Role of Lipid Composition in the Interaction and Activity of the Antimicrobial Compound Fengycin with Complex Membrane Models.

Fengycins are compounds produced by bacteria of the Bacillus genus with strong antifungal activity. In this work, lipids extracted from fungal and oomycetal molds were used to assess the ability of fengycin to bind and insert into complex membrane models prepared as Langmuir lipid monolayers. In addition, fengycin-induced leakage in liposomes prepared from these complex lipid extracts was also evaluated. Fengycin's ability to bind and incorporate into these membranes seemed to be mainly related to ergosterol content. Other membrane characteristics such as phospholipid fatty acyl chain length played a more peripheral role. A high ergosterol concentration appeared to allow other membrane characteristics generally associated with fengycin binding and/or insertion, such as higher proportion of phosphatidylcholine head groups or increased fatty acyl unsaturation, to be present without adversely affecting membrane integrity. Increased membrane leakage was also generally associated with the presence of low or no ergosterol. Leakage was also correlated with the previously reported biological activity of fengycin on these molds.

Supported lipid bilayers using extracted microbial lipids: domain redistribution in the presence of fengycin.

Fengycin is an antimicrobial cyclic lipopeptide known to interact with microbial cell membranes. To gain insight into the role of lipids in fengycin sensitivity, lipids extracted from Alternaria solani, Fusarium sambucinum, and Pythium sulcatum were analyzed and used in the preparation of supported lipid bilayers (SLBs). Total Internal Reflection Fluorescence Microscopy (TIRFM) was used to evaluate lipid phase separation within the SLBs and changes in domain distribution with the application of fengycin. A. solani lipid extract contained the highest quantity of ergosterol while P. sulcatum contained no ergosterol. Sterol content of SLBs was strongly correlated with increases in phase separation, suggesting ergosterol may play a role in promoting ordering of the lipid phases in the bilayer. A. solani experienced the least change in domain characteristics following fengycin exposure, suggesting ergosterol may be buffering effects of the antimicrobial compound. Factors such as lipid headgroup charge and unsaturation levels may impact fengycin's effects on domain phase separation, but these effects were generally overshadowed by the role of ergosterol. In the absence of ergosterol, in the P. sulcatum bilayers, fengycin caused an increase in lipid phase ordering.

Domain redistribution within ergosterol-containing model membranes in the presence of the antimicrobial compound fengycin.

The cyclic lipopeptide fengycin, produced by Bacillus subtilis, exhibits its antimicrobial capabilities by altering the integrity of the cell membrane of plant pathogens. Previous work has correlated fengycin activity with membrane characteristics, such as sterol content. This work focused on the influence of fengycin on supported lipid bilayers containing varying levels of ergosterol. Total internal reflection fluorescence (TIRF) microscopy was used to visualize and distinguish ordered (Lß/Lo) and disordered (Lα/Ld) domains in the model membranes following exposure to low (50 µg) and high (500 µg) fengycin doses. Application of an initial low dose of fengycin to 0% and 3% ergosterol-containing bilayers resulted in redistribution of Lα/Lß and Lo/Ld domains, respectively, which the bilayers compensated and corrected for over time. These membranes were unable to tolerate a second 50 µg dose or a single high fengycin dose. The 6% ergosterol bilayers were able to tolerate sequential low doses of fengycin. Exposure of these bilayers to the high fengycin dose caused a decrease in the number of Lo domains, albeit less than that seen in the 0% and 3% ergosterol bilayers. Bilayers containing 12% ergosterol, exhibited the least amount of change after fengycin exposure. These were the only bilayer to exhibit an increase in area taken up by ordered domains. These results suggest fengycin may preferentially act on the Lß or Lo phase, the area in which ergosterol resides. Bilayers containing low levels of ergosterol appear to be more sensitive to the lipopeptide, suggesting ergosterol plays a role in buffering perturbations caused by fengycin.

Experimental Parameters Leading to Optimal Bilayers for Total Internal Reflection Fluorescence Microscopy Visualization.

Supported lipid bilayer systems were evaluated following various experimental procedures in an effort to determine their appropriateness for visualization using total internal reflection fluorescence (TIRF) microscopy. The incorporation and distribution of Texas Red® 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (TR-DHPE) was studied when incorporated into bilayers of variable lipid composition using different forms of mechanical shearing. Results showed that 0.8 mol% TR-DHPE provides the most optimum TIRF images. At this concentration, a sufficient level of photostability can be achieved without an undesirable increase in TR-DHPE aggregates caused by excess probe molecules. Solutions composed of a 3:1 molar ratio of DOPC:DPPC with 0.8 mol% TR-DHPE produce bilayers that consistently display clear, distinct, rounded domains, whereas other lipid compositions did not. This optimum phase separation appears to be influenced by an increase in mechanical shearing during the vesicle formation process, when the lipid solutions were exposed to sonication and extrusion processes. The combination of a sonication and extrusion process also helped with eliminating the presence of TR-DHPE aggregates within the model membranes. It was also shown that bilayers formed on conditioned glass, placed on a slide, produced more highly detailed bilayers in which distinct lipid phase separation could be optimally visualized using TIRF microscopy.

Effect of tea tree (Melaleuca alternifolia) oil as a natural antimicrobial agent in lipophilic formulations.

There has been increased interest surrounding the use of tea tree oil (TTO) as a natural antimicrobial. In this study, the antimicrobial activity of TTO and its components were investigated in vitro and in a predominantly lipid-based personal care formulation. In vitro, TTO showed minimal inhibitory concentrations of 0.2% (for Saccharomyces cerevisiae and Pythium sulcatum), 0.4% (for Escherichia coli, Bacillus subtilis, and Rhizopus stolonifer), and 0.8% (for Botrytis cinerea). TTO at 0.08%-0.8% was often as efficient as parabens. Comparison of the antimicrobial activities of TTO components showed that terpinen-4-ol and γ-terpinene were generally most effective in inhibiting microbial growth. TTO activity in a personal care product was evaluated through air and water exposure, artificial inoculation, and shelf life studies. While TTO did not increase shelf life of unopened products, it decreased microbial load in products exposed to water and air. Results from this study support that antimicrobial activity of TTO can be attributed to varying levels of its components and that low levels of TTO were effective in reducing microbial growth during the use of the product. This study showed that TTO can act as a suitable preservative system within an oil-based formulation.