Visualization of Lipid Membrane Reorganization Induced by a Pore-Forming Toxin Using High-Speed Atomic Force Microscopy

Abstract

We examined the effect of a sphingomyelin (SM)-binding pore-forming toxin (PFT), lysenin, on the dynamics of a phase-separated membrane of SM, where SM formed liquid-ordered (Lo) domains with cholesterol (Chol) within a phosphatidylcholine-rich liquid-disordered (Ld) phase. We visualized the lysenin-induced membrane reorganization using high-speed atomic force microscope (HS-AFM). Lysenin oligomerized on the SM-rich Lo domain and simultaneously its oligomers assembled into a hexagonal close-packed (hcp) structure. The phase boundary was stable during the assembling of lysenin on the SM-rich domain, indicating that lysenin did not affect the line tension between Lo and Ld phases. After the full coverage of the SM-rich domain by oligomers, their hcp assembly gradually expanded into the Ld phase and eventually covered the entire membrane. Our results suggest that pore formation, i.e., insertion of lysenin into the membrane in its oligomeric state, induced the exclusion of SM and Chol from the SM-rich domain, which was followed by further binding and oligomerization of lysenin.

Intramembrane congestion effects on lysenin channel voltage-induced gating

“…However, lysenin channels prefer insertion into lipid rafts (Ishitsuka and Kobayashi 2004; Kulma et al 2010), which promote localized accumulation and increased local channel density. Lysenin clustering into SM-rich domains and formation of 2-D arrays in supported lipid membranes has been clearly demonstrated by using high-speed Atomic Force Microscopy (Yilmaz and Kobayashi 2015; Yilmaz et al 2013) and these studies revealed that rafts are the first target for oligomerization. However, at very high lysenin concentration, pore assembly has been shown to gradually expand into the disordered phase following exclusion of SM and Chol from the SM-rich domain.…”

“…Lysenin’s ability to self-insert stable channels into artificial membranes facilitates establishing congested conditions by successively increasing the number of channels inserted into the BLM, which is expected to influence the voltage-induced gating. In addition, lysenin has been shown to favor insertion into SM-rich lipid rafts (Abe and Kobayashi 2014; Kulma et al 2010; Yamaji-Hasegawa et al 2003; Yamaji et al 1998; Yilmaz and Kobayashi 2015; Yilmaz et al 2013), which facilitates further self-congestion conditions by manipulating the surface area of the rafts through changes in the SM amount in the membrane (Abe and Kobayashi 2014; Jin et al 2008; Mitsutake et al 2011). …”

In Situ Atomic Force Microscopy Studies on Nucleation and Self-Assembly of Biogenic and Bio-Inspired Materials

“…Lysenin recognized SM-rich domain and self-assembled into hexagonal close-packed (hcp) structure on the SM-rich domain, which did not perturb the phase boundary between SM and Chol. After the SM-rich domain is fully covered by lysenin, the hcp assembly started to occur in Chol liquid-disordered phase and eventually covered the entire membrane [160].…”

AnIn silicoAlgorithm for Identifying Amino Acids that Stabilize Oligomeric Membrane-Toxin Pores through Electrostatic Interactions

“…To target PFTs directly, small molecules, peptides and liposome decoys that inhibit the binding of PFTs to host receptors, oligomerization of the pores on the host plasma membrane, or block already formed pores have been designed (recently reviewed in Escajadillo & Nizet, 2018) to protect against infection propagated by PFTs of Bacillus anthracis (anthrax) (Nestorovich & Bezrukov, 2014; Rai et al, 2006), Staphylococcus aureaus (pneumonia, gut and skin infections) (Ragle et al, 2010), Escherichia coli (gut and urinary tract infections) (Mandal et al, 2016), Vibrio cholerae (cholera) (Rai et al, 2006), Streptococcus pneumoniae (pneumonia) (Henry et al, 2015), and many other bacteria. While inhibitors that block fully formed pores may yield some therapeutic benefit, blocked pores can nonetheless cause other disruptive effects such as bending and deformation of the plasma membrane (Tzokov et al, 2006; Drücker et al, 2019) and alteration of the lateral organization of lipid domains (Yilmaz & Kobayashi, 2015) as well as lipid dynamics (Ponmalar et al, 2019). Further, we have also shown through experiments and modelling that not just fully formed pores but oligomeric intermediates along the pore formation pathway of the α -PFT, Cytolysin A, are also capable of spontaneously compromising membrane integrity by causing leakage (Agrawal et al, 2017; Desikan, Maiti, & Ayappa, 2017).…”