Synergistic Biophysical Techniques Reveal Structural Mechanisms of Engineered Cationic Antimicrobial Peptides in Lipid Model Membranes.

In the quest for new antibiotics, two novel engineered cationic antimicrobial peptides (eCAPs) have been rationally designed. WLBU2 and D8 (all 8 valines are the d-enantiomer) efficiently kill both Gram-negative and -positive bacteria, but WLBU2 is toxic and D8 nontoxic to eukaryotic cells. We explore protein secondary structure, location of peptides in six lipid model membranes, changes in membrane structure and pore evidence. We suggest that protein secondary structure is not a critical determinant of bactericidal activity, but that membrane thinning and dual location of WLBU2 and D8 in the membrane headgroup and hydrocarbon region may be important. While neither peptide thins the Gram-negative lipopolysaccharide outer membrane model, both locate deep into its hydrocarbon region where they are primed for self-promoted uptake into the periplasm. The partially α-helical secondary structure of WLBU2 in a red blood cell (RBC) membrane model containing 50 % cholesterol, could play a role in destabilizing this RBC membrane model causing pore formation that is not observed with the D8 random coil, which correlates with RBC hemolysis caused by WLBU2 but not by D8.

Bacteriocin enterocin CRL35 is a modular peptide that induces non-bilayer states in bacterial model membranes.

The mechanism of action of the anti-Listeria peptide enterocin CRL35 was studied with biophysical tools by using lipid mixtures that mimicked Gram-positive plasma membranes. Langmuir monolayers and infrared spectroscopy indicated that the peptide readily interacted with phospholipid assembled in monolayers and bilayers to produce a dual effect, depending on the acyl chains. Indeed, short chain mixtures were disordered by enterocin CRL35, but the gel-phases of membranes composed by longer acyl chains were clearly stabilized by the bacteriocin. Structural and functional studies indicated that non-bilayer states were formed when liposomes were co-incubated with enterocin CRL35, whereas significant permeabilization could be detected when bilayer and non-bilayer states co-existed. Results can be explained by a two-step model in which the N-terminal of the peptide firstly docks enterocin CRL35 on the lipid surface by means of electrostatic interactions; then, C-terminal triggers membrane perturbation by insertion of hydrophobic α-helix.

Elastic behavior of model membranes with antimicrobial peptides depends on lipid specificity and d-enantiomers.

In an effort to provide new treatments for the global crisis of bacterial resistance to current antibiotics, we have used a rational approach to design several new antimicrobial peptides (AMPs). The present study focuses on 24-mer WLBU2 and its derivative, D8, with the amino acid sequence, RRWVRRVRRWVRRVVRVVRRWVRR. In D8, all of the valines are the d-enantiomer. We use X-ray low- and wide-angle diffuse scattering data to measure elasticity and lipid chain order. We show a good correlation between in vitro bacterial killing efficiency and both bending and chain order behavior in bacterial lipid membrane mimics; our results suggest that AMP-triggered domain formation could be the mechanism of bacterial killing in both Gram-positive and Gram-negative bacteria. In red blood cell lipid mimics, D8 stiffens and orders the membrane, while WLBU2 softens and disorders it, which correlate with D8's harmless vs. WLBU2's toxic behavior in hemolysis tests. These results suggest that elasticity and chain order behavior can be used to predict mechanisms of bactericidal action and toxicity of new AMPs.

Membrane order and ionic strength modulation of the inhibition of the membrane-bound acetylcholinesterase by epigallocatechin­3­gallate.

In the present work, we analyzed how external factors can modulate the efficiency of epigallocatechin­3­O­gallate (EGCG) inhibition of a membrane-bound isoform of the acetylcholinesterase. Increasing the ionic strength but not the osmolarity of the bulk medium proved to be an important factor. In addition, we verified a clear correlation between the inhibitory activity with the order degree of the membranes by using cholesterol-partially depleted red blood cell ghosts. These two factors i.e. high salt concentration in the bulk medium and less viscous membranes, allow a deeper insertion of the EGCG into the lipid bilayer, thus leading to a greater inhibition of AChE. As a corollary, we propose that any treatment or process that leads to a slight decrease in cholesterol content in the membranes can efficiently enhance the inhibitory activity of EGCG, which can have important consequences in all the pathologies where the inhibition of AChE is recommended.

