Non-Polar Lipids as Regulators of Membrane Properties in Archaeal Lipid Bilayer Mimics

The modification of archaeal lipid bilayer properties by the insertion of apolar molecules in the lipid bilayer midplane has been proposed to support cell membrane adaptation to extreme environmental conditions of temperature and hydrostatic pressure. In this work, we characterize the insertion effects of the apolar polyisoprenoid squalane on the permeability and fluidity of archaeal model membrane bilayers, composed of lipid analogues. We have monitored large molecule and proton permeability and Laurdan generalized polarization from lipid vesicles as a function of temperature and hydrostatic pressure. Even at low concentration, squalane (1 mol%) is able to enhance solute permeation by increasing membrane fluidity, but at the same time, to decrease proton permeability of the lipid bilayer. The squalane physicochemical impact on membrane properties are congruent with a possible role of apolar intercalants on the adaptation of Archaea to extreme conditions. In addition, such intercalant might be used to cheaply create or modify chemically resistant liposomes (archeaosomes) for drug delivery.

New method for high-throughput measurements of viscosity in submicrometer-sized membrane systems

In order to unravel the underlying principles of membrane adaptation in small systems like bacterial cells, robust approaches to characterize membrane fluidity are needed. Currently available relevant methods require advanced instrumentation and are not suitable for high throughput settings needed to elucidate the biochemical pathways involved in adaptation. We developed a fast, robust, and financially accessible quantitative method to measure microviscosity of lipid membranes in bulk suspension using a commercially available plate reader. Our approach, which is suitable for high-throughput screening, is based on the simultaneous measurements of absorbance and fluorescence emission of a viscosity-sensitive fluorescent dye DCVJ incorporated into a lipid membrane. We validated our method using artificial membranes with various lipid compositions over a range of temperatures and observed values that were in good agreement with previously published results. Using our approach, we were able to detect a lipid phase transition in the ruminant pathogen Mycoplasma mycoides.

Slow andad hoc: Unravelling the two features characterizing the development of bacterial resistance to membrane active peptides

Membrane disrupting drugs such as antimicrobial peptides are being considered as a solution to counter the problem of antibiotic resistance. Although it can be intuitively imagined that bacteria will eventually develop resistance to this class of drugs as well, the concern has largely been ignored. Drawing upon the experimental data from the resistance ofStaphylococcus aureusto antimicrobial peptides, we theoretically model the membrane adaptation under drug pressure. Using our model, we simulate the serial passage experiments with and without the drug pressure, and use the comparisons with experiments to estimate the unknown kinetic parameters. While the development of resistance to enzyme or membrane targeting drugs are both driven by spontaneous mutations, an additional lysylation step required in the latter slows the development of resistance. By quantifying the tradeoff between the gain in fitness under drug pressure and a loss in growth due to membrane modification, our model shows a fast reversal of membrane composition in drug free conditions, re-sensitizing the bacterium to the drugs.

Disrupting Membrane Adaptation Restores In Vivo Efficacy of Antibiotics Against Multidrug-Resistant Enterococci and Potentiates Killing by Human Neutrophils

Daptomycin resistance in enterococci is often mediated by the LiaFSR system, which orchestrates the cell membrane stress response. Activation of LiaFSR through the response regulator LiaR generates major changes in cell membrane function and architecture (membrane adaptive response), permitting the organism to survive the antibiotic attack. Here, using a laboratory strain of Enterococcus faecalis, we developed a novel Caenorhabditis elegans model of daptomycin therapy and showed that disrupting LiaR-mediated cell membrane adaptation restores the in vivo activity of daptomycin. The LiaR effect was also seen in a clinical strain of daptomycin-resistant Enterococcus faecium, using a murine model of peritonitis. Furthermore, alteration of the cell membrane response increased the ability of human polymorphonuclear neutrophils to readily clear both E. faecalis and multidrug-resistant E. faecium. Our results provide proof of concept that targeting the cell membrane adaptive response restores the in vivo activity of antibiotics, prevents resistance, and enhances the ability of the innate immune system to kill infecting bacteria.

One drug multiple targets: An approach to predict drug efficacies on bacterial strains differing in membrane composition

Rational design methodologies such as quantitative structure activity relationships (QSAR) have conventionally focused on screening through several drugs for their activity against a single target, either a bacterial protein or membrane. Recent concerns in drug design such as the development of drug resistance by membrane adaptation, or the undesirable damage to gut microbiota require a paradigm shift in activity prediction. A complementary approach capable of predicting the activity of a single drug against diverse targets, the diversity arising from bacterial adaptation or a heterogeneous composition with other helpful or harmful bacteria, is needed. As a first predictive step towards this goal, we develop a quantitative model for the activity of daptomycin onStreptococcus aureusstrains with different membrane compositions, mainly varying in lysylation. The results of the predictions are good, and within the limits of the scarcely available data, hint at an interaction of daptomycin with the inner membrane. The complementary approach may in principle be extended to estimate the activity against gut bacterial membranes, when systematic data can be curated for training the model.

Lipidomic and biophysical homeostasis of mammalian membranes in response to dietary lipids is essential for cellular fitness

ABSTRACTBiological membranes form the functional, dynamic interface that hosts a major fraction of all cellular bioactivity. Proper membrane physiology requires maintenance of a narrow range of physicochemical properties, which must be buffered from external perturbations. While homeostatic adaptation of membrane fluidity to temperature variation is a ubiquitous design feature of ectothermic organisms, such responsive membrane adaptation to external inputs has not been directly observed in mammals. Here, we report that challenging mammalian membrane homeostasis by dietary lipids leads to robust lipidomic remodeling to preserve membrane physical properties. Specifically, exogenous polyunsaturated fatty acids (PUFAs) are rapidly and extensively incorporated into membrane lipids, inducing a reduction in membrane packing. These effects are rapidly compensated both in culture and in vivo by lipidome-wide remodeling, most notably upregulation of saturated lipids and cholesterol. These lipidomic changes result in recovery of membrane packing and permeability. This lipidomic and biophysical compensation is mediated in part by lipid regulatory machinery, whose pharmacological or genetic abrogation results in cytotoxicity when membrane homeostasis is challenged by dietary lipids. These results reveal an essential mammalian mechanism for membrane homeostasis wherein lipidome remodeling in response to dietary lipid inputs preserves functional membrane phenotypes.