Growth of Staphylococcus aureus in the presence of oleic acid shifts the glycolipid fatty acid profile and increases resistance to antimicrobial peptides.

Staphylococcus aureus readily adapts to various environments and quickly develops antibiotic resistance, which has led to an increase in multidrug-resistant infections. Hence, S. aureus presents a significant global health issue and its adaptations to the host environment are crucial for understanding pathogenesis and antibiotic susceptibility.

Lipidomics of homeoviscous adaptation to low temperatures in utilizing exogenous straight-chain unsaturated fatty acids.

Unlabelled: It is well established that can incorporate exogenous straight-chain unsaturated fatty acids (SCUFAs) into membrane phospho- and glyco-lipids from various sources in supplemented culture media and when growing during infection. Given the enhancement of membrane fluidity when oleic acid (C18:1Δ9) is incorporated into lipids, we were prompted to examine the effect of medium supplementation with C18:1Δ9 on growth at low temperatures. C18:1Δ9 supported the growth of a cold-sensitive, branched-chain fatty acid (BCFA)-deficient mutant at 12°C.

Growth of in the presence of oleic acid shifts the glycolipid fatty acid profile and increases resistance to antimicrobial peptides.

readily adapts to various environments and quickly develops antibiotic resistance, which has led to an increase in multidrug-resistant infections. Hence, presents a significant global health issue and its adaptations to the host environment are crucial for understanding pathogenesis and antibiotic susceptibility. When is grown conventionally, its membrane lipids contain a mix of branched-chain and straight-chain saturated fatty acids.

Lipidomics of homeoviscous adaptation to low temperatures in utilizing exogenous straight-chain unsaturated fatty acids over biosynthesized endogenous branched-chain fatty acids.

It is well established that can incorporate exogenous straight-chain unsaturated fatty acids (SCUFAs) into membrane phospho- and glyco-lipids from various sources in supplemented culture media, and when growing in an infection. Given the enhancement of membrane fluidity when oleic acid (C18:1Δ9) is incorporated into lipids, we were prompted to examine the effect of medium supplementation with C18:1Δ9 on growth at low temperatures. C18:1Δ9 supported the growth of a cold-sensitive, branched-chain fatty acid (BCFA)-deficient mutant at 12°C.

A genetically encoded far-red fluorescent indicator for imaging synaptically released Zn.

Synaptic zinc ion (Zn) has emerged as a key neuromodulator in the brain. However, the lack of research tools for directly tracking synaptic Zn in the brain of awake animals hinders our rigorous understanding of the physiological and pathological roles of synaptic Zn. In this study, we developed a genetically encoded far-red fluorescent indicator for monitoring synaptic Zn dynamics in the nervous system.

The Antibiotic Peptide Daptomycin Functions by Reorganizing the Membrane.

The mechanism of the antimicrobial peptide daptomycin is reviewed and discussed. Daptomycin is a last-resort antibiotic in current use against drug-resistant bacterial infections. Many models have been proposed for its function, most based on the observation that it increases membrane permeability and causes leakage of contents, such as ions and small molecules from bacterial cells and lipid vesicles.

Lipidomic and Ultrastructural Characterization of the Cell Envelope of Staphylococcus aureus Grown in the Presence of Human Serum.

can incorporate exogenous straight-chain unsaturated and saturated fatty acids (SCUFAs and SCFAs, respectively) to replace some of the normally biosynthesized branched-chain fatty acids and SCFAs. In this study, the impact of human serum on the lipidome and cell envelope structure was comprehensively characterized. When was grown in the presence of 20% human serum, typical human serum lipids, such as cholesterol, sphingomyelin, phosphatidylethanolamines, and phosphatidylcholines, were present in the total lipid extracts.

A Quantitative Model of Daptomycin Binding to Lipid Bilayers.

Daptomycin is a cyclic lipopeptide of clinical importance in the treatment of multidrug resistant infections, including those caused by methicillin-resistant S. aureus strains. Similar to many other antimicrobial peptides, daptomycin binds with preference to anionic membranes such as those typically found in prokaryotes.

Daptomycin-Phosphatidylglycerol Domains in Lipid Membranes.

Daptomycin is an acidic, 13-amino acid, cyclic polypeptide that contains a number of nonproteinogenic residues and is modified at its N-terminus with a decanoyl chain. It has been in clinical use since 2003 against selected drug-resistant Staphylococcus aureus and Enterococcus spp infections. In vitro, daptomycin is active against Gram-positive pathogens at low concentrations but its antibiotic activity depends critically on the presence of calcium ions.

