1887

Abstract

haemolysin A (HlyA), an RTX toxin, is secreted probably as an unfolded intermediate, by the type I (ABC transporter-dependent) pathway, utilizing a C-terminal secretion signal. However, the mechanism of translocation and post-translocation folding is not understood. We identified a mutation () at the extreme C terminus, which is dominant in competition experiments, blocking secretion of the wild-type toxin co-expressed in the same cell. This suggests that unlike recessive mutations which affect recognition of the translocation machinery, the mutation interferes with some later step in secretion. Indeed, the mutation reduced haemolytic activity of the toxin and the activity of -lactamase when the latter was fused to a C-terminal 23 kDa fragment of HlyA carrying the mutation. A second mutant (), lacking the six C-terminal residues of HlyA, also showed reduced haemolytic activity and neither mutant protein regained normal haemolytic activity in unfolding/refolding experiments. Tryptophan fluorescence spectroscopy indicated differences in structure between the secreted forms of wild-type HlyA and the HlyA Del6 mutant. These results suggested that the mutations affected the correct folding of both HlyA and the -lactamase fusion. Thus, we propose a dual function for the HlyA C terminus involving an important role in post-translocation folding as well as targeting HlyA for secretion.

Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.038562-0
2010-08-01
2024-04-18
Loading full text...

Full text loading...

/deliver/fulltext/micro/156/8/2495.html?itemId=/content/journal/micro/10.1099/mic.0.038562-0&mimeType=html&fmt=ahah

