Host defence peptide plectasin targets bacterial cell wall precursor lipid II by a calcium-sensitive supramolecular mechanism.

Antimicrobial resistance is a leading cause of mortality, calling for the development of new antibiotics. The fungal antibiotic plectasin is a eukaryotic host defence peptide that blocks bacterial cell wall synthesis. Here, using a combination of solid-state nuclear magnetic resonance, atomic force microscopy and activity assays, we show that plectasin uses a calcium-sensitive supramolecular killing mechanism. Efficient and selective binding of the target lipid II, a cell wall precursor with an irreplaceable pyrophosphate, is achieved by the oligomerization of plectasin into dense supra-structures that only form on bacterial membranes that comprise lipid II. Oligomerization and target binding of plectasin are interdependent and are enhanced by the coordination of calcium ions to plectasin's prominent anionic patch, causing allosteric changes that markedly improve the activity of the antibiotic. Structural knowledge of how host defence peptides impair cell wall synthesis will likely enable the development of superior drug candidates.

Repurposing DNase I and alginate lyase to degrade the biofilm matrix of dual-species biofilms of Staphylococcus aureus and Pseudomonas aeruginosa grown in artificial sputum medium: In-vitro assessment of their activity in combination with broad-spectrum antibiotics.

BACKGROUND: Biofilm-associated pulmonary infections pose therapeutic challenges in cystic fibrosis patients, especially when involving multiple bacterial species. Enzymatic degradation of the biofilm matrix may offer a potential solution to enhance antibiotic efficacy. This study investigated the repurposing of DNase I, commonly used for its mucolytic activity in cystic fibrosis, to target extracellular DNA within biofilms, as well as potential synergies with alginate lyase and broad-spectrum antibiotics in dual-species biofilms of Pseudomonas aeruginosa and Staphylococcus aureus. METHODS: Dual-species biofilms were grown in artificial sputum medium using S. aureus and P. aeruginosa isolated by pairs from the same patients and exposed to various combinations of enzymes, meropenem, or tobramycin. Activity was assessed by measuring biofilm biomass and viable counts. Matrix degradation and decrease in bacterial load were visualized using confocal microscopy. Biofilm viscoelasticity was estimated by rheology. RESULTS: Nearly complete destruction of the biofilms was achieved only if combining the enzymatic cocktail with the two antibiotics, and if using supratherapeutic levels of DNase I and high concentrations of alginate lyase. Biofilms containing non-pigmented mucoid P. aeruginosa required higher antibiotic concentrations, despite low viscoelasticity. In contrast, for biofilms with pigmented mucoid P. aeruginosa, a correlation was observed between the efficacy of different treatments and the reduction they caused in elasticity and viscosity of the biofilm. CONCLUSIONS: In this complex, highly drug-tolerant biofilm model, enzymes prove useful adjuvants to enhance antibiotic activity. However, the necessity for high enzyme concentrations emphasizes the need for thorough concentration-response evaluations and safety assessments before considering clinical applications.

Two separate mechanisms are involved in membrane permeabilization during lipid oxidation.

Lipid oxidation is a universal degradative process of cell membrane lipids that is induced by oxidative stress and reactive oxygen and nitrogen species (RONS) in multiple pathophysiological situations. It has been shown that certain oxidized lipids alter membrane properties, leading to a loss of membrane function. Alteration of membrane properties is thought to depend on the initial membrane lipid composition, such as the number of acyl chain unsaturations. However, it is unclear how oxidative damage is related to biophysical properties of membranes. We therefore set out to quantify lipid oxidation through various analytical methods and determine key biophysical membrane parameters using model membranes containing lipids with different degrees of lipid unsaturation. As source for RONS, we used cold plasma, which is currently developed as treatment for infections and cancer. Our data revealed complex lipid oxidation that can lead to two main permeabilization mechanisms. The first one appears upon direct contact of membranes with RONS and depends on the formation of truncated oxidized phospholipids. These lipids seem to be partly released from the bilayer, implying that they are likely to interact with other membranes and potentially act as signaling molecules. This mechanism is independent of lipid unsaturation, does not rely on large variations in lipid packing, and is most probably mediated via short-living RONS. The second mechanism takes over after longer incubation periods and probably depends on the continued formation of lipid oxygen adducts such as lipid hydroperoxides or ketones. This mechanism depends on lipid unsaturation and involves large variations in lipid packing. This study indicates that polyunsaturated lipids, which are present in mammalian membranes rather than in bacteria, do not sensitize membranes to instant permeabilization by RONS but could promote long-term damage.

Lipid Membranes as Key Targets for the Pharmacological Actions of Ginsenosides.

In this review, we will focus on the activity of ginsenosides on membranes and their related effects, from physicochemical, biophysical, and pharmacological viewpoints. Ginsenosides are a class of saponins with a large structural diversity and a wide range of pharmacological effects. These effects can at least partly be related to their activity on membranes which results from their amphiphilic character. Some ginsenosides are able to interact with membrane lipids and associate into nanostructures, making them possible adjuvants for vaccines. They are able to modulate membrane biophysical properties such as membrane fluidity, permeability or the formation of lateral domains with some degree of specificity towards certain cell types such as bacteria, fungi, or cancer cells. In addition, they have shown antioxidant properties which protect membranes from lipid oxidation. They further displayed some activity on membrane proteins either through direct or indirect interaction. We investigate the structure activity relationship of ginsenosides on membranes and discuss the implications and potential use as anticancer, antibacterial, and antifungal agents.

