Structure of the Bacterial Cell Envelope and Interactions with Antimicrobials: Insights from Molecular Dynamics Simulations.

Bacteria have evolved over 3 billion years, shaping our intrinsic and symbiotic coexistence with these single-celled organisms. With rising populations of drug-resistant strains, the search for novel antimicrobials is an ongoing area of research. Advances in high-performance computing platforms have led to a variety of molecular dynamics simulation strategies to study the interactions of antimicrobial molecules with different compartments of the bacterial cell envelope of both Gram-positive and Gram-negative species. In this review, we begin with a detailed description of the structural aspects of the bacterial cell envelope. Simulations concerned with the transport and associated free energy of small molecules and ions through the outer membrane, peptidoglycan, inner membrane and outer membrane porins are discussed. Since surfactants are widely used as antimicrobials, a section is devoted to the interactions of surfactants with the cell wall and inner membranes. The review ends with a discussion on antimicrobial peptides and the insights gained from the molecular simulations on the free energy of translocation. Challenges involved in developing accurate molecular models and coarse-grained strategies that provide a trade-off between atomic details with a gain in sampling time are highlighted. The need for efficient sampling strategies to obtain accurate free energies of translocation is also discussed. Molecular dynamics simulations have evolved as a powerful tool that can potentially be used to design and develop novel antimicrobials and strategies to effectively treat bacterial infections.

Martini-3 Coarse-Grained Models for the Bacterial Lipopolysaccharide Outer Membrane of Escherichia coli.

The outer lipopolysaccharide (LPS) membrane of Gram-negative bacteria forms the main barrier for transport of antimicrobial molecules into the bacterial cell. In this study we develop coarse-grained models for the outer membrane of Escherichia coli in the Martini-3 framework. The coarse-grained model force field was parametrized and validated using all-atom simulations of symmetric membranes of lipid A and rough LPS as well as a complete asymmetric membrane of LPS with the O-antigen. The bonded parameters were obtained using an iterative refinement procedure with target bonded distributions obtained from all-atom simulations. The membrane thickness, area of the LPS, and density distributions for the different regions as well as the water and ion densities in Martini-3 simulations show excellent agreement with the all-atom data. Additionally the solvent accessible surface area for individual molecules in water was found to be in good agreement. The binding of calcium ions with phosphate and carboxylate moieties of LPS is accurately captured in the Martini-3 model, indicative of the integrity of the highly negatively charged LPS molecules in the outer membranes of Gram-negative bacteria. The melting transition of the coarse-grained lipid A membrane model was found to occur between 300 and 310 K, and the model captured variations in area per LPS, order parameter, and membrane thickness across the melting transition. Our study reveals that the proposed Martini-3 models for LPS are able to capture the physicochemical balance of the complex sugar architecture of the outer membrane of Escherichia coli. The coarse-grained models developed in this study would be useful for determining membrane protein interactions and permeation of potential antimicrobials through bacterial membranes at mesoscopic spatial and temporal scales.

Differentiating interactions of antimicrobials with Gram-negative and Gram-positive bacterial cell walls using molecular dynamics simulations.

Developing molecular models to capture the complex physicochemical architecture of the bacterial cell wall and to study the interaction with antibacterial molecules is an important aspect of assessing and developing novel antimicrobial molecules. We carried out molecular dynamics simulations using an atomistic model of peptidoglycan to represent the architecture for Gram-positive S. aureus. The model is developed to capture various structural features of the Staphylococcal cell wall, such as the peptide orientation, area per disaccharide, glycan length distribution, cross-linking, and pore size. A comparison of the cell wall density and electrostatic potentials is made with a previously developed cell wall model of Gram-negative bacteria, E. coli, and properties for both single and multilayered structures of the Staphylococcal cell wall are studied. We investigated the interactions of the antimicrobial peptide melittin with peptidoglycan structures. The depth of melittin binding to peptidoglycan is more pronounced in E. coli than in S. aureus, and consequently, melittin has greater contacts with glycan units of E. coli. Contacts of melittin with the amino acids of peptidoglycan are comparable across both the strains, and the D-Ala residues, which are sites for transpeptidation, show enhanced interactions with melittin. A low energetic barrier is observed for translocation of a naturally occurring antimicrobial thymol with the four-layered peptidoglycan model. The molecular model developed for Gram-positive peptidoglycan allows us to compare and contrast the cell wall penetrating properties with Gram-negative strains and assess for the first time binding and translocation of antimicrobial molecules for Gram-positive cell walls.

