Antimicrobial peptides are small, cationic proteins that can induce lysis of bacterial cells through interaction with their membranes. Different mechanisms for cell lysis have been proposed, but these models tend to neglect the role of the chemical composition of the membrane, which differs between bacterial species and can be heterogeneous even within a single cell. Moreover, the cell envelope of Gram-negative bacteria such as E. coli contains two membranes with differing compositions. To this end, we report the first molecular dynamics simulation study of the interaction of the antimicrobial peptide, polymyxin B1 with complex models of both the inner and outer membranes of E. coli. The results of >16 microseconds of simulation predict that polymyxin B1 is likely to interact with the membranes via distinct mechanisms. The lipopeptides aggregate in the lipopolysaccharide headgroup region of the outer membrane with limited tendency for insertion within the lipid A tails. In contrast, the lipopeptides readily insert into the inner membrane core, and the concomitant increased hydration may be responsible for bilayer destabilization and antimicrobial function. Given the urgent need to develop novel, potent antibiotics, the results presented here reveal key mechanistic details that may be exploited for future rational drug development.
Antimicrobial peptides (AMPs) are small cationic membrane-active peptides; they can be found in most living organisms and play an essential part in innate immunity [1–3]. These peptides exhibit broad-spectrum antimicrobial activity against bacteria, fungi and viruses, making them of great biomedical interest, particularly in the field of novel antibiotic design. Polymyxin B1 (PMB1) is a small antimicrobial lipopeptide first derived from the bacterial species Bacilus Polymyxa in 1947[2,4, 5]. It is composed of a cyclic polypeptide ring and a branched fatty acid tail, and among the amino acids forming the peptide segment are the irregular amino acids D-Phenylalanine (DPhe) and α, γ-Diamino Butyric acid (DAB). Its full sequence is thus: DABC-Thr-Leu-DPhe-DAB-DABC-DAB-Thr-DAB-CO (CH2) 4CH (CH3) CH2CH3, where DABC represents the cyclic linkage. The five non-cyclized DAB amino acids each carry a charge of +1, and thus the cationic peptide has a total charge of +5 [6]. PMB1 is a highly potent antimicrobial peptide and is selective predominantly towards all Gram-negative bacterial species, with the exception of the Proteus groups [7]. Unfortunately, treatment of patients with PMB1 has been shown to have adverse side effects on the renal and nervous system [8–10], and therefore clinical use of PMB1 has been limited to topical treatment as well as “last resort” therapy of patients infected with multidrug-resistant bacteria or with chronic conditions who suffer from recurring respiratory infections [11]. However, given the alarming rise in the number of bacterial strains exhibiting multidrug-resistance, there has recently been renewed interest in PMB1 [2]. PMB1 lipopolypeptides are known to permeate across the bacterial outer membrane (OM) by self-promoted uptake, while it is disruption of the inner membrane (IM) that subsequently leads to cell death. The peptides are thought to fulfil the initial stages of their bactericidal activity by anchoring themselves to the bacterial membrane via the DAB amino acids[12,13]. While the precise mode of action subsequent to peptide anchoring to the membrane is still unclear, it has been established that the polypeptide ring is responsible for causing an increased permeability of the bacterial membrane[4]. It has been proposed that the observed permeabilization is caused by membrane insertion of the polypeptide ring and fatty acid tail, resulting in bilayer disruption and an outflow of intracellular components, followed by cell death [4]. Because of experimental difficulties associated with characterizing dynamic, heterogeneous systems such as membrane-bound AMPs, molecular dynamics (MD) simulations provide a complementary approach to studying their modes of action, in unprecedented detail [14,15]. Here, we have used a series of MD simulations (Table 1) over microsecond timescales to study, the interaction of an AMP with accurate models of both membrane components of a Gram-negative bacterial cell, there has previously been only one report of a computational study of an AMP interacting with a model OM[16]. In particular, the former membrane is represented by a realistic mixture of phospholipids representative of the bacterial IM, whilst the latter is modelled as an asymmetric bilayer containing a phospholipid mixture in the inner leaflet and rough lipopolysaccharide (LPS) in the outer leaflet. For comparison, we also study the interactions of the AMP with a symmetric lipid A membrane. Computational work on AMPs is well documented [17–19] and simulations of complex models of the OM are also available [20–23], though we report one of the first combinations of the two aspects in atomistic detail. We thus investigate the molecular-level mechanisms of PMB1 binding, insertion, and bilayer disruption for both IM and OM models of the envelope of the archetypal Gram-negative bacterial species, E. coli.