Structure of gel phase DPPC determined by X-ray diffraction.

High resolution low angle x-ray data are reported for the gel phase of DPPC lipid bilayers, extending the previous q range of 1.0 Å-1 to 1.3 Å-1, and employing a new technique to obtain more accurate intensities and form factors |F(q)| for the highest orders of diffraction. Combined with previous wide angle x-ray and volumetric data, a space filling model is employed to obtain gel phase structure at a mesoscopic level. This analysis provides direct evidence that the hydrocarbon chains from opposing monolayers are mini-interdigitated, consistent with the previously well-established result that the opposing monolayers are strongly coupled with respect to their chain tilt directions. Even more detailed structural features are described that have not been obtained from experiment but that could, in principle, be obtained from simulations that would first be validated by agreement with the wide angle and the new low angle |F(q)| x-ray data.

Selective Interaction of Colistin with Lipid Model Membranes.

Although colistin's clinical use is limited due to its nephrotoxicity, colistin is considered to be an antibiotic of last resort because it is used to treat patients infected with multidrug-resistant bacteria. In an effort to provide molecular details about colistin's ability to kill Gram-negative (G(-)) but not Gram-positive (G(+)) bacteria, we investigated the biophysics of the interaction between colistin and lipid mixtures mimicking the cytoplasmic membrane of G(+), G(-) bacteria as well as eukaryotic cells. Two different models of the G(-) outer membrane (OM) were assayed: lipid A with two deoxy-manno-octulosonyl sugar residues, and Escherichia coli lipopolysaccharide mixed with dilaurylphosphatidylglycerol. We used circular dichroism and x-ray diffuse scattering at low and wide angle in stacked multilayered samples, and neutron reflectivity of single, tethered bilayers mixed with colistin. We found no differences in secondary structure when colistin was bound to G(-) versus G(+) membrane mimics, ruling out a protein conformational change as the cause of this difference. However, bending modulus KC perturbation was quite irregular for the G(-) inner membrane, where colistin produced a softening of the membranes at an intermediate lipid/peptide molar ratio but stiffening at lower and higher peptide concentrations, whereas in G(+) and eukaryotic mimics there was only a slight softening. Acyl chain order in G(-) was perturbed similarly to KC. In G(+), there was only a slight softening and disordering effect, whereas in OM mimics, there was a slight stiffening and ordering of both membranes with increasing colistin. X-ray and neutron reflectivity structural results reveal colistin partitions deepest to reach the hydrocarbon interior in G(-) membranes, but remains in the headgroup region in G(+), OM, and eukaryotic mimics. It is possible that domain formation is responsible for the erratic response of G(-) inner membranes to colistin and for its deeper penetration, which could increase membrane permeability.

Impairment of the class IIa bacteriocin receptor function and membrane structural changes are associated to enterocin CRL35 high resistance in Listeria monocytogenes.