Binding of Daptomycin to Anionic Lipid Vesicles Is Reduced in the Presence of Lysyl-Phosphatidylglycerol.

The cytoplasmic membrane of Staphylococcus aureus contains ∼20 mol% of the net cationic lipid lysyl-phosphatidylglycerol (LPG). Elevated fractions of LPG are associated with increased resistance to cationic antibiotics, including the lipopeptide daptomycin (DAP). Although the surface charge of the bacterial cytoplasmic membrane is altered by LPG, surface binding of DAP was found to be only moderately affected in anionic vesicles containing 20 mol% LPG.

Branched phospholipids render lipid vesicles more susceptible to membrane-active peptides.

Iso- and anteiso-branched lipids are abundant in the cytoplasmic membranes of bacteria. Their function is assumed to be similar to that of unsaturated lipids in other organisms - to maintain the membrane in a fluid state. However, the presence of terminally branched membrane lipids is likely to impact other membrane properties as well.

Peptides with the same composition, hydrophobicity, and hydrophobic moment bind to phospholipid bilayers with different affinities.

We investigated the dependence of membrane binding on amino acid sequence for a series of amphipathic peptides derived from δ-lysin. δ-Lysin is a 26 amino acid, N-terminally formylated, hemolytic peptide that forms an amphipathic α-helix bound at membrane-water interfaces. A shortened peptide, lysette, was derived from δ-lysin by deletion of the four N-terminal amino acid residues.

Lysylated phospholipids stabilize models of bacterial lipid bilayers and protect against antimicrobial peptides.

Aminoacylated phosphatidylglycerols are common lipids in bacterial cytoplasmic membranes. Their presence in Staphylococcus aureus has been linked to increased resistance to a number of antibacterial agents, including antimicrobial peptides. Most commonly, the phosphatidylglycerol headgroup is esterified to lysine, which converts anionic phosphatidylglycerol into a cationic lipid with a considerably increased headgroup size.

On the origin of multiphasic kinetics in peptide binding to phospholipid vesicles.

We critically examined a series of exact kinetic models for their ability to describe binding of a typical α-helical amphipathic peptide to lipid bilayers. Binding of the model peptide lysette-26 was measured through fluorescence resonance energy transfer from a Trp residue on the peptide to a fluorescently labeled acceptor lipid included in vesicles composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine. Experimental data was collected varying peptide and lipid concentrations over an order of magnitude.

Molecular dynamics studies of transportan 10 (tp10) interacting with a POPC lipid bilayer.

We performed a series of molecular dynamics simu lations to study the nature of interactions between transportan 10 (tp10) and a zwitterionic POPC bilayer. Tp10 is an amphipathic cell-penetrating peptide with a net positive charge of +5 and is known to adopt an α-helical secondary structure on the surface of POPC membranes. The study showed that tp10 preferentially binds to the membrane surface with its hydrophobic side facing the hydrophobic lipid core.

Lysyl-phosphatidylglycerol attenuates membrane perturbation rather than surface association of the cationic antimicrobial peptide 6W-RP-1 in a model membrane system: implications for daptomycin resistance.

The presence of the cationic phospholipid lysyl-phosphatidylglycerol (lysyl-PG) in staphylococcal cytoplasmic membranes has been linked to increased resistance to cationic compounds, including antibiotics such as daptomycin as well as host defense antimicrobial peptides. We investigated the effects of lysyl-PG on binding of 6W-RP-1, a synthetic antimicrobial peptide, to lipid vesicles and on peptide-induced membrane permeabilization. Unexpectedly, physiological lysyl-PG concentrations only minimally reduced membrane binding of 6W-RP-1.

Binding and permeabilization of model membranes by amphipathic peptides.

Measurement of binding and activity of antimicrobial and cytolytic amphipathic peptides on membranes is essential to understanding their function and cell specificity. The use of model systems has provided a wealth of information on the interactions of amphipathic peptides with membranes. Binding of peptides to membranes can be monitored by measuring Förster resonance energy transfer from a Trp residue on the peptide to a lipid fluorophore incorporated in the membrane.

Mechanisms of antimicrobial, cytolytic, and cell-penetrating peptides: from kinetics to thermodynamics.

