Staphylococcal leukocidin pores are formed by the obligatory interaction
of two distinct polypeptides, one of class F and one of class S, making them
unique in the family of β-barrel pore-forming toxins (β-PFTs). By contrast, other
β-PFTs form homooligomeric pores. For example, the staphylococcal α-
hemolysin is a homoheptamer. Limited and controversial data exist on the
assembly and molecular architecture of the leukocidin pore.
In this work, biochemical and biophysical methods were used to
characterize the leukocidin pore produced by the LukF (HlgB) and LukS (HlgC)
components encoded by Staphylococcus aureus. I demonstrate that LukF and
LukS assemble to form an SDS-stable pore on rabbit erythrocyte membranes. In
addition, the pore-forming properties of recombinant leukocidin were
investigated with planar lipid bilayers. Although leukocidins and
staphylococcal α-hemolysin share partial sequence identity and related folds,
LukF and LukS produce a pore with a unitary conductance of 2.5 nS (1 M KCl, 5
mM HEPES, pH 7.4), which is over three times greater than that of α-hemolysin
measured under the same conditions.
The subunit composition and stoichiometry of a leukocidin pore were
determined by two independent methods, gel shift electrophoresis and sitespecific
chemical modification during single channel recording. Four LukF and
four LukS subunits were shown to co-assemble into an octameric
transmembrane structure. The existence of an additional subunit in part
explains properties of the leukocidin pore, such as its high conductance.
Additionally, this is the first time that either technique has been applied
successfully to assess the composition of a heteromeric membrane protein. It is
also relevant to understanding the mechanism of assembly of β-PFT pores, and
suggests new possibilities for engineering these proteins.
In additional studies, the HlyII pore encoded by Bacillus cereus was found
to form a homoheptameric transmembrane pore with properties conforming in
general with those of other members of the class of β-PFTs. HlyII possesses
additional properties which make it an attractive candidate for applications in
biotechnology, such as an oligomer with a high thermal stability in the presence
of SDS and the ability of the pore to remain open at high transmembrane
potentials.
| Identifer | oai:union.ndltd.org:tamu.edu/oai:repository.tamu.edu:1969.1/3952 |
| Date | 16 August 2006 |
| Creators | Miles, Jr., George Emmett |
| Contributors | Bayley, Hagan |
| Publisher | Texas A&M University |
| Source Sets | Texas A and M University |
| Language | en_US |
| Detected Language | English |
| Type | Book, Thesis, Electronic Dissertation, text |
| Format | 4243996 bytes, electronic, application/pdf, born digital |
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