Graduation Year

2019

Document Type

Dissertation

Degree

Ph.D.

Degree Name

Doctor of Philosophy (Ph.D.)

Degree Granting Department

Physics

Major Professor

Jianjun Pan, Ph.D.

Committee Member

Jianfeng Cai, Ph.D.

Committee Member

Sagar Pandit, Ph.D.

Committee Member

Zhimin Shi, Ph.D.

Keywords

AFM, Colistin, Endophilin H0, GUV, Lipid Bilayer, Peptidomimetics

Abstract

Understanding the cellular membrane interaction with membrane active biomolecules and antimicrobial agents provides an insight in their working mechanism. Here, we studied the effect of antimicrobial agents; a recently developed peptidomimetics E107-3 and colistin as well as the N-terminal helix H0, of Endophilin A1 on the lipid bilayer.

It is important to discern the interaction mechanism of antimicrobial peptides with lipid membranes in battling multidrug resistant bacterial pathogens. We study the modification of structural and mechanical properties with a recently reported peptidomimetic on lipid bilayer. The compound referred to as E107-3 is synthesized based on the acylated reduced amide scaffold and has been shown to exhibit good antimicrobial potency. This compound increases lipid bilayer permeability as indicated by our vesicle leakage essay. Micropipette aspiration experiment shows that exposure of GUV to the compound causes the protrusion length Lp to spontaneously increase and then decrease, followed by GUV rupture. Solution atomic force microscopy (AFM) is used to visualize lipid bilayer structural modulation within a nanoscopic regime. This compound induces nanoscopic heterogeneous structures rather than pore like structures as produced by melittin. Finally, we use AFM-based force spectroscopy to study the impact of the compound on lipid bilayer’s mechanical properties. With the incremental addition of this compound, we found the bilayer puncture force decreases moderately and a 39% decrease of the bilayer area compressibility modulus KA. To explain our experimental data, we propose a membrane interaction model encompassing disruption of lipid chain packing and extraction of lipid molecules. The later action mode is supported by our observation of a double-bilayer structure in the presence of fusogenic calcium ions.

Polyanionic Lipopolysaccharides LPS are important in regulating the permeability of outer membrane (OM) of gram-negative bacteria. To initiate the bactericidal activity of polymyxins, it is essential to impair the LPS-enriched OM. Here, we study the mechanism of membrane permeability caused by colistin (Polymyxin E) of LPS/phospholipid bilayers. Our vesicle leakage experiment showed that colistin binding enhanced bilayer permeability; the maximum increase in the bilayer permeability was positively correlated with the LPS fraction. Addition of magnesium ions abolished the effect of LPS in enhancing bilayer permeabilization. Solution atomic force microscopy (AFM) measurements on planar lipid bilayers shows the formation of nano- and macro clusters which protruded from the bilayer by ~2nm. Moreover, increasing the fraction of LPS or colistin enhances the formation of clusters but inhibits by magnesium ions addition. To explain our experimental data, we proposed a lipid-clustering model where colistin binds to LPS to form large-scale complexes segregated from zwitterionic phospholipids. The discontinuity (and thickness mismatch) at the edge of LPS-colistin clusters will create a passage that allows solutes to permeate through. The proposed model is consistent with all data obtained from our leakage and AFM experiments. Our results of LPS-dependent membrane restructuring provided useful insights into the mechanism that could be used by polymyxins in impairing the permeability barrier of the OM of Gram-negative bacteria.

Also, we studied the effect of helix H0 of a membrane modification inducing protein endophilin, on planar bilayer. We obtained transmembrane defects on the bilayer when scanned.with AFM.

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