The mechanisms of six different antimicrobial, cytolytic, and cell-penetrating peptides, including some of their variants, are discussed and compared. The specificity of these polypeptides varies; however, they all form amphipathic alpha-helices when bound to membranes, and there are no striking differences in their sequences. We have examined the thermodynamics and kinetics of their interaction with phospholipid vesicles, namely, binding and peptide-induced dye efflux.

Wasp mastoparans follow the same mechanism as the cell-penetrating peptide transportan 10.

We have been examining the mechanism and kinetics of the interactions of a selected set of peptides with phospholipid membranes in a quantitative manner. This set was chosen to cover a broad range of physical-chemical properties and cell specificities. Mastoparan (masL) and mastoparan X (masX) are two similar peptides from the venoms of the wasps Vespula lewisii and Vespa xanthoptera, respectively, and were chosen to complete the set.

Magainin 2 revisited: a test of the quantitative model for the all-or-none permeabilization of phospholipid vesicles.

The all-or-none kinetic model that we recently proposed for the antimicrobial peptide cecropin A is tested here for magainin 2. In mixtures of phosphatidylcholine (PC)/phosphatidylglycerol (PG) 50:50 and 70:30, release of contents from lipid vesicles occurs in an all-or-none fashion and the differences between PC/PG 50:50 and 70:30 can be ascribed mainly to differences in binding, which was determined independently and is approximately 20 times greater to PC/PG 50:50 than to 70:30. Only one variable parameter, beta, corresponding to the ratio of the rates of pore opening to pore closing, is used to fit dye release kinetics from these two mixtures, for several peptide/lipid ratios ranging from 1:25 to 1:200.

The activity of the amphipathic peptide delta-lysin correlates with phospholipid acyl chain structure and bilayer elastic properties.

Release of lipid vesicle content induced by the amphipathic peptide delta-lysin was investigated as a function of lipid acyl chain length and degree of unsaturation for a series of phosphatidylcholines. Dye efflux and peptide binding were examined for three homologous lipid series: di-monounsaturated, di-polyunsaturated, and asymmetric phosphatidylcholines, with one saturated and one monounsaturated acyl chain. Except for the third series, peptide activity correlated with the first moment of the lateral pressure profile, which is a function of lipid acyl chain structure.

Small changes in the primary structure of transportan 10 alter the thermodynamics and kinetics of its interaction with phospholipid vesicles.

The kinetics and thermodynamics of binding of transportan 10 (tp10) and four of its variants to phospholipid vesicles, and the kinetics of peptide-induced dye efflux, were compared. Tp10 is a 21-residue, amphipathic, cationic, cell-penetrating peptide similar to helical antimicrobial peptides. The tp10 variants examined include amidated and free peptides, and replacements of tyrosine by tryptophan.

A quantitative model for the all-or-none permeabilization of phospholipid vesicles by the antimicrobial peptide cecropin A.

The mechanism of the all-or-none release of the contents of phospholipid vesicles induced by the antimicrobial peptide cecropin A was investigated. A detailed experimental study of the kinetics of dye release showed that the rate of release increases with the ratio of peptide bound per vesicle and, at constant concentration, with the fraction of the anionic lipid phosphatidylglycerol in neutral, phosphatidylcholine membranes. Direct measurement of the kinetics of peptide binding and dissociation from vesicles revealed that the on-rate is almost independent of vesicle composition, whereas the off-rate decreases by orders of magnitude with increasing content of anionic lipid.

Investigation of domain formation in sphingomyelin/cholesterol/POPC mixtures by fluorescence resonance energy transfer and Monte Carlo simulations.

We have recently proposed a phase diagram for mixtures of porcine brain sphingomyelin (BSM), cholesterol (Chol), and 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) on the basis of kinetics of carboxyfluorescein efflux induced by the amphipathic peptide delta-lysin. Although that study indicated the existence of domains, phase separations in the micrometer scale have not been observed by fluorescence microscopy in BSM/Chol/POPC mixtures, though they have for some other sphingomyelins (SM). Here we examine the same BSM/Chol/POPC system by a combination of fluorescence resonance energy transfer (FRET) and Monte Carlo simulations.

Mechanism of the cell-penetrating peptide transportan 10 permeation of lipid bilayers.

The mechanism of the interaction between the cell-penetrating peptide transportan 10 (tp10) and phospholipid membranes was investigated. Tp10 induces graded release of the contents of phospholipid vesicles. The kinetics of peptide association with vesicles and peptide-induced dye efflux from the vesicle lumen were examined experimentally by stopped-flow fluorescence.