Amphiphilic membrane intercalators : functionality and mechanisms of action in microbial systems

• Main Author: Chia, Geraldine Wan Ni
• Other Authors: Stefan Wuertz
• Format: Thesis-Doctor of Philosophy
• Language: English
• Published: Nanyang Technological University 2022
• Subjects: Engineering::Environmental engineering
• Online Access: https://hdl.handle.net/10356/156062
• Institution: Nanyang Technological University

The cell membrane is an essential component of the bacteria, that is primarily made up of a lipid bilayer. To function properly, its physicochemical properties must remain in a narrow range. Unlike the cells of higher organisms, the bacterial cell is often presented with unpredictable and hostile environments e.g., temperature extreme or antibiotics. When these factors challenge the membrane properties beyond the physiological and physical limits of the bacteria, membrane integrity is compromised, and the cell dies. Synthetic conjugated oligoelectrolytes (COE) and naturally occurring carotenoids are amphiphilic molecules that comprise a rigid hydrophobic backbone with hydrophilic functional groups at the backbone termini. They spontaneously intercalate the bacterial membrane and change its properties. Amphiphiles that are shorter in molecular length relative to the lipid bilayer (≈ 4 nm) generally destabilize the membrane whereas molecules that are similar or longer than the lipid bilayer stabilize the membrane. The membrane disrupting molecules, COE2-3C and COE-D8, are shorter (≈ 3 nm) than the lipid bilayer. The different susceptibility towards COE2-3C between Gram-positive and Gram-negative bacteria was found to be associated with (1) higher COE uptake i.e., not impeded by outer membrane (OM), (2) greater leakage caused in cardiolipin-rich membranes and (3) damage in membranes of lower curvature. These insights suggested a linear molecular topology for COEs to better penetrate membranes, which was validated by COE-D8 with a high antimicrobial activity against Enterococcus faecalis and Escherichia coli. The optimum lipophilicity in COE-D8 conferred the molecule high membrane affinity. Scanning electron microscopy also revealed extensive membrane damage by COE-D8 in Gram-negative E. coli. In contrast, COE-S6 and polar carotenoids (lutein and zeaxanthin) are relatively long amphiphiles (≈ 4–5 nm) that span the lipid bilayer with little hydrophobic mismatch. Their rigid hydrophobic backbone is postulated to maintain membrane stability against butanol, which is known to fluidize the cytoplasmic membrane and disrupt the OM by releasing lipooligosaccharides (LOS). COE-S6 comprise of six phenylenevinylene units in its conjugated core, which is longer compared to its predecessor COE1-5C (five units). Fluorescence microscopy showed that COE-S6 mitigated the perturbation caused by butanol in giant unilamellar vesicles (GUV) while COE-S6 treated E. coli had a lower amount of LOS released and improved growth rates at 1.8% (v/v) butanol compared to the untreated control and COE1-5C treated cells. The elongated backbone in COE-S6 likely favored membrane stabilization given the larger hydrophobic surface area compared to COE1-5C. Further, lutein and zeaxanthin are naturally occurring molecular rivets known to stabilize membranes in thylakoids and retina. Biophysical experiments revealed that the fluidization effect of butanol and butanol partitioning constant were reduced in carotenoid-rich membranes i.e., a lower butanol permeability. Carotenoid treated E. coli also demonstrated a two-log fold increase in cell survival upon 3.5% (v/v) butanol exposure. The study with carotenoids and COE-S6 have demonstrated the proof of concept to supplement these molecules as exogeneous chemical additives into bacterial membranes and provide the structural support to increase membrane tolerance against environmental stresses such as solvents and heat. In sum, the interactions of amphiphilic COEs and carotenoids with the bacterial membrane were investigated and the potential of short COEs as novel membrane-targeting antimicrobials was demonstrated, together with the utility of longer COEs and carotenoids in biotechnologically useful microorganisms for membrane stabilization.