Interactions of Surfactants with the Bacterial Cell Wall and Inner Membrane: Revealing the Link between Aggregation and Antimicrobial Activity.

Surfactants with their intrinsic ability to solubilize lipid membranes are widely used as antibacterial agents, and their interactions with the bacterial cell envelope are complicated by their differential aggregation tendencies. We present a combined experimental and molecular dynamics investigation to unravel the molecular basis for the superior antimicrobial activity and faster kill kinetics of shorter-chain fatty acid surfactant, laurate, when compared with the longer-chain surfactants studied in contact time assays with live Escherichia coli (E. coli). From all-atom molecular dynamics simulations, translocation events across peptidoglycan were the highest for laurate followed by sodium dodecyl sulfate, myristate, palmitate, oleate, and stearate. The translocation kinetics were positively correlated with the critical micellar concentration, which determined the free monomer surfactant concentration available for translocation across peptidoglycan. Interestingly, aggregates showed a lower propensity to translocate across the peptidoglycan layer and longer translocation times were observed for oleate, thereby revealing an intrinsic sieving property of the bacterial cell wall. Molecular dynamics simulations with surfactant-incorporated bacterial inner membranes revealed the greatest hydrophobic mismatch and membrane thinning in the presence of laurate when compared with the other surfactants. The enhanced antimicrobial efficacy of laurate over oleate was further verified by experiments with giant unilamellar vesicles, and electroporation molecular dynamics simulations revealed greater inner membrane poration tendency in the presence of laurate when compared with the longer-chain surfactants. Our study provides molecular insights into surfactant translocation across peptidoglycan and chain length-induced structural disruption of the inner membrane, which correlate with contact time kill efficacies observed as a function of chain length with E. coli. The insights gained from our study uncover unexplored barrier properties of the bacterial cell envelope to rationalize the development of antimicrobial formulations and therapeutics.

A generic force field for simulating native protein structures using dissipative particle dynamics.

A coarse-grained force field for molecular dynamics simulations of the native structures of proteins in a dissipative particle dynamics (DPD) framework is developed. The parameters for bonded interactions are derived by mapping the bonds and angles for 20 amino acids onto target distributions obtained from fully atomistic simulations in explicit solvent. A dual-basin potential is introduced for stabilizing backbone angles, to cover a wide spectrum of protein secondary structures. The backbone dihedral potential enables folding of the protein from an unfolded initial state to the folded native structure. The proposed force field is validated by evaluating the structural properties of several model peptides and proteins including the SARS-CoV-2 fusion peptide, consisting of α-helices, ß-sheets, loops and turns. Detailed comparisons with fully atomistic simulations are carried out to assess the ability of the proposed force field to stabilize the different secondary structures present in proteins. The compact conformations of the native states were evident from the radius of gyration and the high intensity peaks of the root mean square deviation histograms, which were found to be within 0.4 nm. The Ramachandran-like energy landscape on the phase space of backbone angles (θ) and dihedrals (ϕ) effectively captured the conformational phase space of α-helices at ∼(ϕ = 50°,θ = 90°) and ß-strands at ∼(ϕ = ±180°,θ = 90-120°). Furthermore, the residue-residue native contacts were also well reproduced by the proposed DPD model. The applicability of the model to multidomain complexes was assessed using lysozyme and a large α-helical bacterial pore-forming toxin, cytolysin A. Our study illustrates that the proposed force field is generic, and can potentially be extended for efficient in silico investigations of membrane bound polypeptides and proteins using DPD simulations.

Developing a Coarse-Grained Model for Bacterial Cell Walls: Evaluating Mechanical Properties and Free Energy Barriers.

