Crystal structure of listeriolysin O reveals molecular details of oligomerization and pore formation

Abstract

Listeriolysin O (LLO) is an essential virulence factor of Listeria monocytogenes that causes listeriosis. Listeria monocytogenes owes its ability to live within cells to the pH-and temperature-dependent pore-forming activity of LLO, which is unique among cholesteroldependent cytolysins. LLO enables the bacteria to cross the phagosomal membrane and is also involved in activation of cellular processes, including the modulation of gene expression or intracellular Ca 2 þ oscillations. Neither the pore-forming mechanism nor the mechanisms triggering the signalling processes in the host cell are known in detail. Here, we report the crystal structure of LLO, in which we identified regions important for oligomerization and pore formation. Mutants were characterized by determining their haemolytic and Ca 2 þ uptake activity. We analysed the pore formation of LLO and its variants on erythrocyte ghosts by electron microscopy and show that pore formation requires precise interface interactions during toxin oligomerization on the membrane.

Stonefish toxin defines an ancient branch of the perforin-like superfamily

“…S1). These data are consistent with suggestions that charge complementarity between perforin monomers may drive initial oligomerization events (33,34).…”

Perfringolysin O and related cholesterol-dependent cytolysins

“…It was shown that at neutral pH, the charge repulsion of these carboxylates caused the premature unfurling of the ɑHBs into β-hairpins in the soluble LLO monomers: restraining the unfurling of αHB1 by tethering it to D2 through an engineered disulfide slowed the pH-dependent inactivation. Recently, the predicted acidic triad carboxylate structure was confirmed when LLO was crystalized and its structure was solved [4]. Thus, unfolding of the ɑHBs serves dual functions in LLO: in the context of the phagosome, the ɑHBs unfold and transition to TMHs following the normal pore-forming mechanism.…”

“…Although D4 was initially shown to interact between PFO monomers [54,55], subsequent studies indicated that D4 remains surrounded by water throughout the assembly of the pore [19,56]. More recently, however, Pokrajac et al [57] and Koster et al [4] showed that purified D4 from pyolysin and LLO, respectively, was capable of forming linear oligomeric arrays on liposomes. In contrast to the earlier work of Tweten et al [54] and Iwamoto et al [58] who showed an inhibitory effect of D4 on native toxin, Pokrajac et al showed that D4 increased pyolysin activity.…”

“…The severe bend in the β-strands that span D1 and D3 is probably relieved when the β-hairpins are extended from their α-helical structure in the monomer to form the pore complex [25,26,56]. The recently solved structure of LLO [4] showed that a Glu-Lys pair at the monomermonomer interface of D1 is important to its activity and that the loss of either charge abolishes activity. Interestingly, substitution of the lysine with tryptophan abolished activity, but the same substitution of the glutamate had no effect on pore-forming activity.…”

“…The crystal structures of six CDCs in the water-soluble monomer state have been solved, which revealed that CDCs also exhibit a high degree of tertiary structural similarity [2][3][4][5][6][7]. The homology between the primary and crystal structures of the CDCs predicts that CDC family members share many similar features and a common pore-forming mechanism, although it is clear there are some variations on this theme.…”