Abstract
The eukaryotic actin cytoskeleton is an evolutionarily well-established pathogen target, as a large number of bacterial factors disturb its dynamics to alter the function of the host cells. These pathogenic factors modulate or mimic actin effector proteins or they modify actin directly, leading to an imbalance of the precisely regulated actin turnover. Here, we show that the pore-forming, cholesterol-dependent cytolysin pneumolysin (PLY), a major neurotoxin of Streptococcus pneumoniae, has the capacity to bind actin directly and to enhance actin polymerisation in vitro. In cells, the toxin co-localised with F-actin shortly after exposure, and this direct interaction was verified by Förster resonance energy transfer. PLY was capable of exerting its effect on actin through the lipid bilayer of giant unilamellar vesicles, but only when its pore competence was preserved. The dissociation constant of G-actin binding to PLY in a biochemical environment was 170–190nM, which is indicative of a high-affinity interaction, comparable to the affinity of other intracellular actin-binding factors. Our results demonstrate the first example of a direct interaction of a pore-forming toxin with cytoskeletal components, suggesting that the cross talk between pore-forming cytolysins and cells is more complex than previously thought.
Highlights
Bacteria produce effector molecules, such as protein toxins, to modulate host cell function and host defence, thereby increasing bacterial pathogenic potential
The toxin co-localised with F-actin shortly after exposure, and this direct interaction was verified by Förster resonance energy transfer
Confocal zstack reconstruction revealed that the toxin puncta were positioned close to the surface of the cells. To study whether this co-localisation represented a direct interaction, we utilised a Förster resonance energy transfer (FRET) approach, namely, acceptor photobleaching (Fig. 2b), which was able to show the interaction of both molecules
Summary
Bacteria produce effector molecules, such as protein toxins, to modulate host cell function and host defence, thereby increasing bacterial pathogenic potential. Many bacterial effector molecules are poreforming proteins, which build holes in membranes, thereby damaging cells. The cholesterol-binding step is followed by a penetration step, in which components of the oligomerised toxin molecules unfold and penetrate the membrane to form the pore.[2] PLY comprises four domains. Upon oligomerisation in a pre-pore ring, a complex conformational change occurs.[2,3] Domain 2 bends towards the membrane, allowing the subsequent partial refolding of domain 3. In this process, the helical regions of domain 3, hidden inside the
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