The bacterial cell envelope of Gram-negative bacteria is a complex biological barrier with multiple layers consisting of the inner membrane, periplasm of peptidoglycan, and the outer membrane with lipopolysaccharides (LPS). With rising antimicrobial resistance there is increasing interest in understanding interactions of small molecules with the cell membrane to aid in the development of novel drug molecules. Hence suitable representations of the bacterial membrane are required to carry out meaningful molecular dynamics simulations. Given the complexity of the cell envelope, fully atomistic descriptions of the cell membrane with explicit solvent are computationally prohibitive, allowing limited sampling with small system sizes. However, coarse-grained (CG) models such as MARTINI allow one to study phenomena at physiologically relevant length and time scales. Although MARTINI models for lipids and the LPS are available in literature, a suitable CG model of peptidoglycan is lacking. Using an all-atom model described by Gumbart et al. [PLoS Comput. Biol. 2014, 10, e1003475], we develop a CG model of the peptidoglycan network within the MARTINI framework. The model is parametrized to reproduce the end-to-end distance of glycan strands. The structural properties such as the equilibrium angle between adjacent peptides along the strands, area per disaccharide, and cavity size distributions agree well with the atomistic simulation results. Mechanical properties such as the area compressibility and the bending modulus are accurately reproduced. While developing novel antibiotics it is important to assess barrier properties of the peptidogylcan network. We evaluate and compare the free energy of insertion for a thymol molecule using umbrella sampling on both the MARTINI and all-atom peptidoglycan models. The insertion free energy was found to be less than kBT for both the MARTINI and all-atom models. Additional restraint free simulations reveal rapid translocation of thymol across peptidogylcan. We expect that the proposed MARTINI model for peptidoglycan will be useful in understanding phenomena associated with bacterial cell walls at larger length and time scales, thereby overcoming the current limitations of all-atom models.

Establishing an Electrostatics Paradigm for Membrane Electroporation in the Framework of Dissipative Particle Dynamics.

With an exclusive aim to looking into a mechanism of membrane electroporation on mesoscopic length and time scales, we report the dissipative particle dynamics (DPD) simulation results for systems with and without electrolytes. A polarizable DPD model of water is employed for accurate modeling of long-range electrostatics near the water-lipid interfaces. A great deal of discussion on field induced change in dipole moments of water and lipids together with the special variation of electric field is made in order to understand the dielectrophoretic movement of water, initiating a pore formation via an intrusion through the bilayer core. The presence of salt alters the dipolar arrangement of lipids and water, and thereby it reduces the external field required to create a pore in the membrane. The species fluxes through the pore, distributions for bead density, electrostatic potential, stresses across the membrane, etc. are used to answer some of the key questions pertaining to mechanism of electroporation. The findings are compared with the molecular dynamics simulation results found in the literature, and the comparison successfully establishes an electrostatics paradigm for biomembrane studies using DPD simulations.

Electroporation Using Dissipative Particle Dynamics with a Novel Protocol for Applying Electric Field.

In molecular dynamics simulations of membrane electroporation, the bilayer is subjected to an electric field E either by direct addition of a force f = qE on the charge-bearing species or by imposing an ion imbalance in the salt solutions on the two sides of the bilayer. The former is believed to mimic electroporation with high fields over nanosecond pulse period, during which the membrane is almost uncharged, especially in the low salt limit. Conversely, the ion imbalance method elucidates a low electric field-induced poration over a longer period of micro- to milliseconds with a fully charged membrane. Both these methods of applying electric field have disadvantages while investigating electroporation using dissipative particle dynamics (DPD) simulations. The method involving direct addition of force fails to address the presence of a nonuniform dielectric background for ions embedded in nonpolarizable DPD water and those found in the core of the bilayer. The ion imbalance method in DPD simulations suffers from its unavoidable use of a wall potential to prevent the movement of ions across the periodic boundaries. To address the above issues, we propose a simple method for imposing a desired transmembrane potential (TMV) by placing oppositely but uniformly charged plates on either side of the bilayer. Our DPD simulations demonstrate that the profiles for bead density, mechanical stress, electrical potential, as well as the transient responses in the dipole moment and species fluxes obtained from the proposed method utilizing charged plates are quite similar to those obtained using the ion imbalance method. The proposed protocol is free from the aforementioned drawbacks of the direct force addition and ion imbalance methods.

Electrostatic interactions in dissipative particle dynamics-Ewald-like formalism, error analysis, and pressure computation.

An accurate time evolution of charged species having exponentially smeared out charge density (Slater type charge distribution) in dissipative particle dynamic (DPD) simulations necessitates the optimal choice of the Ewald splitting parameter (α), charge smearing length (λ), and real space cutoff (c) when the Ewald summation or its variant such as particle-particle particle-mesh or particle-mesh Ewald is employed for long range electrostatics. The present article offers the error estimates in the electrostatic energy and the force as a function of α and ß(1/λ) on account of spherical truncation c in real space. These error estimate formulae are validated by our DPD simulation results. We also give here an Ewald-like derivation for electrostatic energy and force for the Slater type charge density. A quick estimate of the electrostatic pressure without the use of the tedious expression which involves three dimensional Fourier transforms is also presented, and its range of validity is discussed. The basis for the proposed formula for pressure is the fact that the minimum-image truncation in many cases allows one to compute the thermodynamic quantities with reasonable accuracy.