Induction of iron regulated proteins during normal growth of Neisseria meningitidis in a chemically defined medium

Brandileone, Maria Cristina de Cunto; Zanella, Rosemeire Cobo; Vieira, Vera Simonsen Dias; Sacciii, Claudio Tavares; Milagres, Lucimar Gonçalves; Frasch, Carl Eduard

doi:10.1590/S0036-46651994000400002

Abstracts

The expression of iron regulated proteins (IRPs) in vitro has been obtained in the past by adding iron chelators to the culture after bacterial growth, in the presence of an organic iron source. We have investigated aspects concerning full expression of the meningococcal IRPs during normal growth, in defined conditions using Catlin medium, Mueller Hinton and Tryptic Soy Broth (TSB). The expression of IRPs varied between different strains with respect to Ethylenediamine Di-ortho-Hidroxy-phenyl-acetic acid (EDDA) concentrations, and according to culture medium, and also between different lots of TSB. For each strain, a specific set of IRPs were expressed and higher EDDA concentrations, or addition of glucose, or use of different culture media did not resulted in a differential expression of IRPs. We were not able to grow N. meningitidis under normal growth conditions using Desferal. We looked for a good yield of outer membrane vesicles (OMVs) expressing IRPs in iron-deficient Catlin medium containing EDDA and Hemin. Culture for 32 h at 30ºC after growing for 16 h at 37ºC supported good bacterial growth. Bacterial lysis was noted after additional 24 h at 30ºC. Approximately 4 times more OMVs was recoverable from a culture supernatant after 24 h at 30ºC than from the cells after 16 h at 37ºC. The IRP were as well expressed in OMVs from culture supernatant obtained after 24 h at 30ºC as from the cells after 16 h at 37ºC.

Neisseria meningitidis; Iron regulated proteins

A expressão das proteínas reguladas pelo ferro (IRPs), in vitro, tem sido obtida pela adição de quelantes de ferro ao meio de cultura, após o crescimento bacteriano, na presença de fonte de ferro orgânico. Neste estudo foram investigados aspectos da máxima expressão das IRPs de meningococo durante o crescimento normal, em condições de cultura definidas, utilizando-se o meio de Catlin e os caldos Mueller-Hinton e Tryptic Soja (TSB). Foram avaliadas as melhores condições para se obter vesículas de membrana externa (OMVs) contendo IRPs para uso em vacina de meningococo B. A expressão das IRPs variou entre as diferentes cepas com relação as diferentes concentrações de Etilenediamine Di-orto-Hidroxifenil-ácido acético (EDDA), de acordo com o meio de cultura e também entre os diferentes lotes de TSB. Para cada cepa, um específico padrão de ERPs foi expresso e altas concentrações de EDDA, adição de glicose ou o uso de diferentes meios de cultura não resultou em expressão diferencial das IRPs. Não foi possível cultivar meningococo em condições normais de crescimento utilizando-se Desferral. Foi investigado condições de crescimento para se obter bom rendimento de OMVs com expressão de IRPs em meio de Catlin deficiente em ferro, contendo EDDA e hemina. Cultura de 32 h a 30ºC, após incubação por 16 h a 37ºC, manteve bom crescimento, porém, lise da bactéria foi observada após 46 h a 30ºC. Aproximadamente 4 vezes mais OMVs foram recuperadas do sobrenadante de cultura após 24 h a 30ºC do que das células após 16 h a 37ºC. As IRPs foram bem expressas nas OMVs obtidas do sobrenadante de cultura após 24 h a 30ºC como também nas OMVs obtidas das células crescidas por 16 h a 37ºC.

MICROBIOLOGY

Induction of iron regulated proteins during normal growth of Neisseria meningitidis in a chemically defined medium

Indução das proteínas reguladas pelo ferro durante o crescimento normal de Neisseria meningitidis em meio quimicamente definido

Maria Cristina de Cunto Brandileone^I; Rosemeire Cobo Zanella^I; Vera Simonsen Dias Vieira^I; Claudio Tavares Sacciii^I; Lucimar Gonçalves Milagres^I; Carl Eduard Frasch^II

^IBacteriology Division, Adolfo Lutz Institute, Av. Dr. Arnaldo, 351, São Paulo 01246-902, Brazil

^IICenter for Biologics Evaluation and Research, Food and Drugs Administration, Bethesda, MD, USA

^{Address for correspondence} Address for correspondence: M.C.C. Brandileone Bacteriology Division, Adolfo Lutz Institute Av. Dr. Arnaldo, 351 São Paulo, 01246-902, Brazil