To study the initial stages of AMP interaction with the envelope of Gram-negative bacteria, we simulated PMB1 in the presence of a realistic model of the asymmetric E. coli OM, composed of Re LPS in the outer leaflet and a mixture of phospholipids (including phosphatidylethanolamine, phosphatidylglycerol, and cardiolipin) in the inner leaflet. The molecular compositions of the simulated systems are given in Table 1. We performed simulations of simplified OM model systems, containing lipid A in both leaflets (Sim_LipA). The reasons for doing this are two-fold; firstly this setup more easily enables the peptides to permeate into the lipid part of the membrane, allowing interrogation of these membrane-peptide interactions over a tractable timescale, and secondly it abrogates the problem of peptides moving across periodic boundaries to interact with the phospholipid portion of the asymmetric bilayer. While the lack of sugars in lipid A provides a simplified model, it does enable us to study the behaviour of the peptide at the lipid headgroup/tail interface in realistic detail. Two independent simulation systems, each comprising eight PMB1 placed ~0. 5 nm above one leaflet of the membrane, were run for 3 microseconds. Here, we report only the behaviour that differs from the asymmetric bilayer studies described above. We next turned our attention to the E. coli IM. We performed simulations of a symmetrical phospholipid membrane model (composed of phosphatidylethanolamine, phosphatidylglycerol, and cardiolipin phospolipids), and exposed it to PMB1 lipopeptides (Table 1).
Our results reveal contrasting behaviour of PMB1 in the presence of different bacterial membrane models. In the case of the LPS simulations, our observations support data from previous fluorescence studies suggesting that DAB residues are key to PMB1 binding and antimicrobial function [12,13]. Strikingly, we observed aggregation of PMB1 on the LPS membrane surface (Fig 1 and S2 Fig), in which monomers arranged themselves in a micelle-like conformation to bury their hydrophobic tails from the polar sugar rings. This aggregation, coupled with the tendency for PMB1 to cross-link the sugar hydroxyl groups resulted in formation of an immobile, “protein membrane cluster”. The adoption of this fatty acid tail orientation and aggregation may represent a necessary prelude to pore formation, in a manner that reduces the energetic cost for translocation of the PMB1 “micelle” across the polar environment of the LPS sugar groups. Intriguingly, two attempts to speed-up the process of PMB1 insertion into the outer membrane firstly by loosening the membrane by replacing the LPS cross-linking divalent cations with monovalent ions, and secondly by performing steered MD simulations to' pull' a peptide, also provided strong support for the hypothesis that insertion of PMB1 into the outer membrane is not a facile molecular process. Because of the slow dynamics of the LPS system [23], we also studied a simplified OM model consisting only of lipid A. Unlike the LPS system, PMB1 appeared to have no effect upon intra-lipid A hydrogen bonding, but instead the DAB residues interacted with phosphate groups, pushing them apart, leading to local membrane deformation and creating a region of reduced membrane-surface charge density that may no longer inhibit fatty acid tail insertion. During this process, Mg2+ ions were intermittently displaced and rebound to phosphate groups, but no long-term ion displacement was observed. As a result, we only witnessed complete insertion of a single PMB1 molecule on the timescales feasible with atomistic simulations. Nevertheless, it has been widely documented that divalent cations are essential for maintenance of OM stability, and PMB1 has been shown to cause destabilisation via displacement of these divalent cations [6,31,32]. For the IM model, our results are again in agreement with previous experimental work [12,13,26,33] highlighting the importance of the DAB residues in protein binding, confirming that electrostatic interactions between the DAB and the lipid headgroups are the initial driving force for PMB1 adsorption. However, in contrast with the OM, the distinguishing behaviour of the PMB1 molecules in the IM system was the lack of PMB1 aggregation as well as the striking result that the fatty acid tails of every PMB1 molecule spontaneously penetrated into the hydrophobic core of the membrane, some as far as the lower leaflet (Fig 3). Unlike the OM models, the IM exhibited a large increase in the disorder of the acyl tails upon fatty acid tail insertion. Indeed, PMB1 insertion seemed to be driven largely by hydrophobic interactions, not only due to fatty acid tails but also the D-Phe sidechain. The converged, inserted PMB1 conformation (S4 Fig) structurally resembles that observed in NMR studies performed by Mares et al [26] which focused on the interaction between LPS and PMB1. In both cases, the majority of the ring structure remained bound to the phospholipid headgroups on the membrane surface, while the hydrophobic portion of the ring (residues D-Phe and Leu) adopted an embedded orientation. In the case of the IM, we also observed a further possible role of the DAB residues in antimicrobial action, namely attraction of water towards the membrane core. Unlike the OM models, upon loss of the surface-bound state and PMB1-membrane insertion, the lipid lateral diffusion rate increased beyond the PMB1-free rate, correlating with peptide/water membrane penetration. Concomitant with this, we observed a remarkable thinning effect of >0. 