Vishwesh Haricharan Rai

Unravelling Early Stage Dynamics of Cytolysin A – Lipid Membrane Interactions

The pore-forming toxins (PFTs) released by pathogenic bacteria1 disrupt the cell membranes by pore formation leading to osmotic imbalance due to selective ion transfer. The two broad classes of the PFTs – α-PFTs and β-PFTs – based on the mechanism of pore formation employed by the PFT, differ in the secondary structures of their transmembrane pore domains. In this context, one of the most commonly studied α-PFT – Cytolysin A – comprising a five α-helix bundle and a β-hairpin, transitions from inactive monomer to active protomeric form upon membrane association. The monomer employs the hydrophobic β-tongue region as the starting point for the assembly process upon membrane binding. Benke et al. proposed an off-pathway reaction model for the formation of the potent form of the monomer. The model proposed that the addition of DDM at its CMC to the soluble monomers triggers the formation of protomers, along with the reversible formation of a molten-globule-like intermediate. Although the presence of the non-native intermediates at the early stage of the assembly pathway is evident, very little is known about the role of these intermediate species in its assembly, host membrane pore formation, and bilayer remodeling. Researchers believe that the protein behaves identically in both environments – in detergents and in lipid membranes. Here, we measured the activity of the monomer, the intermediate, the protomer, the pre-pore, and the pore forms of ClyA using fluorescence microscopy. We tracked the detergent-triggered temporal evolution in the helicity of these forms via circular dichroism, FTIR and FRET measurements. Recent studies have established the role of cholesterol in the assembly of the ClyA on the membrane as cholesterol stabilizes the intermediates by interacting with two protomers and enhancing pore formation. To account for the effect of phase separation in lipid bilayers on the activity of ClyA, we investigated the ClyA actions on bilayers by varying the lipid compositions. Interestingly, our results with DDM-induced intermediates showed that the bilayer components greatly influence membrane disruption activities ClyA species during the assembly pathway. However, all our studies using lipid membranes essentially pointed towards prevalence of entirely different pathway, for which further studies are going on.