SUMMARY

The expression of iron regulated proteins (IRPs) in vitro has been obtained in the past by adding iron chelators to the culture after bacterial growth, in the presence of an organic iron source. We have investigated aspects concerning full expression of the meningococcal IRPs during normal growth, in defined conditions using Catlin medium, Mueller Hinton and Tryptic Soy Broth (TSB). The expression of IRPs varied between different strains with respect to Ethylenediamine Di-ortho-Hidroxy-phenyl-acetic acid (EDDA) concentrations, and according to culture medium, and also between different lots of TSB. For each strain, a specific set of IRPs were expressed and higher EDDA concentrations, or addition of glucose, or use of different culture media did not resulted in a differential expression of IRPs. We were not able to grow N. meningitidis under normal growth conditions using Desferal. We looked for a good yield of outer membrane vesicles (OMVs) expressing IRPs in iron-deficient Catlin medium containing EDDA and Hemin. Culture for 32 h at 30ºC after growing for 16 h at 37ºC supported good bacterial growth. Bacterial lysis was noted after additional 24 h at 30ºC. Approximately 4 times more OMVs was recoverable from a culture supernatant after 24 h at 30ºC than from the cells after 16 h at 37ºC. The IRP were as well expressed in OMVs from culture supernatant obtained after 24 h at 30ºC as from the cells after 16 h at 37ºC.

Keywords:Neisseria meningitidis; Iron regulated proteins.

RESUMO

A expressão das proteínas reguladas pelo ferro (IRPs), in vitro, tem sido obtida pela adição de quelantes de ferro ao meio de cultura, após o crescimento bacteriano, na presença de fonte de ferro orgânico. Neste estudo foram investigados aspectos da máxima expressão das IRPs de meningococo durante o crescimento normal, em condições de cultura definidas, utilizando-se o meio de Catlin e os caldos Mueller-Hinton e Tryptic Soja (TSB). Foram avaliadas as melhores condições para se obter vesículas de membrana externa (OMVs) contendo IRPs para uso em vacina de meningococo B. A expressão das IRPs variou entre as diferentes cepas com relação as diferentes concentrações de Etilenediamine Di-orto-Hidroxifenil-ácido acético (EDDA), de acordo com o meio de cultura e também entre os diferentes lotes de TSB. Para cada cepa, um específico padrão de ERPs foi expresso e altas concentrações de EDDA, adição de glicose ou o uso de diferentes meios de cultura não resultou em expressão diferencial das IRPs. Não foi possível cultivar meningococo em condições normais de crescimento utilizando-se Desferral. Foi investigado condições de crescimento para se obter bom rendimento de OMVs com expressão de IRPs em meio de Catlin deficiente em ferro, contendo EDDA e hemina. Cultura de 32 h a 30ºC, após incubação por 16 h a 37ºC, manteve bom crescimento, porém, lise da bactéria foi observada após 46 h a 30ºC. Aproximadamente 4 vezes mais OMVs foram recuperadas do sobrenadante de cultura após 24 h a 30ºC do que das células após 16 h a 37ºC. As IRPs foram bem expressas nas OMVs obtidas do sobrenadante de cultura após 24 h a 30ºC como também nas OMVs obtidas das células crescidas por 16 h a 37ºC.

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Recebido para publicação em 09/11/1993

