Part:BBa_K554002
HlyA secretion signal peptide
The HlyA is a signal peptide found in the C-terminal signal sequence of alpha-hemolysin (HlyA). It is used to target proteins for secretion via the Type I secretion pathway of gram-negative bacteria. Fusion of the HlyA signal peptide to the target protein may result in transport of the protein from the cytoplasm to the extracellular medium in a single step. HlyA, the signal sequence, seems to interact with the cytoplasmic region of the pre-formed HlyB–D complex. After the binding of the HlyA secretion signal by the HlyB–D complex, HlyD induces the interaction with TolC. To be effective, only the last 50-60 aminoacids of the C-terminal of Hly-A are required, so we can use it as a fusion protein that is attached to the interleukins (such as IL-12 and IL-10) and allows it to be secreted. Besides, HlyA is itself a weak antigen, turning it harmeless to the immune responses we are trying to interfere with.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Usage and Biology
You can see a representation of this device acting in the schema below: HlyB gene and product are shown as a symbolic cilinder in orange.
Representation of device 3, the protein secretion system, in a Jedi bacteria that contains Device 1 (Adrenaline sensor/IL-12 producer). To export a protein, the bacteria must have the HlyD, HlyB and TolC proteins and the target protein must have a signal sequence (HlyA tail). In this case, the target protein to be secreted is IL-12.
A more realistic schema of ABC transport system is shown below:
MIT_MAHE 2020
Biology
The HlyA is a signal peptide found in the C-terminal signal sequence of alpha-hemolysin (HlyA). It is used to target proteins for secretion via the Type I secretion pathway of gram-negative bacteria. Fusion of the HlyA signal peptide to the target protein may result in transport of the protein from the cytoplasm to the extracellular medium in a single step. The molecular functions of HlyA include adenylate cyclase activity, calcium ion binding as well as toxin activity, while the biological functions include cytolysis and pathogenesis. HlyA has been utilized to conduct nanopore sequencing of DNA. It has also been used to form pores on cellular plasma membrane to deplete cellular nucleotides (Kou Qin et al., 2011) (Kou Qin et al., 2008).
The structure of several hemolysins has been solved by X-ray crystallography in the soluble and pore-forming conformations. For example, alpha-hemolysin of Staphylococcus aureus forms a homo-heptameric β-barrel in biological membranes (L. Song et al., 1996). The heptamer of α-hemolysin from Staphylococcus aureus has a mushroom-like shape and measures up to 100 Å in diameter and 100 Å in height. A membrane-spanning, solvent-accessible channel runs along the sevenfold axis and ranges from 14 Å to 46 Å in diameter. On the exterior of the 14-strand antiparallel β-barrel there is a hydrophobic belt approximately 30 Å in width that provides a surface complementary to the non-polar portion of the lipid bilayer. The interfaces are composed of both salt-links and hydrogen bonds, as well as hydrophobic interactions, and these contacts provide a molecular stability for the heptamer in SDS solutions even up to 65°C (E. Gouaux, 1998).
Hemolysin is a potential virulence factor produced by microorganisms, which can put a human's health at risk. A recent study showed that Alpha-hemolysin from uropathogenic Escherichia coli produces extra-intestinal infections and can cause cystitis, pyelonephritis and may result in permanent renal scarring and failure (Changying Wang et al., 2020). Another example includes the pneumonia produced by Staphylococcus aureus. In this case, it has been proven that alpha-hemolysin takes part in inducing necrotic pulmonary injury by the use of the NLRP3 inflammasome, which is responsible for inflammatory processes and of pyroptosis (Chahnaz Kebaier et al., 2012).
References
Song, L., Hobaugh, M. R., Shustak, C., Cheley, S., Bayley, H., & Gouaux, J. E. (1996). Structure of staphylococcal alpha-hemolysin, a heptameric transmembrane pore. Science (New York, N.Y.), 274(5294), 1859–1866. https://doi.org/10.1126/science.274.5294.1859
Gouaux E. Alpha-Hemolysin from Staphylococcus aureus: an archetype of beta-barrel, channel-forming toxins. J Struct Biol. 1998;121(2):110-22. doi: 10.1006/jsbi.1998.3959. PMID: 9615434.
Wang, C., Li, Q., Lv, J., Sun, X., Cao, Y., Yu, K., Miao, C., Zhang, Z. S., Yao, Z., & Wang, Q. (2020). Alpha-hemolysin of uropathogenic Escherichia coli induces GM-CSF-mediated acute kidney injury. Mucosal immunology, 13(1), 22–33. https://doi.org/10.1038/s41385-019-0225-6
Kebaier, C., Chamberland, R. R., Allen, I. C., Gao, X., Broglie, P. M., Hall, J. D., Jania, C., Doerschuk, C. M., Tilley, S. L., & Duncan, J. A. (2012). Staphylococcus aureus α-hemolysin mediates virulence in a murine model of severe pneumonia through activation of the NLRP3 inflammasome. The Journal of infectious diseases, 205(5), 807–817. https://doi.org/10.1093/infdis/jir846
Qin, K., Dong, C., Wu, G., & Lambert, N. A. (2011). Inactive-state preassembly of G(q)-coupled receptors and G(q) heterotrimers. Nature chemical biology, 7(10), 740–747. https://doi.org/10.1038/nchembio.642
Qin, K., Sethi, P. R., & Lambert, N. A. (2008). Abundance and stability of complexes containing inactive G protein-coupled receptors and G proteins. FASEB journal: official publication of the Federation of American Societies for Experimental Biology, 22(8), 2920–2927. https://doi.org/10.1096/fj.08-105775
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