BACKGROUND: Enterocin CRL35 is a class IIa bacteriocin with anti-Listeria activity. Resistance to these peptides has been associated with either the downregulation of the receptor expression or changes in the membrane and cell walls. The scope of the present work was to characterize enterocin CRL35 resistant Listeria strains with MICs more than 10,000 times higher than the MIC of the WT sensitive strain. METHODS: Listeria monocytogenes INS7 resistant isolates R2 and R3 were characterized by 16S RNA gene sequencing and rep-PCR. Bacterial growth kinetic was studied in different culture media. Plasma membranes of sensitive and resistant bacteria were characterized by FTIR and Langmuir monolayer techniques. RESULTS: The growth kinetic of the resistant isolates was slower as compared to the parental strain in TSB medium. Moreover, the resistant isolates barely grew in a glucose-based synthetic medium, suggesting that these cells had a major alteration in glucose transport. Resistant bacteria also had alterations in their cell wall and, most importantly, membrane lipids. In fact, even though enterocin CRL35 was able to bind to the membrane-water interface of both resistant and parental sensitive strains, this peptide was only able to get inserted into the latter membranes. CONCLUSIONS: These results indicate that bacteriocin receptor is altered in combination with membrane structural modifications in enterocin CRL35-resistant L. monocytogenes strains. GENERAL SIGNIFICANCE: Highly enterocin CRL35-resistant isolates derived from Listeria monocytogenes INS7 have not only an impaired glucose transport but also display structural changes in the hydrophobic core of their plasma membranes.

Cholesterol induces surface localization of polyphenols in model membranes thus enhancing vesicle stability against lysozyme, but reduces protection of distant double bonds from reactive-oxygen species.

The main scope of the present study was to analyze the membrane interaction of members of different classes of polyphenols, i.e. resveratrol, naringenin, epigallocatechin gallate and enterodiol, in model systems of different compositions and phase states. In addition, the possible association between membrane affinity and membrane protection against both lipid oxidation and bilayer-disruptive compounds was studied. Gibbs monolayer experiments indicated that even though polyphenols showed poor surface activity, it readily interacted with lipid films. Actually, a preferential interaction with expanded monolayers was observed, while condensed and cholesterol-containing monolayers decreased the affinity of these phenolic compounds. On the other hand, fluorescence anisotropy studies showed that polyphenols were able to modulate membrane order degree, but again this effect was dependent on the cholesterol concentration and membrane phase state. In fact, cholesterol induced a surface rather than deep into the hydrophobic core localization of phenolic compounds in the membranes. In general, the polyphenolic molecules tested had a better antioxidant activity when they were allowed to get inserted into the bilayers, i.e. in cholesterol-free membranes. On the other hand, a membrane-protective effect against bilayer permeabilizing activity of lysozyme, particularly in the presence of cholesterol, could be assessed. It can be hypothesized that phenolic compounds may protect membrane integrity by loosely covering the surface of lipid vesicles, once cholesterol push them off from the membrane hydrophobic core. However, this cholesterol-driven distribution may lead to a reduced antioxidant activity of linoleic acid double bonds.

Interfacial stabilization of the antitumoral drug Paclitaxel in monolayers of GM1 and GD1a gangliosides.

Molecular interactions between the anti-cancer agent Paclitaxel (Ptx), and two gangliosides with different sialic acid content, GM1 and GD1a, were investigated using the Langmuir film balance technique. Ptx showed interfacial activity reducing the air/water surface tension by 18 mN·m(-1). However, the drug was able to insert into preformed ganglioside monolayers at much higher surface pressures, indicating a preferential interaction of Ptx with GM1 and GD1a. Compression isotherms of binary mixtures of Ptx and GM1 or GD1a also indicated non-ideal mixed monolayers in which the drug became stabilized at the interface in the presence of gangliosides. Ptx reached much higher surface pressure values in the mixed monolayers than those sustained in pure Ptx, although partial desorption of the drug from the interface into the subphase was also observed at high Ptx contents. The mean molecular area of the mixtures showed condensation, mainly in the case of GD1a, whereas Ptx induced a decrease in the compressibility of monolayers when mixed with either GM1 or GD1a. Additionally, Brewster angle microscopy analysis indicated that higher amounts of Ptx are present at the mixed ganglioside/Ptx interface when compared to pure drug monolayers. Finally, GD1a micelles increased in size in the presence of Ptx, whereas GM1 micelles kept their diameter, according to dynamic light scattering measurements, which could be explained by the different properties of ganglioside monolayers. The results obtained on ganglioside-Ptx interactions allowed interpreting the different Ptx loading capacity of GM1 and GD1a, enabling them to act as potential drug carriers.