Aceito para publicação em 23/05/1994

1. ARCHIBALD, F.S. & DEVOE, I.N. - Expression of Neisseria meningitidis in vitro. Infect. Immun., 27: 322-334, 1980.
2. BANERJEE-BHATNAGAR, N. & FRASCH, C.E. - Expression Neisseria meningitidis iron-regulated outer membrane proteins, including a 70-Kilodaltons transferrin receptor, and their potencial for use as vaccine. Infect. Immun., 58: 2875-2881, 1990.
3. BLACK, J.R. ; DYER, D.W. ; THOMPSON, M.K. & SPARLING, P.F. - Human immune response to iron-repressible outer membrane proteins of Neisseria meningitidis. Infect. Immun., 54: 710-713, 1986.
4. BULLEN, J.J. - The significance of iron in infection. Rev. infect. Dis., 3: 1127-1138, 1981.
5. CATLIN, W.B. - Nutritional profiles of Neisseria gonorrhoeae, Neisseria meningitidis and Neisseria lactamica in chemically for gonococcal typing. J. infect. Dis., 128: 178-194, 1973.
6. DYER, D.W.; WEST E.P.; MCKENNA, W.; THOMPSON, S.H. & SPARLING, P.F. - A pleiotropic iron-uptake mutant of Neisseria meningitidis lacks a 70-Kilodalton iron - regulated protein. Infect. Immun., 56: 977-983, 1988.
7. FINKELSTEIN, R.A.; SCIORTINO, C.V. & MCINTOSH, M.A. - Role of iron in microbe-host interactions. Rev. infect. Dis., 5: S759-S777, 1983.
8. GOTSCHLICH, E.C.; CORNELISSEN, C.; HILL, S.A. et al. - The mechanisms of genetic variation of gonococcal pili. Iron-inducible proteins of Neisseria. In: ACHTMAN, M.; KOHL, P.; MARCHAL, C. et al., ed. Neisseriae. New York, Walter de Gruyter, 1990. p.405-414.
9. HOLBEIN, B.E. - Differences in virulence for mice between disease and carrier strains of Neisseria meningitidis. Canad. J. Microbiol., 27: 738-741, 1981.
10. LAEMMLI, U.K. - Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227: 680-685, 1970.
11. LEE, B.C. & SCHRYVERS, A.B. - Specificity of the lactoferrin and transferrin receptors in Neisseria gonorrhoeae. Molec. Microbiol., 2: 827-829, 1988.
12. LOWRY, O.H.; ROSEBROUGH, N.J.; FARR, A.L. & RANDALL, R.H. - Protein measurement with the Folin phenol reagent. J. biol. Chem., 193: 265-275, 1951.
13. MASSON, P.L.; HEREMANS, J.F. & DIVE, C.H. - An iron-binding protein common to many external secretions. Clin. chim. Acta, 14: 729-734, 1966.
14. MCKENNA, W.R.; MICHELSEN, P.A.; SPARLING, P.F. & DYER, D.W. - Iron uptake from lactoferrin and transferrin by Neisseria gonorrhoeae. Infect. Immun., 56: 785-791, 1988.
15. MICKELSEN, P.A. & SPARLING, F.P. - Ability of Neisseria gonorrhoeae, Neisseria meningitidis, and comensal Neisseria species to obtain iron from transferrin and iron compounds. Infect. Immun., 33: 555-564, 1981.
16. MICKELSEN, P.A.; BLACKMAN, E. & SPARLING, F.P. - Ability of Neisseria gonorrhoeae, Neisseria meningitidis, and comensal Neisseria species to obtain iron from lactoferrin. Infect. Immun., 35: 915-920, 1982.
17. MOCCA, L.F. & FRASCH, C.E. - Sodium dodecyl sulfate polyacrylamide gel typing system for characterization of Neisseria meningitidis isolates. J. clin. Microbiol., 16: 240-244, 1982.
18. NEILANDS, J.B. - Microbiol envelope proteins related to iron. Ann. Rev. Microbiol., 36: 285-309, 1982.
19. NORQUIST, A.J.; DAVIS, L.; NORLANDER, L. & NORMARK, S. - The effect of iron starvation on the outer membrane protein composition of Neisseria gonorrhoeae. FEMS Microbiol. Lett., 4: 71-75, 1978.
20. OSBORN, M.J. - Studies on the Gram- negative cell wall. 1. Evidence for the role of 2-Keto-3-deoxyoc-tonate in the lipopolysaccharide of Salmonella typhimurium. Proc. nat. Acad. Sci. (Wash.), 50: 499-506, 1963.
21. PAYNE, S.M. & FINSKELSTEIN, R.A. - A critical role of iron in host-bacterial interactions. J. clin. Invest., 61: 1428-1440, 1978.
22. PETTERSSON, A,; KUIPERS, B.; PELZER, M., et al. - Monoclonal antibodies against the 70-Kilodalton iron regulated protein of Neisseria meningitidis are bactericidal and strain specific. Infect. Immun., 58: 3036-3041, 1990.
23. SCHRYVERS, A.B. - Characterization of the human transferrin and lactoferrin- receptors in Haemophilus infuenzae. Molec. Microbiol., 2: 467-472, 1988.
24. SCHRYVERS, A.B. & GONZALEZ, G.C. - Comparison of the abilities of different proteins sources of iron to enhance Neisseria meingitidis infection in mice. Infect. Immun., 57: 2425-2429, 1989.
25. SCHRYVERS, A.B. & GONZALEZ, G.C. - Receptors for transferrin in pathogenic bacteria are specific for the host's protein. Canad. J. Microbiol., 36: 145-147, 1990.
26. SCHRYVERS, A.B. & LEE, B.C. - Comparative analysis of the transferrin and lactoferrin binding proteins in the family Neisseriaceae. Canad. J. Microbiol., 35: 409-415, 1989.
27. SCHRYVERS, A.B. & MORRIS, L.J. - Identification and characterization of the lactoferrin-binding protein from Neisseria meningitidis. Infect. Immun., 56: 1144-1149, 1988.
28. SCHRYVERS, A.B. & MORRIS, L.J. - Identification and characterization of the transferrin receptor from Neisseria meningitidis. Molec. Microbiol., 2: 281-288, 1988.
29. SIMONSON, C.; BRENER, D. & DE VOE, I.W. - Expression of a high affinity mechanism for acquisition of transferrin iron by Neisseria meningitidis. Infect. Immun., 36: 107-113, 1982.
30. TSAI, C.M. & FRASCH, C.E. - Chemical analysis of major outer membrane proteins of Neisseria meningitidis: comparison of serotypes 2 and 11. J. Bact., 141: 169-176, 1980.
31. WEINBERG, E.D. - Iron and infection. Microbiol. Rev., 42: 45-66, 1978.
32. WEST, S.E. & SPARLING, P.F. - Response of Neisseria gonorrhoeae to iron limitation: alterations in expression of membrane proteins without apparent siderophore production. Infect. Immun., 47: 388-394, 1985.

