Gram-negative Bacterial Outer Membrane Research Articles

Reaction-Induced Self-Assembly of Polymyxin Mitigates Cytotoxicity and Reverses Drug Resistance.

Polymyxins have been regarded as an efficient therapeutic against many life-threatening, multidrug resistant Gram-negative bacterial infections; however, the cytotoxicity and emergence of drug resistance associated with polymyxins have greatly hindered their clinical potential. Herein, the reaction-induced self-assembly (RISA) of polymyxins and natural aldehydes in aqueous solution is presented. The resulting assemblies effectively mask the positively charged nature of polymyxins, reducing their cytotoxicity. Moreover, the representative PMBA4 (composed of polymyxin B (PMB) and (E)-2-heptenal (A4)) assemblies demonstrate enhanced binding to Gram-negative bacterial outer membranes and exhibit multiple antimicrobial mechanisms, including increased membrane permeability, elevated bacterial metabolism, suppression of quorum sensing, reduced ATP synthesis, and potential reduction of bacterial drug resistance. Remarkably, PMBA4 assemblies reverse drug resistance in clinically isolated drug-resistant strains of Gram-negative bacteria, demonstrating exceptional efficacy in preventing and eradicating bacterial biofilms. PMBA4 assemblies efficiently eradicate Gram-negative bacterial biofilm infections in vivo and alleviate inflammatory response. This RISA strategy offers a practical and clinically applicable approach to minimize side effects, reverse drug resistance, and prevent the emergence of resistance associated with free polymyxins.

Arginine-linked HPV-associated E7 displaying bacteria-derived outer membrane vesicles as a potent antigen-specific cancer vaccine

BackgroundBacteria-based cancer therapy have demonstrated innovative strategies to combat tumors. Recent studies have focused on gram-negative bacterial outer membrane vesicles (OMVs) as a novel cancer immunotherapy strategy due to its intrinsic properties as a versatile carrier.MethodHere, we developed an Human Papillomavirus (HPV)-associated E7 antigen displaying Salmonella-derived OMV vaccine, utilizing a Poly(L-arginine) cell penetrating peptide (CPP) to enhance HPV16 E7 (aa49-67) H-2 Db and OMV affinity, termed SOMV-9RE7.ResultsDue to OMV’s intrinsic immunogenic properties, SOMV-9RE7 effectively activates adaptive immunity through antigen-presenting cell uptake and antigen cross-presentation. Vaccination of engineered OMVs shows immediate tumor suppression and recruitment of infiltrating tumor-reactive immune cells.ConclusionThe simplicity of the arginine coating strategy boasts the versatility of immuno-stimulating OMVs that can be broadly implemented to personalized bacterial immunotherapeutic applications.

β-lactam-induced OMV release promotes polymyxin tolerance in Salmonella enterica sv. Typhi.

The rise of multidrug-resistant bacteria is a global concern, leading to a renewed reliance on older antibiotics like polymyxins as a last resort. Polymyxins, cationic cyclic peptides synthesized nonribosomally, feature a hydrophobic acyl tail and positively charged residues. Their antimicrobial mechanism involves initial interaction with Gram-negative bacterial outer-membrane components through polar and hydrophobic interactions. Outer membrane vesicles (OMVs), nano-sized proteoliposomes secreted from the outer membrane of Gram-negative bacteria, play a crucial role in tolerating harmful molecules, including cationic peptides such as polymyxins. Existing literature has documented environmental changes' impact on modulating OMV properties in Salmonella Typhimurium. However, less information exists regarding OMV production and characteristics in Salmonella Typhi. A previous study in our laboratory showed that S. Typhi ΔmrcB, a mutant associated with penicillin-binding protein (PBP, a β-lactam antibiotic target), exhibited hypervesiculation. Consequently, this study investigated the potential impact of β-lactam antibiotics on promoting polymyxin tolerance via OMVs in S. Typhi. Our results demonstrated that sub-lethal doses of β-lactams increased bacterial survival against polymyxin B in S. Typhi. This phenomenon stems from β-lactam antibiotics inducing hypervesiculation of OMVs with higher affinity for polymyxin B, capturing and diminishing its biologically effective concentration. These findings suggest that β-lactam antibiotic use may inadvertently contribute to decreased polymyxin effectivity against S. Typhi or other Gram-negative bacteria, complicating the effective treatment of infections caused by these pathogens. This study emphasizes the importance of evaluating the influence of β-lactam antibiotics on the interaction between OMVs and other antimicrobial agents.

Characterization of a Myeloid Differentiation Factor 2-Derived Peptide that Facilitates THP-1 Macrophage-Mediated Phagocytosis of Gram-Negative Bacteria.

