Investigation of cell membrane dynamics: A potential marker for lipid response towards membrane-active proteins and peptides

Abstract

The cell membrane is made up of lipids and proteins held together with intermolecular hydrophobic/hydrophilic interactions. These physical non-covalent interactions help in maintaining the integrity of the cell membrane, yet allowing them to have a flexible and fluidic nature, exploited by the cell to cr›ntro1 the transport of nutrients and cell signaling events. The interaction between a membrane targeting molecule and lipid bilayer is a crucial step when a pathogen tries to infect a host cell. Likewise, the cell envelope of most virulent pathogens is the primary target for common drug molecules and antibiotics. Understanding the underlying physical interactions between membrane targeting molecules of both prokaryotic and eukaryotic cell membranes is an important aspect of developing strategies to mitigate virulent diseases. In this thesis, I will emphasize the importance of understanding the dynamics of membrane lipids, which provides a unique marker to identify and characterize protein/peptide-lipid interaction. In the first part, I have described our results on the interaction of membrane targeting pore-forming toxin, listeriolysin O (LLO) used by the bacteria Listeria monocytogenes to attack and disrupt the host cellular machinery. LLO oligomerizes upon binding to the lipid bilayer forming either complete or incomplete rings that are observed to exist in an inserted transmembrane pore state or an un-inserted pre-pore state. The significant outcome from this part of the study is our ability to identify the signatures of different structural states of LLO, which have a distinct effect on lipid bilayer diffusivity. Using vesicle leakage as an indicator of pore function in combination with insights regarding membrane-bound structure from Förster resonance energy transfer (FRET) and lipid dynamics from fluorescence correlation spectroscopy (FCS) measurements on single giant vesicles exposed to LLO, we found that the un-inserted state of LLO decreases the lipid mobility whereas the inserted state enhances the same. For providing a better understanding of the significance of the PFT oligomeric structures (arcs and rings) on lipid dynamics, the results from a concentration- dependent study to correlate arcs and rings with the lipid dynamics will be discussed in the second part of my thesis. The experiments were carried out on supported lipid bilayers using a combination of FRET and FCS coupled with the micro-rheological measurements. The lipid dynamics undergo a dynamical cross-over, which is correlated with transitions of LLO oligomeric state populations from rings to arc-like pore complexes, due to interplay of lipid ejection and crowding by membrane bound oligomers. The proposed two-state free area based diffusion model predicts the oligomeric state of the protein, which reiterates the importance of utilizing lipid diffusion as a marker for exploring the structural effects on protein-lipid interactions. In the last part of this thesis, I have shifted focus to understand lipid response from the pathogens' point of view. Although the mammalian cell membrane is well studied and characterized, bacterial and viral cell envelopes are comparatively less understood. Nevertheless, targeting the pathogenic bacterial cell membranes be- come essential to find antimicrobial molecules to combat pathogens in this emerging antibiotic-resistant era. In this part of the study, we tracked the lipid mobility in the live bacterial cell envelope and observed that the lipid mobility alters significantly in the presence of membrane targeting antibiotics. Interestingly, we could correlate this change in lipid mobility as a signature of antibiotic action rather than a stress induced physical response of the cell. To conclude, lipid mobility is observed to yield unique response towards mem- brane targeting molecules, which in turn can be used to monitor physical changes happening with the cell membrane. Our results confirm that the lipid dynamics are sensitive to the proteins/antibiotics and their different oligomeric structures that associate with phospholipid bilayers. Depending on the type of molecules interacting with the membrane, we observe unique lipid response in the form of change in mobility. The lipid dynamics reciprocated as a result of membrane-protein or membrane-peptide interactions provides insight on the physical attributes that could eventually help in protecting the host cells against pathogenic virulence factors.