Address for correspondence:

M.C.C. Brandileone

Bacteriology Division, Adolfo Lutz Institute

Av. Dr. Arnaldo, 351

São Paulo, 01246-902, Brazil

Publication Dates

Publication in this collection
20 Sept 2006
Date of issue
Aug 1994

History

Accepted
23 May 1994
Received
09 Nov 1993

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

[1] 1. ARCHIBALD, F.S. & DEVOE, I.N. - Expression of Neisseria meningitidis in vitro. Infect. Immun., 27: 322-334, 1980.

[2] 2. BANERJEE-BHATNAGAR, N. & FRASCH, C.E. - Expression Neisseria meningitidis iron-regulated outer membrane proteins, including a 70-Kilodaltons transferrin receptor, and their potencial for use as vaccine. Infect. Immun., 58: 2875-2881, 1990.

[3] 3. BLACK, J.R. ; DYER, D.W. ; THOMPSON, M.K. & SPARLING, P.F. - Human immune response to iron-repressible outer membrane proteins of Neisseria meningitidis. Infect. Immun., 54: 710-713, 1986.

[4] 4. BULLEN, J.J. - The significance of iron in infection. Rev. infect. Dis., 3: 1127-1138, 1981.

[5] 5. CATLIN, W.B. - Nutritional profiles of Neisseria gonorrhoeae, Neisseria meningitidis and Neisseria lactamica in chemically for gonococcal typing. J. infect. Dis., 128: 178-194, 1973.

[6] 6. DYER, D.W.; WEST E.P.; MCKENNA, W.; THOMPSON, S.H. & SPARLING, P.F. - A pleiotropic iron-uptake mutant of Neisseria meningitidis lacks a 70-Kilodalton iron - regulated protein. Infect. Immun., 56: 977-983, 1988.

[7] 7. FINKELSTEIN, R.A.; SCIORTINO, C.V. & MCINTOSH, M.A. - Role of iron in microbe-host interactions. Rev. infect. Dis., 5: S759-S777, 1983.

[8] 8. GOTSCHLICH, E.C.; CORNELISSEN, C.; HILL, S.A. et al. - The mechanisms of genetic variation of gonococcal pili. Iron-inducible proteins of Neisseria. In: ACHTMAN, M.; KOHL, P.; MARCHAL, C. et al., ed. Neisseriae. New York, Walter de Gruyter, 1990. p.405-414.

[9] 9. HOLBEIN, B.E. - Differences in virulence for mice between disease and carrier strains of Neisseria meningitidis. Canad. J. Microbiol., 27: 738-741, 1981.

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

[11] 11. LEE, B.C. & SCHRYVERS, A.B. - Specificity of the lactoferrin and transferrin receptors in Neisseria gonorrhoeae. Molec. Microbiol., 2: 827-829, 1988.

[12] 12. LOWRY, O.H.; ROSEBROUGH, N.J.; FARR, A.L. & RANDALL, R.H. - Protein measurement with the Folin phenol reagent. J. biol. Chem., 193: 265-275, 1951.

