugation in sucrose density gradients. The sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of outer membrane isolated by the different methods resulted in similar protein patterns. The measurement of NADH-dehydrogenase and succinate dehydrogenase (inner membrane enzymes) indicated that the outer membrane preparations obtained by the three methods were pure enough for analytical studies. In addition, preliminary evidences on the potential use of outer membrane proteins for the identification of geographic variants of Y. pestis wild isolates are presented.

Yersinia pestis; outer membrane proteins; membrane isolation techniques

No presente estudo três técnicas para isolamento de frações enriquecidas em membrana externa de Y. pestis foram avaliadas. As técnicas utilizadas foram: centrifugação em gradiente de densidade em sacarose e solubilização diferencial com Sarkosyl ou Triton X-100. A análise por eletroforese em gel de poliacrilamida na presença de dodecil sulfato de sódio (SDS-PAGE) das membranas externas extraídas pelos diferentes métodos evidenciou perfis protéicos semelhantes. A determinação das atividades de NADH-desidrogenase e succinato-desidrogenase (enzimas de membrana interna) indicou que todas as preparações estudadas eram adequadas a estudos analíticos. Obteve-se evidências preliminares sobre o possível uso de perfis protéicos de membrana externa na identificação de variantes geográficos entre isolados selvagens de Y. pestis.

ORIGINAL ARTICLES

Comparative studies of Yersinia pestis outer membrane isolation techniques and their potential use in plague epidemiology

Estudo comparativo de técnicas de isolamento de membrana externa de Yersinia pestis e seu uso na epidemiologia da peste

Frederico Guilherme Coutinho Abath^I; Luís Carlos de Sousa Ferreira^II

^ICentro de Pesquisas Aggeu Magalhães (Fundação Oswaldo Cruz). Caixa Postal 7472. CEP 59.739 Recife, Pernambuco, Brazil

^IILaboratório de Imunopatologia Keizo Asami e Departamento de Biofísica e Radiobiologia da Universidade Federal de Pernambuco. CEP 59739 Recife, Pernambuco, Brazil

^{Present address} Present address: Dr. Luiz Carlos de Souza Ferreira Universidade Federal do Rio de Janeiro, Instituto de Biofísica Carlos Chagas Filho CEP 21.941 Rio de Janeiro, Brasil

SUMMARY

In the present study three techniques for obtaining outer membrane enriched fractions from Yersinia pestis were evaluated. The techniques analysed were: differential solubilization of the cytoplasmic membrane with Sarkosyl or Triton X-100, and centrifugation in sucrose density gradients. The sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of outer membrane isolated by the different methods resulted in similar protein patterns. The measurement of NADH-dehydrogenase and succinate dehydrogenase (inner membrane enzymes) indicated that the outer membrane preparations obtained by the three methods were pure enough for analytical studies. In addition, preliminary evidences on the potential use of outer membrane proteins for the identification of geographic variants of Y. pestis wild isolates are presented.

Key words: Yersinia pestis, outer membrane proteins, membrane isolation techniques.

RESUMO

No presente estudo três técnicas para isolamento de frações enriquecidas em membrana externa de Y. pestis foram avaliadas. As técnicas utilizadas foram: centrifugação em gradiente de densidade em sacarose e solubilização diferencial com Sarkosyl ou Triton X-100. A análise por eletroforese em gel de poliacrilamida na presença de dodecil sulfato de sódio (SDS-PAGE) das membranas externas extraídas pelos diferentes métodos evidenciou perfis protéicos semelhantes. A determinação das atividades de NADH-desidrogenase e succinato-desidrogenase (enzimas de membrana interna) indicou que todas as preparações estudadas eram adequadas a estudos analíticos. Obteve-se evidências preliminares sobre o possível uso de perfis protéicos de membrana externa na identificação de variantes geográficos entre isolados selvagens de Y. pestis.

Full text available only in PDF format.

Texto completo disponível apenas em PDF.

ACKNOWLEDGMENTS

We are acknowledged to Dra. A. M. P. Almeida for helpful, advice, incentive and bacterial strains; Dr. R. R. Brubaker for the kind gift of strains A1122 and KIM and Mr. H. S. Almeida and Mrs. Y. Nakasawa for the helpful technical assistance. Research supported by the Japanese Cooperation Agency.

Recebido para publicação em 18/5/1989.