7 nm and increase in area per lipid in the IM as a result of PMB1 insertion, whilst the resultant cross-leaflet interdigitation of acyl tails is consistent with data from spin-labelling and X-ray diffraction reported by Boggs et al [29] and Theretz et al [30]. Since the PMB1 does not aggregate within the IM, but instead inserts as monomers, we may hypothesise on the basis of our collective observations that PMB1 disrupts the IM not through traditional mechanisms of pore formation but through membrane insertion, bilayer thinning, and water penetration. Such effects are certainly indicative of AMP permeation and the early stages of membrane disruption [34]. The limitations of the current study arise from the slow lateral diffusion of LPS, such that the long simulation times required to witness complete PMB1 translocation within the asymmetric LPS membrane is not presently feasible with atomistic simulations. Indeed it has recently been shown that even for simple, phospholipid bilayers, the simulation times required for convergence have historically been seriously underestimated [35]). The slow reorganization of ionic interactions involving zwitterionic phospholipid headgroups when solutes penetrate the lipid-water interface are a particular problem, which is accentuated in the complex LPS headgroups we are studying. Other studies have suggested that atomistic molecular dynamics simulations of AMPs require multi-microsecond timescales [36]. Our simulations reported here show that even non-equilibrium methods such as steered MD are not able to access insertion behaviour into membranes and thus another approach is needed. The large molecular systems and extended timescales accessible to coarse-grain molecular dynamics (CG-MD) simulations provide an alternative and complementary route to studying antimicrobial peptides. Indeed, CG-MD studies have been shown to provide insights into the action of antimicrobial peptides in flat lipid bilayers and spherical vesicles [15,37,38]. To study the OM of Gram-negative bacteria using this approach, a coarse-grain model of LPS is urgently needed. Nevertheless, the simulations we have performed have helped to identify the initial stages of PMB1 action on the OM of Gram-negative bacteria, and in particular highlight the regions of the peptide that form strongest interactions with the LPS molecules of the outer membrane. In future, a multiscale simulation approach based on these studies may provide further insights regarding mechanism of action. In conclusion, in this study we have shed light on the potential mechanisms for bacterial envelope disruption by PMB1. The aggregation witnessed in the OM models is suggestive of the possible early stages of self-regulated translocation / pore formation, whilst the fatty acid tail insertion in the lipid A environment also appears to be dependent upon aggregation in order to create a charge-free area to allow PMB1 penetration, providing further evidence for a pore model of self-regulated uptake. We may speculate that a ‘ladder’ type mechanism occurs in the context of the full LPS membrane, with the PMB1 molecules initially aggregating on the surface, prior to further penetration through the sugar region by the DAB residues, subsequently disrupting the high surface charge density of the counterion-cross-linked lipid A moieties and resulting in fatty acid tail penetration within the hydrophobic membrane core. A stepwise process for PMB1 disruption of the IM has also been established, beginning with DAB-based adsorption and followed by rapid fatty acid tail insertion within the bilayer, supported by the hydrophobic D-Phe. In this case, deep penetration of monomeric PMB1 molecules enables the DAB residues to drag water into the membrane, suggesting an alternative antimicrobial mechanism for IM destabilisation. Nevertheless, it is possible that other mechanisms (e. g. carpet model) may apply at higher concentrations of PMB1.
All simulations systems are summarised in Table 1. All simulations performed in this work used the GROMACS molecular dynamics software [53,54], version 4. 5. 1 [55]. Standard parameters taken from the GROMOS 53A6 force field [56] were used to model the polymyxin B1 molecule in its fully ionized state. The parameters for the LPS molecules were as described and used previously [27,57] and the GROMOS-CKP (Chandrasekhar[58]-Kukol[59]-Piggot[27,60]) parameters were used for the phospholipids. The SPC water model [61] was used in all simulations. During the simulations, the LPS, phospholipids and solvent (water plus counterions) were maintained at a constant temperature of 313 K using the Nosé-Hoover thermostat [62,63] with a time constant of 0. 5 ps. The only exception being the lipid A bilayer simulations which were performed at a temperature of 323 K. These temperatures were chosen as they are above the gel to liquid crystal phase transition temperatures of all the lipids used in the simulations [64–68]. A pressure of 1 bar was maintained using anisotropic pressure coupling with the Parrinello-Rhaman barostat [69,70] and a time constant of 5 ps. Electrostatic interactions were treated using the smooth particle mesh Ewald (PME) algorithm [71] with a short-range cutoff of 0. 9 nm. The van der Waals interactions were truncated at 1. 4 nm with a long-range dispersion correction applied to the energy and pressure. The neighbor list was updated every five steps during the simulations. All bonds were constrained using the P-LINCS algorithm [72] allowing a 2 fs time step to be applied. All LPS-containing membrane systems were neutralized with Mg2+ ions, whereas the inner membrane model was neutralized with Na+ ions.
Antimicrobial peptides have the ability to kill harmful bacteria through interaction with bacterial membranes. This manuscript describes the first reported computational study of antimicrobial peptide interaction with both membranes of a Gram-negative bacterium. While antimicrobial peptides have been the topic of many simulation studies, these studies have not incorporated the biochemical heterogeneity of natural membranes. Our simulations add the missing biochemical details and in doing so, reveal that the mechanisms of interaction of polymyxin B1 with the inner and outer membranes of E. coli, are really rather different. The peptides insert readily into the inner membrane, whereas the interaction with the LPS-containing outer membrane is more complex. In summary our results represent a key finding for future drug development that targets these bacteria.
Interaction of the Antimicrobial Peptide Polymyxin B1 with Both Membranes of E. coli: A Molecular Dynamics Study