Staphylococcus aureus is an opportunistic pathogen related to a variety of life-threatening infections but for which antimicrobial resistance is liming the treatment options. We report here that myricetin, but not its glycosylated form, can remarkably decrease the production of several S. aureus virulence factors, including adhesion, biofilm formation, hemolysis and staphyloxanthin production, without interfering with growth. Myricetin affects both surface proteins and secreted proteins which indicate that its action is unrelated to inhibition of the agr quorum sensing system. Analysis of virulence related gene expression and computational simulations of pivotal proteins involved in pathogenesis demonstrate that myricetin downregulates the saeR global regulator and interacts with sortase A and α-hemolysin. Furthermore, Myr confers a significant degree of protection against staphylococcal infection in the Galleria mellonella model. The present findings reveal the potential of Myr as an alternative multi-target antivirulence candidate to control S. aureus pathogenicity.

Lipopolysaccharides (LPS) are the primary constituent of the outer membrane of Gram-negative bacteria such as Pseudomonas aeruginosa. Gram-negative bacteria can synthesize modified forms of LPS in response to environmental stimuli or due to genetic mutations, a process known as outer membrane remodeling. Chemical modifications of the LPS modulate the integrity and antibiotic susceptibility of bacterial outer membranes. It also governs microbial adhesion to tissues and artificial material surfaces. We have extended a previous model of the rough LPS to include four novel chemotypes rmlC, galU, LPS Re, and Lipid-A. Atomistic molecular dynamics (MD) simulations were performed for outer membrane models constituted of each LPS chemotypes and 1,2-dipalmitoyl-3-phosphatidylethanolamine. It is shown that the decrease in the LPS polysaccharide chain length leads to a significant increase in the diffusion coefficients for the Ca(2+) counterions, increase in acyl chain packing (decrease in membrane fluidity), and attenuation of the negative potential across the LPS surface as positive counterions becomes more exposed to the solvent. The electrostatic potential on the LPS surfaces reflects heterogeneous charge distributions with increasingly larger patches of positive and negative potentials as the polysaccharide chain length decreases. Such a pattern originates from the spatial arrangement of charged phosphate-Ca(2+) clusters in the LPS inner-core that becomes exposed in the membrane surface as monosaccharide units are lost in the shortest chemotypes LPS Re and Lipid-A. These MD-derived conformational ensembles reproduce experimental trends and provide atom-level structural information on the rough LPS chemotypes that can help to rationalize antibiotic resistance and bacterial adhesion processes.

Surfactants are molecular structures with remarkable physicochemical properties and applications. Most of their characteristics are due to their ability to promote aggregation and interactions with different interfaces. The scarcity of theoretical studies dedicated to evaluating the forces involved in these interactions prompted us to propose other models capable of reproducing the experimental data in better ways. We carried out molecular dynamics (MD) simulations to obtain a model for cetyltrimethylammonium bromide (CTAB), selected from gromos54a7 force field parameters, that better describes most of its behaviors in aqueous solution (micellar structure, counterion dissociation, etc.) and its adsorption pattern on a gold surface. The parameters adopted for one of the models were able to mimic several characteristics suggested by experimental measurements of the CTAB micelles, as well their adsorption pattern on a gold surface. Indeed, this model was able to obtain quasi-spherical micelles, as well as a pattern of adjacent cylindrical micelles with alkyl chain interactions on a gold surface.Keywords: molecular dynamics, micelles, interface interaction, cetyltrimethylammonium bromide, gold IntroductionSurfactants are a class of compounds containing a polar group, charged or neutral, attached to a long hydrophobic tail.1,2 These are remarkably versatile compounds with a broad variety of important applications in the pharmaceutical, medical, and food industries and for nanomaterial synthesis.3,4 Above a certain temperature in solution (the Krafft temperature), surfactants tend to aggregate to minimize unfavorable interactions between the surfactants and the surrounding environment. 5 The minimum concentration required for surfactant aggregation is defined as the critical micellar concentration (CMC), and most of the characteristics of these aggregates are controlled by factors such as the solvent type, chemical structure of the surfactant, and solution conditions (e.g., concentration, temperature, presence of additives, and ionic strength). 6Variations of these factors yield aggregates with different morphologies such as spherical or ellipsoidal micelles, cylindrical or thread-like micelles, disk-like micelle, membranes and vesicles.7 These self-assembled structures have been characterized by a number of techniques, such as dynamic light scattering (DLS), 8 nuclear magnetic resonance (NMR), 9,10 fluorescence spectroscopy, 11 quasielastic neutron scattering (QENS), 12,13 small-angle X-ray scattering (SAXS), 14,15 and small-angle neutron scattering (SANS). 16,17 Computational simulations have also been employed to explore the structures and dynamical behaviors of micelles for different surfactants. 18Considering that no holes exist within a micelle, its radius is estimated as the maximum extension of a hydrocarbon chain and can be evaluated by using the following equation:where l max is the maximum length in nm and n C is the number of carbon atoms in the chain. 19 Indeed, under the previously mentioned conditions, ...