[13] 13. MASSON, P.L.; HEREMANS, J.F. & DIVE, C.H. - An iron-binding protein common to many external secretions. Clin. chim. Acta, 14: 729-734, 1966.

[14] 14. MCKENNA, W.R.; MICHELSEN, P.A.; SPARLING, P.F. & DYER, D.W. - Iron uptake from lactoferrin and transferrin by Neisseria gonorrhoeae. Infect. Immun., 56: 785-791, 1988.

[15] 15. MICKELSEN, P.A. & SPARLING, F.P. - Ability of Neisseria gonorrhoeae, Neisseria meningitidis, and comensal Neisseria species to obtain iron from transferrin and iron compounds. Infect. Immun., 33: 555-564, 1981.

[16] 16. MICKELSEN, P.A.; BLACKMAN, E. & SPARLING, F.P. - Ability of Neisseria gonorrhoeae, Neisseria meningitidis, and comensal Neisseria species to obtain iron from lactoferrin. Infect. Immun., 35: 915-920, 1982.

[17] 17. MOCCA, L.F. & FRASCH, C.E. - Sodium dodecyl sulfate polyacrylamide gel typing system for characterization of Neisseria meningitidis isolates. J. clin. Microbiol., 16: 240-244, 1982.

[18] 18. NEILANDS, J.B. - Microbiol envelope proteins related to iron. Ann. Rev. Microbiol., 36: 285-309, 1982.

[19] 19. NORQUIST, A.J.; DAVIS, L.; NORLANDER, L. & NORMARK, S. - The effect of iron starvation on the outer membrane protein composition of Neisseria gonorrhoeae. FEMS Microbiol. Lett., 4: 71-75, 1978.

[20] 20. OSBORN, M.J. - Studies on the Gram- negative cell wall. 1. Evidence for the role of 2-Keto-3-deoxyoc-tonate in the lipopolysaccharide of Salmonella typhimurium. Proc. nat. Acad. Sci. (Wash.), 50: 499-506, 1963.

[21] 21. PAYNE, S.M. & FINSKELSTEIN, R.A. - A critical role of iron in host-bacterial interactions. J. clin. Invest., 61: 1428-1440, 1978.

[22] 22. PETTERSSON, A,; KUIPERS, B.; PELZER, M., et al. - Monoclonal antibodies against the 70-Kilodalton iron regulated protein of Neisseria meningitidis are bactericidal and strain specific. Infect. Immun., 58: 3036-3041, 1990.

[23] 23. SCHRYVERS, A.B. - Characterization of the human transferrin and lactoferrin- receptors in Haemophilus infuenzae. Molec. Microbiol., 2: 467-472, 1988.

[24] 24. SCHRYVERS, A.B. & GONZALEZ, G.C. - Comparison of the abilities of different proteins sources of iron to enhance Neisseria meingitidis infection in mice. Infect. Immun., 57: 2425-2429, 1989.

[25] 25. SCHRYVERS, A.B. & GONZALEZ, G.C. - Receptors for transferrin in pathogenic bacteria are specific for the host's protein. Canad. J. Microbiol., 36: 145-147, 1990.

[26] 26. SCHRYVERS, A.B. & LEE, B.C. - Comparative analysis of the transferrin and lactoferrin binding proteins in the family Neisseriaceae. Canad. J. Microbiol., 35: 409-415, 1989.

[27] 27. SCHRYVERS, A.B. & MORRIS, L.J. - Identification and characterization of the lactoferrin-binding protein from Neisseria meningitidis. Infect. Immun., 56: 1144-1149, 1988.

[28] 28. SCHRYVERS, A.B. & MORRIS, L.J. - Identification and characterization of the transferrin receptor from Neisseria meningitidis. Molec. Microbiol., 2: 281-288, 1988.

[29] 29. SIMONSON, C.; BRENER, D. & DE VOE, I.W. - Expression of a high affinity mechanism for acquisition of transferrin iron by Neisseria meningitidis. Infect. Immun., 36: 107-113, 1982.

[30] 30. TSAI, C.M. & FRASCH, C.E. - Chemical analysis of major outer membrane proteins of Neisseria meningitidis: comparison of serotypes 2 and 11. J. Bact., 141: 169-176, 1980.

[31] 31. WEINBERG, E.D. - Iron and infection. Microbiol. Rev., 42: 45-66, 1978.

[32] 32. WEST, S.E. & SPARLING, P.F. - Response of Neisseria gonorrhoeae to iron limitation: alterations in expression of membrane proteins without apparent siderophore production. Infect. Immun., 47: 388-394, 1985.

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