Skip to main content

Advertisement

Springer Nature Link
Log in

Permeability Changes of Manduca sexta Midgut Brush Border Membranes Induced by Oligomeric Structures of Different Cry Toxins

  • Published:
The Journal of Membrane Biology Aims and scope Submit manuscript

An Erratum to this article was published on 13 November 2012

Abstract

The pore-formation activity of monomeric and oligomeric forms of different Cry1 toxins (from Cry1A to Cry1G) was analyzed by monitoring ionic permeability across Manduca sexta brush border membrane vesicles. The membrane vesicles were isolated from microvilli structures, showing a high enrichment of apical membrane markers and low intrinsic K+ permeability. A fluorometric assay performed with 3,3′-dipropylthiodicarbocyanine fluorescent probe, sensitive to changes in membrane potential, was used. Previously, it was suggested that fluorescence determinations with this dye could be strongly influenced by the pH, osmolarity and ionic strength of the medium. Therefore, we evaluated these parameters in control experiments using the K+-selective ionophore valinomycin. We show here that under specific ionic conditions changes in fluorescence can be correlated with ionic permeability without effects on osmolarity or ionic strength of the medium. It is extremely important to attenuate the background response due to surface membrane potential and the participation of the endogenous permeability of the membrane vesicles. Under these conditions, we analyzed the pore-formation activity induced by monomeric and oligomeric structures of different Cry1 toxins. The Cry1 toxin samples containing oligomeric structures correlated with high pore activity, in contrast to monomeric samples that showed marginal pore-formation activity, supporting the hypothesis that oligomer formation is a necessary step in the mechanism of action of Cry toxins.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Subscribe and save

Springer+
from $39.99 /Month
  • Starting from 10 chapters or articles per month
  • Access and download chapters and articles from more than 300k books and 2,500 journals
  • Cancel anytime
View plans

Buy Now

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Figure 1.
Figure 2.
Figure 3.
Figure 4.
Figure 5.

Similar content being viewed by others

Explore related subjects

Discover the latest articles, books and news in related subjects, suggested using machine learning.

References

  1. Maagd R.A., Bravo A., Crickmore N. 2001. How Bacillus thuringiensis has evolved specific toxins to colonize the insect world. Trends Genet. 17:193–199

    Article  PubMed  Google Scholar 

  2. Schnepf E., Crickmore N., Van Rie J., Lereclus D., Baum J., Feitelson J., Zeigler D.R., Dean D.H. 1998. Bacillus thuringiensis and its pesticidal crystal proteins. Microbiol. Mol. Biol. Rev. 62:775–806

    PubMed  CAS  Google Scholar 

  3. Bravo A., Jansens S., Peferoen M. 1992. Immunocytochemical localization of Bacillus thuringiensis insecticidal crystal proteins in intoxicated insects. J. Invertebr. Pathol. 60:237–246

    Article  CAS  Google Scholar 

  4. Bravo A., Gómez I., Conde J., Muñoz-Garay C., Sánchez J., Miranda R., Zhuang M., Gill S.S., Soberón M. 2004. Oligomerization triggers binding of a Bacillus thuringiensis Cry1Ab pore-forming toxin to aminopeptidase N receptor leading to insertion into membrane microdomains. Biochim. Biophys. Acta. 1667:38–46

    Article  PubMed  CAS  Google Scholar 

  5. Gómez I., Sánchez J., Miranda R., Bravo A., Soberon M. 2002. Cadherin-like receptor binding facilitates proteolytic cleavage of helix α-1 in domain I and oligomer pre-pore formation of Bacillus thuringiensis Cry1Ab toxin. FEBS Lett. 513:242–246

    Article  PubMed  Google Scholar 

  6. Zhuang M., Oltean D.I., Gomez I., Pullikuth A.K., Soberon M., Bravo A., Gill S.S. 2002. Heliothis virescens and Manduca sexta lipid rafts are involved in Cry1A toxin binding to the midgut epithelium and subsequent pore formation. J. Biol. Chem. 277:13863–13872

    Article  PubMed  CAS  Google Scholar 

  7. Rausell C., Muñoz-Garay C., Miranda-CassoLuengo R., Gómez I., Rudiño-Piñera E., Soberón M., Bravo A. 2004. Tryptophan spectroscopy studies and black lipid bilayer analysis indicate that the oligomeric structure of Cry1Ab toxin from Bacillus thuringiensis is the membrane-insertion intermediate. Biochemistry. 43:166–174

    Article  PubMed  CAS  Google Scholar 

  8. Rausell C., Pardo-López L., Sánchez J., Muñoz-Garay C., Morera C., Soberón M., Bravo A. 2004. Unfolding events in the water-soluble monomeric Cry1Ab toxin during transition to oligomeric pre-pore and membrane inserted pore channel. J. Biol. Chem. 279:55168–55175

    Article  PubMed  CAS  Google Scholar 

  9. Rausell C., García-Robles I., Sánchez J., Muñoz-Garay C., Martínez-Ramírez A.C., Real M.D., Bravo A. 2004. Role of toxin activation on binding and pore formation activity of the Bacillus thuringiensis Cry3 toxins in membranes of Leptinotarsa decemlineata [Say]. Biochem. Biophys. Acta. 1660:99–105

    Article  PubMed  CAS  Google Scholar 

  10. Lorence A., Darszon A., Díaz C., Liévano A., Quintero R., Bravo A. 1995. δ-Endotoxins induce cation channels in Spodoptera frugiperda brush border membranes in suspension and in planar lipid bilayers. FEBS Lett. 360:217–222

    Article  PubMed  CAS  Google Scholar 

  11. Lorence A., Darszon A., Bravo A. 1997. The pore formation activity of Bacillus thuringiensis Cry1Ac toxin on Trichoplusia ni membranes depends on the presence of aminopeptidase N. FEBS Lett. 414:303–307