1. BARENKAMP, S.J.; MUNSON, R.S. & GRANOFF, D.M. Subtyping isolates of Haemophilus influenzae types by outer membrane protein profiles. J. infect. Dis., 143: 668-676, 1981.
2. BOLIN, I.; NORLANDER, L. & WOLF-WATZ, H. Temperature-inducible outer membrane protein of Yersinia pseudotuberculosis and Yersinia enterocolitica is associated with the virulence plasmid. Infect. Immun., 37: 506-512, 1982.
3. BOLIN, I.; PORTNOY, D.A. & WOLF-WATZ, H. Expression of temperature-inducible outer membrane proteins of Yersinia. Infect. Immun., 48: 234-240, 1985.
4. BOLIN, I.; FORSBERG, A.; NORLANDER, L.; SKURNIK, M. & WOLF-WATZ, H. Identification and mapping of the temperature-inducible, plasmid-coded proteins of Yersinia spp. Infect. Immun., 56: 343-348, 1988.
5. BRUBAKER, R.R. Mutation ratio to nonpigmentation in Pasteurella pestis. J. Bact., 98: 1404-1406, 1970.
6. BRAUN, V. & HANTKE, K. Bacterial cell surface receptors. In: GHOSH, B.K. Organization of Prokaryotic cell membranes. Flórida, CRC Press, 1981. v. 2, p. 1-73.
7. FERREIRA, L.C.S. Proteínas da membrana externa e divisăo celular de Escherichia coli K12. Rio de Janeiro, 1985. (Tese de Doutoramento Instituto de Biofísica da Universidade Federal do Rio de Janeiro).
8. HELMUTH, R.; STEPHAN, R.; BUNGE, C.; HOOG, B.; STEINBECK, A. & BULLING, E. Epidemiology of virulence-associated plasmids and outer membrane proteins patterns within seven common Salmonella serotypes. Infect. Immun., 48: 175-182, 1985.
9. HUDSON, B.W.; QUAN, T.J.; SITES, V.R. & MARSHALL, J.D. An electrophoretic and bacteriologic study of Yersinia pestis isolates from Central Java, Ásia and the Western hemisphere. Amer. J. trop. Med. Hyg., 22: 642-653, 1973.
10. ITO, K.; SATO, T. & YURA, T. - Synthesis and assembly of the membrane proteins in E. coli. Cell, 11: 551-559, 1977.
11. JAWETZ, E. & MEYER, K.F. Avirulent strains of Pasteurella pestis. J. infect. Dis., 73: 124-143, 1949.
12. JOHNSTON, K.H.; HOLMES, K.K. & GOTSCHLICH, E.C. The analogic classification of Neisseria gonorrhoea I. Isolation of the outer membrane complex responsible for serotype specificity. J. exp. med., 143: 741-758, 1976.
13. LAEMMLI, U.K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227: 680-685, 1970.
14. LOWRY, O.H.; ROSEBROUGH, N.J.; FARR, A.C. & RANDALL, R.J. Protein measurement with the Folin phenol reagent. J. biol. Chem., 183: 265-275, 1951.
15. LUGTENBERG, B. & ALPHEN, L. VAN. Molecular architecture and functioning of the outer membrane of Escherichia coli and other gram-negative bacteria. Biochim. Biophys. Acta (Amst.), 737: 51-115, 1983.
16. MACKLER, B. DPNH oxidase of heart muscle. Meth. Enzymol., 10: 261-263, 1967.
17. MORRISSEY, J.H. Silver stain for proteins in polyacrylamide gels: a modified procedure with enhanced uniform sensitivity. Analyt. Biochem., 117: 307-310, 1981.
18. OSBORN, M.I.; GANDER, J.E.; PARISI, E. & CARSON, J. Mechanism of assembly of the outer membrane of Salmonella typhimurium: isolation and characterization of cytoplasmic and outer membrane. J. biol. Chem., 247: 3962-3972, 1972.
19. PORTNOY, D.A.; WOLF-WATZ, H.; BOLIN, I.; BEEDER, A.B. & FALKOW, S. Characterization of common virulence plasmids in Yersinia species and their role in the expression of outer membrane proteins. Infect. Immun., 43: 108-114, 1984.
20. STRALEY, S.C. & BRUBAKER, R.R. Localization in Yersinia pestis of peptides associated with virulence. Infect. Immun., 36: 129-135, 1982.
21. STRALEY, S. The plasmid-encoded outer membrane proteins of Yersinia pestis. Rev. infect. Dis., 10: 323-326, 1988.

Present address:

Dr. Luiz Carlos de Souza Ferreira

Universidade Federal do Rio de Janeiro, Instituto de Biofísica Carlos Chagas Filho

CEP 21.941 Rio de Janeiro, Brasil

Publication Dates

Publication in this collection
13 Sept 2006
Date of issue
Apr 1990

History

Received
18 May 1989

This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.