References

  1. Andersen C., Koronakis E., Bokma E., Eswaran J., Humphreys D., Hughes C., Koronakis V. 2002; Transition to the open state of the TolC periplasmic tunnel entrance. Proc Natl Acad Sci U S A 99:11103–11108
    [Google Scholar]
  2. Balakrishnan L., Hughes C., Koronakis V. 2001; Substrate-triggered recruitment of the TolC channel-tunnel during type I export of hemolysin by Escherichia coli. J Mol Biol 313:501–510
    [Google Scholar]
  3. Balsalobre C., Silvan J. M., Berglund S., Mizunoe Y., Uhlin B. E., Wai S. N. 2006; Release of the type I secreted alpha-haemolysin via outer membrane vesicles from Escherichia coli. Mol Microbiol 59:99–112
    [Google Scholar]
  4. Bauer M. E., Welch R. A. 1997; Pleiotropic effects of a mutation in rfaC on Escherichia coli hemolysin. Infect Immun 65:2218–2224
    [Google Scholar]
  5. Baumann U., Wu S., Flaherty K. M., McKay D. B. 1993; Three-dimensional structure of the alkaline protease of Pseudomonas aeruginosa: a two-domain protein with a calcium binding parallel beta roll motif. EMBO J 12:3357–3364
    [Google Scholar]
  6. Benabdelhak H., Kiontke S., Horn C., Ernst R., Blight M. A., Holland I. B., Schmitt L. 2003; A specific interaction between the NBD of the ABC-transporter HlyB and a C-terminal fragment of its transport substrate haemolysin A. J Mol Biol 327:1169–1179
    [Google Scholar]
  7. Chenal A., Guijarro J. I., Raynal B., Delepierre M., Ladant D. 2009; RTX calcium binding motifs are intrinsically disordered in the absence of calcium: implication for protein secretion. J Biol Chem 284:1781–1789
    [Google Scholar]
  8. Chervaux C., Holland I. B. 1996; Random and directed mutagenesis to elucidate the functional importance of helix II and F-989 in the C-terminal secretion signal of Escherichia coli hemolysin. J Bacteriol 178:1232–1236
    [Google Scholar]
  9. Chervaux C., Sauvonnet N., Le Clainche A., Kenny B., Hung A. L., Broome-Smith J. K., Holland I. B. 1995; Secretion of active beta-lactamase to the medium mediated by the Escherichia coli haemolysin transport pathway. Mol Gen Genet 249:237–245
    [Google Scholar]
  10. Delepelaire P. 2004; Type I secretion in Gram-negative bacteria. Biochim Biophys Acta 1694:149–161
    [Google Scholar]
  11. Goni F. M., Ostolaza H. 1998; E. coli alpha-hemolysin: a membrane-active protein toxin. Braz J Med Biol Res 31:1019–1034
    [Google Scholar]
  12. Halegoua S., Inouye M. 1979; Translocation and assembly of outer membrance proteins of Escherichia coli. Selective accumulation of precursors and novel assembly intermediates caused by phenethyl alcohol. J Mol Biol 130:39–61
    [Google Scholar]
  13. Herlax V., Bakas L. 2007; Fatty acids covalently bound to alpha-hemolysin of Escherichia coli are involved in the molten globule conformation: implication of disordered regions in binding promiscuity. Biochemistry 46:5177–5184
    [Google Scholar]
  14. Holland I. B., Benabdelhak H., Young J., de Lima Pimenta A., Schmitt L., Blight M. A. 2003; Bacterial ABC transporters involved in protein translocation. In ABC Proteins: from Bacteria to Man pp 209–241 Edited by Holland I. B., Cole S. P. C., Kuchler K., Higgins C. F. London: Academic Press;
    [Google Scholar]
  15. Holland I. B., Schmitt L., Young J. 2005; Type 1 protein secretion in bacteria, the ABC-transporter dependent pathway. Mol Membr Biol 22:29–39
    [Google Scholar]
  16. Hyland C., Vuillard L., Hughes C., Koronakis V. 2001; Membrane interaction of Escherichia coli hemolysin: flotation and insertion-dependent labeling by phospholipid vesicles. J Bacteriol 183:5364–5370
    [Google Scholar]
  17. Jones H. E., Holland I. B., Campbell A. K. 2002; Direct measurement of free Ca2+ shows different regulation of Ca2+ between the periplasm and the cytosol of Escherichia coli. Cell Calcium 32:183–192
    [Google Scholar]
  18. Kenny B., Chervaux C., Holland I. B. 1994; Evidence that residues −15 to −46 of the haemolysin secretion signal are involved in early steps in secretion, leading to recognition of the translocator. Mol Microbiol 11:99–109
    [Google Scholar]
  19. Koronakis V., Koronakis E., Hughes C. 1989; Isolation and analysis of the C-terminal signal directing export of Escherichia coli hemolysin protein across both bacterial membranes. EMBO J 8:595–605
    [Google Scholar]
  20. Koronakis V., Sharff A., Koronakis E., Luisi B., Hughes C. 2000; Crystal structure of the bacterial membrane protein TolC central to multidrug efflux and protein export. Nature 405:914–919
    [Google Scholar]
  21. Pimenta A. L., Racher K., Jamieson L., Blight M. A., Holland I. B. 2005; Mutations in HlyD, part of the type 1 translocator for hemolysin secretion, affect the folding of the secreted toxin. J Bacteriol 187:7471–7480
    [Google Scholar]
  22. Sanchez-Magraner L., Viguera A. R., Garcia-Pacios M., Garcillan M. P., Arrondo J. L., de la Cruz F., Goni F. M., Ostolaza H. 2007; The calcium-binding C-terminal domain of Escherichia coli alpha-hemolysin is a major determinant in the surface-active properties of the protein. J Biol Chem 282:11827–11835
    [Google Scholar]
  23. Sotomayor Perez A. C., Karst J. C., Davi M., Guijarro J. I., Ladant D., Chenal A. 2010; Characterization of the regions involved in the calcium-induced folding of the intrinsically disordered RTX motifs from the Bordetella pertussis adenylate cyclase toxin. J Mol Biol 397:534–549
    [Google Scholar]
  24. Stanley P., Packman L. C., Koronakis V., Hughes C. 1994; Fatty acylation of two internal lysine residues required for the toxic activity of Escherichia coli hemolysin. Science 266:1992–1996
    [Google Scholar]
  25. Vakharia H., German G. J., Misra R. 2001; Isolation and characterization of Escherichia coli tolC mutants defective in secreting enzymatically active alpha-hemolysin. J Bacteriol 183:6908–6916
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.038562-0
Loading
/content/journal/micro/10.1099/mic.0.038562-0
Loading

Data & Media loading...

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error