The lytic mechanism of Escherichia coli α-hemolysin associated to outer membrane vesicles

Abstract

Alpha-hemolysin (HlyA) is an extracellular toxin secreted by Escherichia coli, targeting to plasma membranes of eukaryotic cells. Recently it was found that this toxin is released to external media associated to bacterial outer membrane vesicles (OMVs), but the hemolytic mechanism in this way has not been studied yet. Our results report that HlyA is the only protein present in OMVs that is responsible for erythrocyte lysis, and show that no fusion event is involved in the lytic mechanism of OMVs-HlyA. Furthermore, the specific hemolytic activity is approximately 10 fold higher than that of purified free-HlyA, showing the same relative lysis efficiency and specificity for erythrocytes from different species. OMVs could be an important auxiliary way of secretion, acting mainly as a concentration mechanism of HlyA near the target cells. Cell lysis would occur after toxin transfer from OMVs to target membranes, as demonstrated by hemolysis kinetic studies, lipid mixing and western blot assays.

Share and Cite:

Herlax, V. , Henning, M. , Bernasconi, A. , Goni, F. and Bakas, L. (2010) The lytic mechanism of Escherichia coli α-hemolysin associated to outer membrane vesicles. Health, 2, 484-492. doi: 10.4236/health.2010.25072.

Conflicts of Interest

The authors declare no conflicts of interest.