The mechanism of action of antimicrobial peptides depends on physicochemical properties such as structure, concentration, and oligomerization. Here, we focused on the effect of dimerization on the mechanism of action of aurein 1.2 (AU). We designed a lysine-linked AU dimer, (AU)2K, and its interaction with membrane mimetics was studied using four biophysical techniques and molecular dynamics simulations. Circular dichroism and molecular dynamics studies showed that AU displayed a typical spectrum for disordered structures in aqueous solution whereas (AU)2K exhibited the typical spectrum of α-helices in a coiled-coil conformation, wherein helices are wrapped around each other. With the addition of large unilamellar vesicles (LUVs), AU adopted an α-helix structure whereas the coiled-coil structure of (AU)2K assumed an extended conformation. Carboxyfluorescein release experiments with LUVs showed that both peptides were able to permeabilize vesicles although the leakage response to increases in peptide concentration differed. Optical microscopy experiments showed that both peptides induced pore opening and the dimer eventually caused the vesicles to burst. Finally, calorimetric traces determined by isothermal titration calorimetry on the LUVs also showed significant differences in peptide-membrane interactions. Together, the results of our study demonstrated that dimerization changes the mechanism of action of AU.

Resistance to antibiotics has made diseases that previously healed easily become more difficult to treat. Staphylococcus aureus is an important cause of hospital-acquired infections and multi-drug resistant. NorA efflux pump, present in bacteria S. aureus, is synthesized by the expression of the norA gene. Menadione, also known as vitamin K3, is one of the synthetic forms of vitamin K. Therefore, the aim of this study is to verify the menadione effect on efflux inhibition through NorA pump gene expression inhibition and assess the effects of menadione in bacterial membrane. The effect of menadione as an efflux pump inhibitor (EPI) was evaluated by the microdilution method, fluorimetry, electron microscopy, and by RT-qPCR to evaluate gene expression. In the molecular docking, association with menadione induces increased fluorescence intensity. Menadione was observed (100% of the clusters) interacting with residues ILE12, ILE15, PHE16, ILE19, PHE47, GLN51, ALA105, and MET109 from NorA. The results showed the norA gene had its expression significantly diminished in the presence of menadione. The simulation showed that several menadione molecules were able to go through the bilayer and allow the entry of water molecules into the hydrophobic regions of the bilayer. When present within membranes, menadione may have caused membrane structural changes resulting in a decline of the signaling pathways involved in norA expression. Menadione demonstrated to be an efflux pump inhibitor with dual mechanism: affecting the efflux pump by direct interaction with protein NorA and indirectly inhibiting the norA gene expression, possibly by affecting regulators present in the membrane altered by menadione.

Understanding the permeation of biomolecules through cellular membranes is critical for many biotechnological applications, including targeted drug delivery, pathogen detection, and the development of new antibiotics. To this end, computer simulations are routinely used to probe the underlying mechanisms of membrane permeation. Despite great progress and continued development, permeation simulations of realistic systems (e.g., more complex drug molecules or biologics through heterogeneous membranes) remain extremely challenging if not intractable. In this work, we combine molecular dynamics simulations with transition-tempered metadynamics and techniques from the variational approach to conformational dynamics to study the permeation mechanism of a drug molecule, trimethoprim, through a multicomponent membrane. We show that collective variables (CVs) obtained from an unsupervised machine learning algorithm called time-structure based Independent Component Analysis (tICA) improve performance and substantially accelerate convergence of permeation potential of mean force (PMF) calculations. The addition of cholesterol to the lipid bilayer is shown to increase both the width and height of the free energy barrier due to a condensing effect (lower area per lipid) and increase bilayer thickness. Additionally, the tICA CVs reveal a subtle effect of cholesterol increasing the resistance to permeation in the lipid head group region, which is not observed when canonical CVs are used. We conclude that the use of tICA CVs can enable more efficient PMF calculations with additional insight into the permeation mechanism.

Antimicrobial peptides (AMPs) are alternatives to conventional antibiotics against multi-drug resistant bacteria with low potential for developing microbial resistance. The design of such molecules requires understanding of the mechanisms of action, particularly the interaction with bacteria cell membranes. In this work, we determine the mechanism responsible for the higher activity against Escherichia coli of the C-terminal lysine dimer of magainin 2, (MG2)K, in comparison to the monomeric peptide magainin 2 (MG2). Langmuir monolayers and vesicles made with the E. coli lipid extract were used to address the two possible states for the peptide-membrane interaction, namely the "binding state" and "pore state", respectively. The incorporation of MG2 and (MG2)K in lipid monolayers at the air-water interface caused slight differences in surface pressure isotherms and polarization-modulated infrared reflection absorption (PM-IRRAS) spectra, and therefore the difference in activity is not associated with the binding state. In contrast, large differences were observed in the leakage experiments where (MG2)K was shown to disrupt the large unilamellar vesicles to a much higher extent owing to efficient pore formation. The binding and penetration of MG2 and (MG2)K were also probed with molecular dynamics (MD) simulations for bilayers made with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine:1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPE:POPG). (MG2)K forms disordered toroidal pores at a significant lower concentration than for MG2. In summary, the combination of experimental and computational simulation results indicated that the "pre-assembling state" of (MG2)K dimer leads to a reduced number of molecules and shorter time being required to kill E. coli.

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