The mode of action of bicomponent γ-haemolysins of Staphylococcus aureus

Mauro Dalla Serra*, Gabriella Viero*, Romina Cunaccia*, Cristina Potrich*, Helga Bastiani*, Christoph Bachmeyer*, Olivier Joubert#, Sandra Werner#, Daniel Keller#, Henri Monteil#, Gilles Prévost# and Gianfranco Menestrina*

*ITC & CNR - Istituto di Biofisica Sezione di Trento, Povo (Italy) #Institut de Bactériologie, UPRES EA-3432, Strasbourg (France). Sponsored by PAT, Fondo Progetti (project Stawars)

The bicomponent γ-haemolysins (γHL) of S. aureus belong to the same protein family of leucocidins and α-haemolysin (αHL). They all oligomerize on cell and model membranes and insert a transmembrane β-barrel that opens pores in the lipid. Alpha-HL forms homoheptameric pores, whereas bicomponent toxins are assembled associating two proteins taken from two different sub-families. They comprise a still debated number of monomers (6-8). To clarify the pore topology we introduced single Cys near the interfaces of γHL monomers and labelled them with a FRET pair probes. The formation of all the γHL interfaces was tested measuring FRET development after adding liposomes. Heterologous couples labelled on facing sites displayed FRET, demonstrating extensive formation of the HlgA-HlgB and HlgB-HlgA interfaces. Heterologous couples labeled on the same side, or the homologous HlgA couple gave no FRET. The homologous HlgB couple, instead, displayed a transient FRET suggesting the formation of an intermediate. We conclude that the pores are assembled by an even number of alternating HlgA and HlgB monomers. The transition of the assembled oligomer to a functional pore requires a proper lipid composition. Maximal activity occurs in the presence of cholesterol and a PC with either unsaturated acyl chains, or saturated acyl chains shorter than 13 carbons. Potentiation by other conical lipids indicates that this shape may be needed to increase cholesterol availability. The β-hairpin of each protomer undergoes the main change required for pore formation, being folded in the soluble form and completely extended in the transmembrane state. Such extension was investigated using double Cys mutants of HlgB in which one Cys was at a fixed position and the other in a variable position along the β-strand. Our results indicate that all the mutated positions passed near enough to the fixed one to form a SS bond, suggesting a mechanism of step-by-step sliding of the β-stretches.

Email: mdalla@itc.it

Address: Mauro Dalla Serra, ITC & CNR-Istituto di Biofisica, Sezione di Trento, via Sommarive 18, I-38050 Povo (Trento) Italy,