References

[1] Coote, J.G. (1992) Structural and fuctional relationships among the RTX toxin determinants of gram-negative bacteria. FEMS Microbiology Reviews, 88, 137-162.
[2] Cavalieri, S., Bohach, G. and Synder, I. (1984) Escherichia coli a-hemolysin characteristics and probable role in pathogenicity. Microbiology Reviews, 48, 326-343.
[3] Welch, R.A. (1991) Pore-forming cytolysins of gramnegative bacteria. Molecular Microbiology, 5(3), 521-528.
[4] Ludwig, A. and Goebel, W. (1999) The family of the multi- genic encoded RTX toxin. In: Alouf, J.E. and Freer, J.H., Eds., The Comprehensive Source-Book of Bacterial Protein Toxins, Academic Press, 330-348.
[5] Stanley, P., Korornakis, V. and Hughes, C. (1998) Acylation of Escherichia coli hemolysin: A unique protein lipidation mechanism underlying toxin fuction. Microbiology and Molecular Biology Reviews, 62(2), 309-333.
[6] Jarchau, T., Chakraborty, T., Garcia, F. and Goebel, W. (1994) Selection for transport competence of C-terminal polypeptides derived from Escherichia coli hemolysin: The shortest peptide capable of autonomous HlyB/HlyD- dependent secretion comprises the C-terminal 62 amino acids of HlyA. Molecular and General Genetics, 245, 53-60.
[7] Koronakis, V., Koronakis, E. and Hughes, C. (1989) Isolation and analysis of the C-terminal signal directing export of Escherichia coli hemolysin protein across both bacterial membranes. European Molecular Biology Organization Journal, 8(2), 595-605.
[8] Balsalobre, C., Silvan, J.M., Berglund, S., Mizunoe, Y., Uhlin, B.E. and Wai, S.N. (2006) Release of the type I secreted alpha-haemolysin via outer membrane vesicles from Escherichia coli. Molecular Microbiology, 59(1), 99-112.
[9] McBroom, A.J., Johnson, A.P., Vemulapalli, S. and Kuehn, M.J. (2006) Outer membrane vesicle production by Escherichia coli is independent of membrane instability. The Journal of Bacteriology, 188(15), 5385-5392.
[10] McBroom, A.J. and Kuehn, M.J. (2007) Release of outer membrane vesicles by Gram-negative bacteria is a novel envelope stress response. Molecular Microbiology, 63(2), 545-558.
[11] Beveridge, T.J. (1999) Structures of Gram-negative cell walls and their derived membrane vesicles. The Journal of Bacteriology, 181(16), 4725-4733.
[12] Horstman, A.L. and Kuehn, M.J. (2000) Enterotoxigenic E. coli secretes active heat-labile enterotoxin via outer mem- brane vesicles. Journal of Biological Chemistry, 275(17), 12489-12496.
[13] Devoe, I.W. and Gilchrist, J.E. (1973) Release of endotoxin in the form of cell wall blebs during in vitro growth of Neisseria meningitis. Journal of Experimental Medicine, 138, 1156-1167.
[14] Keenan, J., Day, T., Neal, S., Cook, B., Perez-Perez, G., Allardyce, R. and Bagshaw, P. (2000) A role for the bacterial outer membrane in the pathogenesis of Helicobacter py- lori infection. FEMS Microbiology Letters, 182(2), 259-264.
[15] Fiocca, R., Necchi, V., Sommi, P., Ricci, V., Telford, J. Cover, T.L. and Solcia, E. (1999) Release of Helicobacter pylori vacuolating cytotoxin by both a specific secretion pathway and budding of outer membrane vesicles. Uptake of released toxin and vesicles by gastric epithelium. Journal of Pathology, 188, 220-226.
[16] Kato, S., Kowashi, Y. and Demuth, D.R. (2002) Outer membrane-like vesicles secreted by Actinobacillus action-mycetemcomitans are enriched in leukotoxin. Microbial Pathogenes, 32, 1-13.
[17] Wai, S.N., Takade, A. and Amako, K. (1995) The release of outer membrane vesicles from the strains of enterotoxigenic Escherichia coli. Microbiology and Immunology, 39(7), 451-456.
[18] Wai, S.N., Lindmark, B., Soderblom, T., Takade, A., Westermark, M., Oscarsson, J., Jass, J., Richter-Dahlfors,V., Mizunoe, Y. and Uhlin, B.E. (2003) Vesicle-mediated export and assembly of pore-forming oligomers of the enterobacte- rial ClyA cytotoxin. Cell, 115(1), 25-35.
[19] Chi, B., Qi, M. and Kuramitsu, H.K. (2003) Role of dentilisin in Treponema denticola epithelial cell layer penetration. Research in Microbiology, 154(9), 637-643.
[20] Kuehn, M. and Kesty, N. (2005) Bacterial outer membrane vesicles and the host-pathogen interaction. Genes and Dev- elopment, 19(22), 2645-2655.
[21] Mashburn-Warren, L. and Whiteley, M. (2006) Special delivery: Vesicle trafficking in prokaryotes. Molecular Mic- robiology, 61(4), 839-846.
[22] McBroom, A. and Kuehn, M.J. (2005) Outer membrane vesicles. In: Curtiss, R. III, et al., Eds., EcoSal-Escherichia coli and Salmonella: Cellular and Molecular Biology, ASM Press, Washington, DC.