ω-3 polyunsaturated fatty acids direct differentiation of the membrane phenotype in mesenchymal stem cells to potentiate osteogenesis.

Mammalian cells produce hundreds of dynamically regulated lipid species that are actively turned over and trafficked to produce functional membranes. These lipid repertoires are susceptible to perturbations from dietary sources, with potentially profound physiological consequences. However, neither the lipid repertoires of various cellular membranes, their modulation by dietary fats, nor their effects on cellular phenotypes have been widely explored. We report that differentiation of human mesenchymal stem cells (MSCs) into osteoblasts or adipocytes results in extensive remodeling of the plasma membrane (PM), producing cell-specific membrane compositions and biophysical properties. The distinct features of osteoblast PMs enabled rational engineering of membrane phenotypes to modulate differentiation in MSCs. Specifically, supplementation with docosahexaenoic acid (DHA), a lipid component characteristic of osteoblast membranes, induced broad lipidomic remodeling in MSCs that reproduced compositional and structural aspects of the osteoblastic PM phenotype. The PM changes induced by DHA supplementation potentiated osteogenic differentiation of MSCs concurrent with enhanced Akt activation at the PM. These observations prompt a model wherein the DHA-induced lipidome leads to more stable membrane microdomains, which serve to increase Akt activity and thereby enhance osteogenic differentiation. More broadly, our investigations suggest a general mechanism by which dietary fats affect cellular physiology through remodeling of membrane lipidomes, biophysical properties, and signaling.

α-Hederin Induces Apoptosis, Membrane Permeabilization and Morphologic Changes in Two Cancer Cell Lines Through a Cholesterol-Dependent Mechanism.

In perspective of reducing the mortality of cancer, there is a high interest in compounds which act on multiple cellular targets and therefore prevent the appearance of cancer resistances. Saponins and α-hederin, an oleanane-type saponin, induce cancer cell death through different pathways, including apoptosis and membrane permeabilization. Unfortunately, the mechanism by which cell death is induced is unknown. We hypothesized that the activity of α-hederin mainly depends on its interaction with membrane cholesterol and therefore investigated the cholesterol and saponin-structure dependency of apoptosis and membrane permeabilization in two malignant monocytic cell lines. Apoptotic cell death and membrane permeabilization were significantly reduced in cholesterol-depleted cells. Permeabilization further depended upon the osidic side chain of α-hederin and led to extracellular calcium influx and nuclear fragmentation, with only the latter being susceptible to caspase inhibitors. Membrane order, measured by laurdan generalized polarization imaging, was neither reduced by α-hederin nor its aglycone hederagenin suggesting that their activity was not related to membrane cholesterol extraction. However, a radical change in morphology, including the disappearance of pseudopodes was observed upon incubation with α-hederin. Our results suggest that the different activities of α-hederin mainly depend on its interaction with membrane cholesterol and consequent pore formation.

Polyunsaturated Lipids Regulate Membrane Domain Stability by Tuning Membrane Order.

The plasma membrane (PM) serves as the functional interface between a cell and its environment, hosting extracellular signal transduction and nutrient transport among a variety of other processes. To support this extensive functionality, PMs are organized into lateral domains, including ordered, lipid-driven assemblies termed lipid rafts. Although the general requirements for ordered domain formation are well established, how these domains are regulated by cell-endogenous mechanisms or exogenous perturbations has not been widely addressed. In this context, an intriguing possibility is that dietary fats can incorporate into membrane lipids to regulate the properties and physiology of raft domains. Here, we investigate the effects of polyunsaturated fats on the organization of membrane domains across a spectrum of membrane models, including computer simulations, synthetic lipid membranes, and intact PMs isolated from mammalian cells. We observe that the ω-3 polyunsaturated fatty acid docosahexaenoic acid is robustly incorporated into membrane lipids, and this incorporation leads to significant remodeling of the PM lipidome. Across model systems, docosahexaenoic acid-containing lipids enhance the stability of ordered raft domains by increasing the order difference between them and coexisting nonraft domains. The relationship between interdomain order disparity and the stability of phase separation holds for a spectrum of different perturbations, including manipulation of cholesterol levels and high concentrations of exogenous amphiphiles, suggesting it as a general feature of the organization of biological membranes. These results demonstrate that polyunsaturated fats affect the composition and organization of biological membranes, suggesting a potential mechanism for the extensive effects of dietary fat on health and disease.

The amphiphilic nature of saponins and their effects on artificial and biological membranes and potential consequences for red blood and cancer cells.

Saponins, amphiphiles of natural origin with numerous biological activities, are widely used in the cosmetic and pharmaceutical industry. Some saponins exhibit relatively selective cytotoxic effects on cancer cells but the tendency of saponins to induce hemolysis limits their anticancer potential. This review focused on the effects of saponin activity on membranes and consequent implications for red blood and cancer cells. This activity seems to be strongly related to the amphiphilic character of saponins that gives them the ability to self-aggregate and interact with membrane components such as cholesterol and phospholipids. Membrane interactions of saponins with artificial membrane models, red blood and cancer cells are reviewed with respect to their molecular structures. The review considered the mechanisms of these membrane interactions and their consequences including the modulation of membrane dynamics, interaction with membrane rafts, and membrane lysis. We summarized current knowledge concerning the mechanisms involved in the interactions of saponins with membrane lipids and examined the structure activity relationship of saponins regarding hemolysis and cancer cell death. A critical analysis of these findings speculates on their potential to further develop new anticancer compounds.