Atomistic Scale Effects of Lipopolysaccharide Modifications on Bacterial Outer Membrane Defenses

Rice, Amy; Wereszczynski, Jeff

doi:10.1016/j.bpj.2018.02.006

Cited by 40 publications

(65 citation statements)

References 77 publications

(99 reference statements)

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“…all of the simulations, large-scale bilayer properties were unaffected by binding of LL-37 and largely consistent with previous results, 8 as detailed in Supplementary Results and Table S1.…”

Section: Ll-37 Does Not Affect Bilayer Properties On the Microsecond supporting

confidence: 90%

Salmonella Membrane Structural Remodeling Increases Resistance to Antimicrobial Peptide LL-37

et al. 2019

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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.

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Section: Ll-37 Does Not Affect Bilayer Properties On the Microsecond supporting

confidence: 90%

Salmonella Membrane Structural Remodeling Increases Resistance to Antimicrobial Peptide LL-37

et al. 2019

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“…chemotype when the -PO − 4 and -PO 2− 4 cases are compared. Overall, these results indicate that, as hypothesized previously,19 lipid A modifications help stabilize the bilayer structure in the absence of divalent cations and may reduce reliance on these cations for stability.…”

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confidence: 83%

“…Figure S4); this difference was less pronounced in mLPS systems, likely due to the already increased ordering that palmitoylation confers. 19 Finally, increased inter-lipid hydrogen bonding was observed in LPS systems with the reduced phosphate charge, while the change in mLPS systems was not statistically significant for this metric.…”

Section: Resultsmentioning

confidence: 89%

“…The number of cations in the bulk solvent during the production portion of simulations is minimal for all systems studied ( Figure S6). 25 with modifications treated as described previously, 19 the C36 force fields for lipids, 40,41 modified Lennard-Jones parameters for sodium ion interactions with certain lipid oxygens, 42 and TIP3P water. 43 Lipid A phosphate groups with a charge of -1 were parameterized by analogy to the C36 general force field, 44 using methylphosphate as a template; the parameters used are given in Table S1.…”

Section: System Preparationmentioning

confidence: 99%

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Lipopolysaccharide Simulations are Sensitive to Phosphate Charge and Ion Parameterization

Rice

Rooney

Greenwood

et al. 2019

Preprint

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The high proportion of lipopolysaccharide (LPS) molecules in the outer membrane of Gram-negative bacteria make it a highly effective barrier to small molecules, antibiotic drugs, and other antimicrobial agents. Given this vital role in protecting bacteria from potentially hostile environments, simulations of LPS bilayers and outer membrane systems represent a critical tool for understanding the mechanisms of bacterial resistance and the development of new antibiotic compounds that circumvent these defenses. The basis of these simulations are parameterizations of LPS, which have been developed for all major molecular dynamics force fields. However, these parameterizations differ in both the protonation state of LPS as well as how LPS membranes behave in the presence of various ion species. To address these discrepancies and understand the effects of phosphate charge on bilayer properties, simulations were performed for multiple distinct LPS chemotypes with different ion parameterizations in both protonated or deprotonated lipid A states. These simulations show that bilayer properties, such as the area per lipid and inter-lipid hydrogen bonding, are highly influenced by the choice of phosphate group charges, cation type, and ion parameterization, with protonated LPS and monovalent cations with modified nonbonded parameters providing the best match to experiments. Additionally, alchemical free energy simulations were performed to determine theoretical pK a values for LPS, and subsequently validated by 31 P solid-state NMR experiments. Results from these complementary computational and experimental studies demonstrate that the protonated state dominates at physiological pH, contrary to the deprotonated form modeled by many LPS force fields.In all, these results highlight the sensitivity of LPS simulations to phosphate charge and ion parameters, while offering recommendations for how existing models should be updated for consistency between force fields as well as to best match experiments.

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“…The results showed that only 5% of the Xcc LOS molecules are released. In the outer membrane of gram negative bacteria the negatively charged LPS molecules cover most of the outer surface and divalent cations such as Mg 2+ and Ca 2+ are essential to neutralize this negative charge and allow strengthening of the lateral interactions between neighboring LPS molecules, which provides enhanced stability for the external bacterial membrane . Similar electrostatic interactions and effects such as increased hydrogen bonding and tighter lipid packing and cross‐linking exerted by divalent cation bridging can be expected to take place in the pathogen‐mimetic mNP–LOS nanostructures to provide the observed stability.…”

Section: Resultsmentioning

confidence: 99%

Cancer Immunotherapy of TLR4 Agonist–Antigen Constructs Enhanced with Pathogen‐Mimicking Magnetite Nanoparticles and Checkpoint Blockade of PD‐L1

Traini

Ruiz‐de‐Angulo

Blanco‐Canosa

et al. 2018

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Despite the tremendous potential of Toll‐like receptor 4 (TLR4) agonists in vaccines, their efficacy as monotherapy to treat cancer has been limited. Only some lipopolysaccharides (LPS) isolated from particular bacterial strains or structures like monophosphoryl lipid A (MPLA) derived from lipooligosaccharide (LOS), avoid toxic overactivation of innate immune responses while retaining adequate immunogenicity to act as adjuvants. Here, different LOS structures are incorporated into nanoparticle‐filled phospholipid micelles for efficient vaccine delivery and more potent cancer immunotherapy. The structurally unique LOS of the plant pathogen Xcc is incorporated into phospholipid micelles encapsulating iron oxide nanoparticles, producing stable pathogen‐mimicking nanostructures suitable for targeting antigen presenting cells in the lymph nodes. The antigen is conjugated via a hydrazone bond, enabling rapid, easy‐to‐monitor and high‐yield antigen ligation at low concentrations. The protective effect of these constructs is investigated against a highly aggressive model for tumor immunotherapy. The results show that the nanovaccines lead to a higher‐level antigen‐specific cytotoxic T lymphocyte (CTL) effector and memory responses, which when combined with abrogation of the immunosuppressive programmed death‐ligand 1 (PD‐L1), provide 100% long‐term protection against repeated tumor challenge. This nanovaccine platform in combination with checkpoint inhibition of PD‐L1 represents a promising approach to improve the cancer immunotherapy of TLR4 agonists.

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Atomistic Scale Effects of Lipopolysaccharide Modifications on Bacterial Outer Membrane Defenses

Cited by 40 publications

References 77 publications

Salmonella Membrane Structural Remodeling Increases Resistance to Antimicrobial Peptide LL-37

Salmonella Membrane Structural Remodeling Increases Resistance to Antimicrobial Peptide LL-37

Lipopolysaccharide Simulations are Sensitive to Phosphate Charge and Ion Parameterization

Cancer Immunotherapy of TLR4 Agonist–Antigen Constructs Enhanced with Pathogen‐Mimicking Magnetite Nanoparticles and Checkpoint Blockade of PD‐L1

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