[1] 1. BARENKAMP, S.J.; MUNSON, R.S. & GRANOFF, D.M. Subtyping isolates of Haemophilus influenzae types by outer membrane protein profiles. J. infect. Dis., 143: 668-676, 1981.

[2] 2. BOLIN, I.; NORLANDER, L. & WOLF-WATZ, H. Temperature-inducible outer membrane protein of Yersinia pseudotuberculosis and Yersinia enterocolitica is associated with the virulence plasmid. Infect. Immun., 37: 506-512, 1982.

[3] 3. BOLIN, I.; PORTNOY, D.A. & WOLF-WATZ, H. Expression of temperature-inducible outer membrane proteins of Yersinia. Infect. Immun., 48: 234-240, 1985.

[4] 4. BOLIN, I.; FORSBERG, A.; NORLANDER, L.; SKURNIK, M. & WOLF-WATZ, H. Identification and mapping of the temperature-inducible, plasmid-coded proteins of Yersinia spp. Infect. Immun., 56: 343-348, 1988.

[5] 5. BRUBAKER, R.R. Mutation ratio to nonpigmentation in Pasteurella pestis. J. Bact., 98: 1404-1406, 1970.

[6] 6. BRAUN, V. & HANTKE, K. Bacterial cell surface receptors. In: GHOSH, B.K. Organization of Prokaryotic cell membranes. Flórida, CRC Press, 1981. v. 2, p. 1-73.

[7] 7. FERREIRA, L.C.S. Proteínas da membrana externa e divisăo celular de Escherichia coli K12. Rio de Janeiro, 1985. (Tese de Doutoramento Instituto de Biofísica da Universidade Federal do Rio de Janeiro).

[8] 8. HELMUTH, R.; STEPHAN, R.; BUNGE, C.; HOOG, B.; STEINBECK, A. & BULLING, E. Epidemiology of virulence-associated plasmids and outer membrane proteins patterns within seven common Salmonella serotypes. Infect. Immun., 48: 175-182, 1985.

[9] 9. HUDSON, B.W.; QUAN, T.J.; SITES, V.R. & MARSHALL, J.D. An electrophoretic and bacteriologic study of Yersinia pestis isolates from Central Java, Ásia and the Western hemisphere. Amer. J. trop. Med. Hyg., 22: 642-653, 1973.

[10] 10. ITO, K.; SATO, T. & YURA, T. - Synthesis and assembly of the membrane proteins in E. coli. Cell, 11: 551-559, 1977.

[11] 11. JAWETZ, E. & MEYER, K.F. Avirulent strains of Pasteurella pestis. J. infect. Dis., 73: 124-143, 1949.

[12] 12. JOHNSTON, K.H.; HOLMES, K.K. & GOTSCHLICH, E.C. The analogic classification of Neisseria gonorrhoea I. Isolation of the outer membrane complex responsible for serotype specificity. J. exp. med., 143: 741-758, 1976.

[13] 13. LAEMMLI, U.K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227: 680-685, 1970.

[14] 14. LOWRY, O.H.; ROSEBROUGH, N.J.; FARR, A.C. & RANDALL, R.J. Protein measurement with the Folin phenol reagent. J. biol. Chem., 183: 265-275, 1951.

[15] 15. LUGTENBERG, B. & ALPHEN, L. VAN. Molecular architecture and functioning of the outer membrane of Escherichia coli and other gram-negative bacteria. Biochim. Biophys. Acta (Amst.), 737: 51-115, 1983.

[16] 16. MACKLER, B. DPNH oxidase of heart muscle. Meth. Enzymol., 10: 261-263, 1967.

[17] 17. MORRISSEY, J.H. Silver stain for proteins in polyacrylamide gels: a modified procedure with enhanced uniform sensitivity. Analyt. Biochem., 117: 307-310, 1981.

[18] 18. OSBORN, M.I.; GANDER, J.E.; PARISI, E. & CARSON, J. Mechanism of assembly of the outer membrane of Salmonella typhimurium: isolation and characterization of cytoplasmic and outer membrane. J. biol. Chem., 247: 3962-3972, 1972.

[19] 19. PORTNOY, D.A.; WOLF-WATZ, H.; BOLIN, I.; BEEDER, A.B. & FALKOW, S. Characterization of common virulence plasmids in Yersinia species and their role in the expression of outer membrane proteins. Infect. Immun., 43: 108-114, 1984.

[20] 20. STRALEY, S.C. & BRUBAKER, R.R. Localization in Yersinia pestis of peptides associated with virulence. Infect. Immun., 36: 129-135, 1982.

[21] 21. STRALEY, S. The plasmid-encoded outer membrane proteins of Yersinia pestis. Rev. infect. Dis., 10: 323-326, 1988.

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