28-mer Fragment Derived from Enterocin CRL35 Displays an Unexpected Bactericidal Effect on Listeria Cells.

Two shorter peptides derived from enterocin CRL35, a 43-mer bacteriocin, were synthesized i.e. the N-terminal fragment spanning from residues 1 to 15, and a 28-mer fragment that represents the C-terminal of enterocin CRL35, the residues 16 to 43. The separate peptides showed no activity when combined. On one hand, the 28-mer peptide displayed an unpredicted antimicrobial activity. On the other, 15- mer peptide had no consistent anti-Listeria effect. The dissociation constants calculated from experimental data indicated that all peptides could bind at similar extent to the sensitive cells. However, transmembrane electrical potential was not dissipated to the same level by the different peptides; whereas the full-length and the C-terminal 28-mer fragment induced almost full dissipation, 15-mer fragment produced only a slow and incomplete effect. Furthermore, a different interaction of each peptide with membranes was demonstrated based on studies carried out with liposomes, which led us to conclude that activity was related to structure rather than to net positive charges. These results open up the possibility of designing new peptides based on the 28-mer fragment with enhanced activity, which would represent a promising approach for combating Listeria and other pathogens.

N-Acyl Chain in Ceramide and Sphingomyelin Determines Their Mixing Behavior, Phase State, and Surface Topography in Langmuir Films.

Sphingolipids are membrane lipids composed by a long chain aminediol base, usually sphingosine, with a N-linked fatty acyl chain whose quality depends on the membrane type. The effect of length and unsaturation of the N-acyl chain on the mixing behavior of different sphingolipids has scarcely been studied, and in this work this issue is addressed employing Langmuir monolayers at the air-water interface, in order to assess the surface mixing in binary mixtures of different species of sphingomyelins and ceramides. The dependence on the monolayer composition of the mean molecular area, perpendicular dipole moment, domain segregation, and surface topography, as well as the film elasticity and optical thickness were studied. The results indicate that composition-dependent favorable interactions among sphingomyelin and ceramide occur as a consequence of complementary lateral packing and increased acyl chain ordering; the phase state of the components appears as a major factor determining miscibility among sphingomyelins and ceramides even in cases where the lipids have a considerable hydrocarbon chain length mismatch.

Proton motive force dissipation precludes interaction of microcin J25 with RNA polymerase, but enhances reactive oxygen species overproduction.

BACKGROUND: Microcin J25 targets the RNA polymerase as well as bacterial membranes. Because there is scarce information on the relationship between the uptake and the activity, a fluorescent microcin J25-derivative was used to further characterize its mechanism of action. METHODS: MccJ25 I13K was labeled with FITC and its uptake by sensitive cells was assessed by fluorescence measurements from supernatants of MccJ25-Escherichia coli suspensions. The interaction of the peptide with bacterial membranes was investigated by fluorescence resonance energy transfer. Oxygen consumption was measured with Clark-type electrode. RNA synthesis was evaluated in vivo by incorporation of [3H]uridine. ROS production was assayed by measuring the fluorescence emission of the ROS-sensitive probe 5(and 6)-carboxy-2',7'-dichlorodihydrofluorescein diacetate. RESULTS: The protonophore 2,4-dinitrophenol decreased 80% of the MccJ25 uptake and prevented inhibition of transcriptional activity, the antibiotic intracellular target. On the other hand, peptide binding to bacterial membranes was not affected and antibacterial activity remained nearly unchanged. Proton gradient dissipation by protonophore accelerated cell oxygen consumption rates and enhanced MccJ25-related reactive oxygen species overproduction. GENERAL SIGNIFICANCE: The deleterious reactive oxygen species would be produced as a consequence of the minor fraction of MccJ25 that interacts with the bacterial plasma membrane from the periplasmic side. These results show the first evidence of the mechanism underlying ROS production in sensitive bacteria.