Lipopolysaccharides (LPS) are a main constituent of the outer membrane of Gram-negative bacteria. Salmonella enterica, like many other bacterial species, are able to chemically modify the structure of their LPS molecules through the PhoPQ pathway as a defense mechanism against the host immune response. These modifications make the outer membrane more resistant to antimicrobial peptides (AMPs), large lipophilic drugs, and cation depletion, and are crucial for survival within a host organism. It is believed that these LPS modifications prevent the penetration of large molecules and AMPs through a strengthening of lateral interactions between neighboring LPS molecules. Here, we performed a series of long-timescale molecular dynamics simulations to study how each of three key S. enterica lipid A modifications affect bilayer properties, with a focus on membrane structural characteristics, lateral interactions, and the divalent cation bridging network. Our results discern the unique impact each modification has on strengthening the bacterial outer membrane through effects such as increased hydrogen bonding and tighter lipid packing. Additionally, one of the modifications studied shifts Ca from the lipid A region, replacing it as a major cross-linking agent between adjacent lipids and potentially making bacteria less susceptible to AMPs that competitively displace cations from the membrane surface. These results further improve our understanding of outer membrane chemical properties and help elucidate how outer membrane modification systems, such as PhoPQ in S. enterica, are able to alter bacterial virulence.
Antimicrobial peptides (AMPs) are key components of the innate immune response and represent promising templates for the development of broad-spectrum alternatives to conventional antibiotics. Most AMPs are short, cationic peptides that interact more strongly with negatively charged prokaryotic membranes than net neutral eukaryotic ones. Both AMPs and synthetic analogues with arginine-like side chains are more active against bacteria than those with lysine-like amine groups, though the atomistic mechanism for this increase in potency remains unclear. To examine this, we conducted comparative molecular dynamics simulations of a model negatively-charged membrane system interacting with two mutants of the AMP KR-12: one with lysine residues mutated to arginines (R-KR12) and one with arginine residues mutated to lysine (K-KR12). Simulations show that both partition analogously to the bilayer and display similar preferences for hydrogen bonding with the anionic POPGs. However, R-KR12 binds stronger to the bilayer than K-KR12 and forms significantly more hydrogen bonds, leading to considerably longer interaction times. Additional simulations with methylated R-KR12 and charge-modified K-KR12 mutants show that the extensive interaction seen in the R-KR12 system is partly due to arginines strong atomic charge distribution, rather than being purely an effect of the greater number of hydrogen bond donors. Finally, free energy simulations reveal that both peptides are disordered in solution but form an amphipathic α-helix when inserted into the bilayer headgroup region. Overall, these results highlight the role of charge and hydrogen bond strength in peptide bilayer insertion, and offer potential insights for designing more potent analogues in the future.
Gram-negative bacteria are protected from their environment by an outer membrane that is primarily composed of lipopolysaccharides (LPSs). Under stress, pathogenic serotypes of Salmonella enterica remodel their LPSs through the PhoPQ two-component regulatory system that increases resistance to both conventional antibiotics and antimicrobial peptides (AMPs). Acquired resistance to AMPs is contrary to the established narrative that AMPs circumvent bacterial resistance by targeting the general chemical properties of membrane lipids. However, the specific mechanisms underlying AMP resistance remain elusive. Here we report a 2-fold increase in bacteriostatic concentrations of human AMP LL-37 for S. enterica with modified LPSs. LPSs with and without chemical modifications were isolated and investigated by Langmuir films coupled with grazing-incidence X-ray diffraction (GIXD) and specular X-ray reflectivity (XR). The initial interactions between LL-37 and LPS bilayers were probed using all-atom molecular dynamics simulations. These simulations suggest that initial association is nonspecific to the type of LPS and governed by hydrogen bonding to the LPS outer carbohydrates. GIXD experiments indicate that the interactions of the peptide with monolayers reduce the number of crystalline domains but greatly increase the typical domain size in both LPS isoforms. Electron densities derived from XR experiments corroborate the bacteriostatic values found in vitro and indicate that peptide intercalation is reduced by LPS modification. We hypothesize that defects at the liquid-ordered boundary facilitate LL-37 intercalation into the outer membrane, whereas PhoPQ-mediated LPS modification protects against this process by having innately increased crystallinity. Since induced ordering has been observed with other AMPs and drugs, LPS modification may represent a general mechanism by which Gram-negative bacteria protect against host innate immunity.
Small-angle X-ray scattering (SAXS) has become an increasingly popular technique for characterizing the solution ensemble of flexible biomolecules. However, data resulting from SAXS is typically low-dimensional and is therefore difficult to interpret without additional structural knowledge. In theory, molecular dynamics (MD) trajectories can provide this information, but conventional simulations rarely sample the complete ensemble. Here, we demonstrate that accelerated MD simulations can be used to produce higher quality models in shorter time scales than standard simulations, and we present an iterative Bayesian Monte Carlo method that is able to identify multistate ensembles without overfitting. This methodology is applied to several ubiquitin trimers to demonstrate the effect of linkage type on the solution states of the signaling protein. We observe that the linkage site directly affects the solution flexibility of the trimer and theorize that this difference in plasticity contributes to their disparate roles in vivo.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.