    Article  CAS  Google Scholar 

  12. Bashford C.L., Smith J.C. 1979. The use of optical probes to monitor membrane potential. Methods Enzymol. 55:569–586

    Article  PubMed  CAS  Google Scholar 

  13. Waggoner A. 1976. Optical probes of membrane potential. J. Membr. Biol. 27:317–334

    Article  PubMed  CAS  Google Scholar 

  14. Cabrini G., Verkman A.S. 1986. Localization of cyanine dye binding to brush border membranes by quenching of n-(9-anthroyloxy) fatty acid probes. Biochim. Biophys. Acta. 862:285–293

    Article  PubMed  CAS  Google Scholar 

  15. Kirouac M., Vachon V., Rivest S., Schwartz J-L., Laprade R. 2003. Analysis of the properties of Bacillus thuringiensis insecticidal toxins using a potential-sensitive fluorescent probe. J. Membr. Biol. 196:51–59

    Article  PubMed  CAS  Google Scholar 

  16. Bravo A., Miranda R., Gómez I., Soberon M. 2002. Pore formation activity of Cry toxins from Bacillus thuringiensis in an improved membrane preparation from Manduca sexta midgut cell microvilli. Biochem. Biophys. Acta. 1562:63–69

    Article  PubMed  CAS  Google Scholar 

  17. Harlow E., Lane D., 1988. Antibodies. A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY

    Google Scholar 

  18. Gómez I., Oltean D.L, Gill S.S., Bravo A., Soberón M. 2001. Mapping the epitope in cadherin-like receptors involved in Bacillus thuringiensis Cry1A toxin interaction using phage display. J. Biol. Chem. 276:28906–28912

    Article  PubMed  Google Scholar 

  19. Güereca L., Bravo A. 1999. The oligomeric state of Bacillus thuringiensis Cry toxins in solution. Biochim. Biophys. Acta. 1429:342–350

    Article  PubMed  Google Scholar 

  20. Höfte H., Grave H., Seurinck J., Jansens S., Mahillon J., Ampe C., Vandekerckhove J., Vanderbruggen H., VanMontagu M., Zabeau M., Vaek M. 1986. Structural and functional analysis of a cloned delta endotoxin of Bacillus thuringiensis berliner 1715. Eur. J. Biochem. 161:273–280

    Article  PubMed  Google Scholar 

  21. Hille, B. 1992. Chapter 16 structure and function In: B. Hille, editor. Ionic Channels of Excitable Membranes, pp. 427–464. Sinauer Associates, Suderland, MA

    Google Scholar 

  22. Watts A., van Gorkom C.M. 1991. Surface organization of lipid bilayers. In: P. Yeagle, editor. The Structure of Biological Membranes. CRC Press, New York

  23. Schwartz J.L., Laprade R. 2000. Membrane permeabilization by Bacillus thuringiensis toxins: Protein insertion and pore formation. In: J. F. Charles, A. Deleeluse, C. Nielsen-LeRoux et al., editors. Entomopathogenic Bacteria: From Laboratory to Field Application. Kluwer Academic, Amsterdam, Dordrecht, The Netherlands, pp. 199–217

  24. Carroll J., Ellar D.J. 1993. An analysis of Bacillus thuringiensis δ-endotoxin action on insect midgut membrane permeability using a light scattering assay. Eur. J. Biochem. 214:771–778

    Article  PubMed  CAS  Google Scholar 

  25. Carroll J., Ellar D.J. 1997. Analysis of the large aqueous pores produced by Bacillus thuringiensis protein insecticide in Manduca sexta midgut brush border membrane vesicles. Eur. J. Biochem. 245:797–804

    Article  PubMed  CAS  Google Scholar 

  26. Tran B.L., Vachon V., Shwartz J.L., Laprade R. 2001. Differential effects on the pore formation properties of Bacillus thuringiensis insecticidal crystal proteins. Appl. Environ. Microbiol. 67:4488–4494

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgment

Our thanks to Lizbeth Cabrera for technical assistance and CONACyT J44962Q for financial support.

Author information

Authors and Affiliations

  1. Instituto de Biotecnología, Universidad Nacional Autónoma de México, Apdo postal 510-3, Cuernavaca, Morelos, 62250, México

    C. Muñoz-Garay, J. Sánchez, A. Darszon, M. Soberón & A. Bravo

  2. Plant Research International, P.O. Box 16, 6700, AA, Wageningen, The Netherlands

    R.A. de Maagd & P. Bakker

Authors
  1. C. Muñoz-Garay
    View author publications

    Search author on:PubMed Google Scholar

  2. J. Sánchez
    View author publications

    Search author on:PubMed Google Scholar

  3. A. Darszon
    View author publications

    Search author on:PubMed Google Scholar

  4. R.A. de Maagd
    View author publications

    Search author on:PubMed Google Scholar

  5. P. Bakker
    View author publications

    Search author on:PubMed Google Scholar

  6. M. Soberón
    View author publications

    Search author on:PubMed Google Scholar

  7. A. Bravo
    View author publications

    Search author on:PubMed Google Scholar

Corresponding author

Correspondence to A. Bravo.

Additional information

An erratum to this article can be found online at http://dx.doi.org/10.1007/s00232-012-9519-2

Rights and permissions

Reprints and permissions

About this article

Cite this article

Muñoz-Garay, C., Sánchez, J., Darszon, A. et al. Permeability Changes of Manduca sexta Midgut Brush Border Membranes Induced by Oligomeric Structures of Different Cry Toxins. J Membrane Biol 212, 61–68 (2006). https://doi.org/10.1007/s00232-006-0003-8

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00232-006-0003-8

Keywords