Controlling pore assembly of staphylococcal γ‐haemolysin by low temperature and by disulphide bond formation in double‐cysteine LukF mutants

Abstract

SummaryStaphylococcal LukF and Hlg2 are water-soluble monomers of g g g g -haemolysin that assemble into oligomeric pores on the erythrocyte membranes. Here, we have created double-cysteine LukF mutants, in which single disulphide bonds connect either the prestem domain and the cap domain (V12C-T136C, CapStem), or two b b b b -strands within the prestem domain (T117C-T136C, Stem-Stem) to control pore assembly of g g g g -haemolysin at intermediate stages. The disulphide-trapped mutants were inactive in erythrocyte lysis, but gained full haemolytic activity if the disulphide bonds were reduced. The disulphide bonds blocked neither the membrane binding ability nor the intermediate prepore oligomerization, but efficiently inhibited the transition from prepores to pores. The prepores of Cap-Stem were dissociated into monomers in 1% SDS. In contrast, the prepores of Stem-Stem were stable in SDS and had ring-shaped structures similar to those of wild-type LukF, as observed by transmission electron microscopy. The transition of both mutants from prepores to pores could even be achieved by reducing disulphide bonds at low temperature (2 ∞ ∞ ∞ ∞ C), whereas prepore oligomerization was effectively inhibited by low temperature. Finally, real-time transition of Stem-Stem from prepores to pores on ghost cells, visualized using a Ca 2 + + + + -sensitive fluorescent indicator (Rhod2), was shown by the sequential appearance of fluorescence spots, indicating pore-opening events. Taken together, these data indicate that the prepores are legitimate intermediates during g g g g -haemolysin pore assembly, and that conformational changes around residues 117 and 136 of the prestem domain are essential for pore formation, but not for membrane binding or prepore oligomerization. We propose a mechanism for g g g ghaemolysin pore assembly based on the demonstrated intermediates.

Crystal structure of the octameric pore of staphylococcal γ-hemolysin reveals the β-barrel pore formation mechanism by two components

“…As the amino-latch showed steric conflict with loop A in both interfaces, loop A is likely to cause the conformation change of the amino-latch. We demonstrated previously that the conformation change of the prestem from a rigid to a flexible structure is essential for prepore formation (11). The transition of the prestem would be coupled with pre- pore formation, to which interprotomer electrostatic interaction of loop A makes a strong contribution.…”

“…A conformational change in the amino-latch was reported to precede those in the prestem and prepore formation (11). Therefore, the release of the amino-latch from the cap domain would occur first in protomer assembly.…”

“…The heterodimer is known to assemble into a ring-shaped nonlytic oligomeric intermediate called a prepore before formation of the pore (11)(12)(13). Formation of the prepore was first identified for staphylococcal αHL (39)(40)(41)(42), and is now recognized as the general mechanism adopted by a wide variety of PFTs (43).…”

“…The soluble forms of F and S components bind sequentially to the target cells and form a heterodimer (9, 10). Each heterodimer assembles into an oligomer on the target cell to form a ring-shaped particle called a prepore, in which the β-barrel pore is not yet formed (11)(12)(13)(14). After forming a stable prepore, the β-barrel pore is formed.…”

Heavy chain-only antibodies and tetravalent bispecific antibody neutralizing Staphylococcus aureus leukotoxins

“…Leukotoxins exert their action by (i) binding of S and F proteins to target cell membranes (21,30); (ii) oligomerization and rapidly increasing intracellular calcium concentrations independently of pore formation and cell activation (12,31); and (iii) reconfiguration of S and F proteins to form functional pores permeable to monovalent cations (32). The capacity of antibodies to inhibit these three steps was tested.…”