[23] Kolling, G.L. and Matthews, K.R. (1999) Export of virulence genes and shiga toxin by membrane vesicles of Escherichia coli O157:H7. Applied and Environmental Microbiology, 65(5), 1843-1848.
[24] Moayeri, M. and Welch, R. (1997) Prelytic and lytic conformation of erythrocyte-associated Escherichia coli hemolysin. Infection and Immunity, 65(6), 2233-2239.
[25] Boehm, D., Welch, R. and Snyder, I. (1990) Calcium is requiered for binding of Escherichia coli hemolysin (HlyA) to erythrocyte membrane. Infection and Immunity, 58(6), 1951-1958.
[26] Markwell, M.A., Haas, S.M., Bieber, L.L. and Tolbert, N.E. (1978) A modification of the lowry procedure to simplify protein determination in membrane and lipoprotein samples. Analytical Biochemistry, 87(1), 206-210.
[27] Karkhanis, Y.D., Zeltner, J.Y., Jackson, J.J. and Carlo, D.J. (1978) A new and improved microassay to determine 2- keto-3-deoxyoctonate in lipopolisaccharide of Gram-nega- tive bacteria. Annals of Clinical Biochemistry, 85, 595-601.
[28] Chen, P., Toribara, T. and Warner, H. (1956) Microdeter- mination of phosphorus. Annals of Chemistry, 28, 1756- 1758.
[29] Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227(5259), 680-685.
[30] Fomsgaard, A., Freudenberg, M. and Galanos, C. (1990) Modification of the silver staining technique to detect lipopolysaccharide in polyacrylamide gels. Journal of Clinical Microbiology, 28(12), 2627-2631.
[31] Towbin, H., Staehelin, T. and Gordon, J. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications. Proceedings of the National Academy of Sciences, 76(9), 4350-4354.
[32] Snyder, I.S. and Zwadyk, P. (1969) Some factors affecting production and assay of Escherichia coli hemolysin. Journal of General Microbiology, 5, 133-143.
[33] Soloaga, A., Ramírez, J.M. and Goñi, F.M. (1998) Reversible denaturation, self-aggregation, and membrane activity of Escherichia coli alpha-hemolysin, a protein stable in 6 M urea. Biochemistry, 37(18), 6387-6393.
[34] Mayer, L.D., Hope, M.J. and Cullis, P.R. (1986) Vesicles of variable sizes produced by a rapid extrusion procedure. Biochimica et Biophysica Acta, 858, 161-168.
[35] Struck, D.K., Hoekstra, D. and Pagano, R.E. (1981) Use of resonance energy transfer to monitor membrane fusion. Biochemistry, 20, 4093-4099.
[36] Herlax, et al, unpublished results.
[37] Rennie, R.P. and Arbuthnott, J.P. (1974) Partial characterization of Escherichia coli haemolysin. Journal of Medical Microbiology, 7(2), 179-188.
[38] Ostolaza, H., Bartolome, B., Ortiz de Zarate, I., de la Cruz, F. and Goñi, F.M. (1993) Release of lipid vesicle contents by the bacterial protein toxin alpha-haemolysin. Biochimica et Biophysica Acta, 1147, 81-88.
[39] Demuth, D.R., James, D., Kowash, Y. and Kato, S. (2003) Interaction of Actinobacillus actinomycetemcomitans outer membrane vesicles with HL60 cells does not require leukotoxin. Cellular Microbiology, 5(2), 111-121.
[40] Lally, E.T., Golub, E.E. and Kieba, I.R. (1991) Structure and function of the B and D genes of the Actinobacillus action-mycetemcomitans leukotoxin complex. Microbial Pathogenesis, 11, 111-121.
[41] Herlax, V., Maté, S., Rimoldi, O. and Bakás, L. (2009) Relevance of fatty acid covalently bound to Escherichia coli alpha-hemolysin and membrane microdomains in the oligomerization process. Journal of Biological Chemistry, 284, 25199-25210.
[42] Cortajarena, H., Goñi, F. and Ostolaza, H. (2001) Glycophorin as a receptor for Escherichia coli alphahemolysin in erythrocytes. Journal of Biological Chemistry, 276(16), 12513-12519.
[43] Valeva, A., Walev, I., Kemmer, H., Weis, S., Siegel, I., Boukhallouk, F., Wassenaar, T., Chavakis, T. and Bhakdi, S. (2005) Binding of Escherichia coli hemolysin and Activa- tion of the target cells is not receptor-dependent. Journal of Biological Chemistry, 280(44), 36657-36663.
[44] Herlax, V., Tacconi de Alani, M. and Bakás, L. (2005) Role of lipopolysaccharide on the structure and function of alpha- hemolysin from Escherichia coli. Chemistry and Physics of Lipids, 135(2), 107-115.
[45] Ostolaza, H., Bartolome, B., Serra, J.L., Cruz, F. and Goñi, F.M. (1991) Alpha-haemolysin from E. coli. Purification and self-aggregation properties. Microbiology Letters, 280(2), 195-198.
[46] Czuprynski, C.J. and Welch, R.A. (1995) Biological effects of RTX toxins: The possible role of lipopolysaccharide. Trends in Microbiology, 3(12), 480-483.

Copyright © 2024 by authors and Scientific Research Publishing Inc.

Creative Commons License

This work and the related PDF file are licensed under a Creative Commons Attribution 4.0 International License.