Myeloid differentiation factor 2 (MD2), the TLR4 coreceptor, has been shown to possess opsonic activity and has been implicated in phagocytosis and intracellular killing of Gram-negative bacteria. However, any MD2 protein segment involved in phagocytosis of Gram-negative bacteria is not yet known. A short synthetic MD2 segment, MD54 (amino acid regions 54 to 69), was shown to interact with a Gram-negative bacterial outer membrane component, LPS, earlier. Furthermore, the MD54 peptide induced aggregation of LPS and facilitated its internalization in THP-1 cells. Currently, it has been investigated if MD2-derived MD54 possesses any opsonic property and role in phagocytosis of Gram-negative bacteria. Remarkably, we observed that MD54 facilitated agglutination of Gram-negative bacteria, Escherichia coli (ATCC 25922) and Pseudomonas aeruginosa (ATCC BAA-427), but not of Gram-positive bacteria, Bacillus subtilis (ATCC 6633) and Staphylococcus aureus (ATCC 25923). The MD54-opsonized Gram-negative bacteria internalized within PMA-treated THP-1 cells and were killed over a longer incubation period. However, both internalization and intracellular killing of the MD54-opsonized Gram-negative bacteria within THP-1 phagocytes were appreciably inhibited in the presence of a phagocytosis inhibitor, cytochalasin D. Furthermore, MD54 facilitated the clearance of Gram-negative bacteria E. coli (ATCC 25922) and P. aeruginosa (ATCC BAA-427) from the infected BALB/c mice whereas an MD54 analog, MMD54, was inactive. Overall, for the first time, the results revealed that a short MD2-derived peptide can specifically agglutinate Gram-negative bacteria, act as an opsonin for these bacteria, and facilitate their phagocytosis by THP-1 phagocytes. The results suggest that the MD54 segment could have a crucial role in MD2-mediated host-pathogen interaction involving the Gram-negative bacteria.

Structural Modeling of T9SS Outer Membrane Proteins and Their Complexes.

The type 9 secretion system (T9SS) is a recently discovered machinery that both transports cargo proteins across the Gram-negative bacterial outer membrane and attaches them to lipopolysaccharides on the extracellular surface. Outer membrane proteins (OMPs) are key components of the T9SS and are involved in both steps. In this chapter, we describe a method for the in silico modeling of T9SS OMPs and their complexes, and model validation. This is useful when the production of recombinant OMPs is difficult, and these protocols can also be applied to OMP complexes outside of the T9SS.

Proteomic profiling of membrane vesicles from Mycobacterium avium subsp. paratuberculosis: Navigating towards an insilico design of a multi-epitope vaccine targeting membrane vesicle proteins

Bacteria typically produce membrane vesicles (MVs) at varying levels depending on the surrounding environments. Gram-negative bacterial outer membrane vesicles (OMVs) have been extensively studied for over 30 years, but MVs from Gram-positive bacteria only recently have been a focus of research. In the present study, we isolated MVs from Mycobacterium avium subsp. paratuberculosis (MAP) and analyzed their protein composition using LC-MS/MS. A total of 316 overlapping proteins from two independent preparations were identified in our study, and topology prediction showed these cargo proteins have different subcellular localization patterns. When MVs were administered to bovine-derived macrophages, significant up-regulation of pro-inflammatory cytokines was observed via qRT-PCR. Proteome functional annotation revealed that many of these proteins are involved in the cellular protein metabolic process, tRNA aminoacylation, and ATP synthesis. Secretory proteins with high antigenicity and adhesion capability were mapped for B-cell and T-cell epitopes. Antigenic, Immunogenic and IFN-γ inducing B-cell, MHC-I, and MHC-II epitopes were stitched together through linkers to form multi-epitope vaccine (MEV) construct against MAP. Strong binding energy was observed during the docking of the 3D structure of the MEV with the bovine TLR2, suggesting that the putative MEV may be a promising vaccine candidate against MAP. However, in vitro and in vivo analysis is required to prove the immunogenic concept of the MEV which we will follow in our future studies. SignificanceJohne's disease is a chronic infection caused by Mycobacterium avium subsp. paratuberculosis that has a potential link to Crohn's disease in humans. The disease is characterized by persistent diarrhea and enteritis, resulting in significant economic losses due to reduced milk yield and premature culling of infected animals. The dairy industry in the United States alone experiences losses of approximately USD 250 million due to Johne's disease. The current vaccine against Johne's disease is limited by several factors, including variable efficacy, limited duration of protection, interference with diagnostic tests, inability to prevent infection, and logistical and cost-related challenges. Nevertheless, a multiepitope vaccine design approach targeting M. avium subsp. paratuberculosis has the potential to overcome these challenges and offer improved protection against Johne's disease.

An attacin antimicrobial peptide, Hill_BB_C10074, from Hermetia illucens with anti-Pseudomonas aeruginosa activity

BackgroundThere is a global need to develop new therapies to treat infectious diseases and tackle the rise in antimicrobial resistance. To date, the larvae of the Black Solider Fly, Hermetia illucens, have the largest repertoire of antimicrobial peptides derived from insects. Antimicrobial peptides are of particular interest in the exploration of alternative antimicrobials due to their potent action and reduced propensity to induce resistance compared with more traditional antibiotics.ResultsThe predicted attacin from H. illucens, Hill_BB_C10074, was first identified in the transcriptome of H. illucens populations that had been fed a plant-oil based diet. In this study, recombinant Hill_BB_C10074 (500 µg/mL), was found to possess potent antimicrobial activity against the serious Gram-negative pathogen, Pseudomonas aeruginosa. Sequence and structural homology modelling predicted that Hill_BB_C10074 formed a homotrimeric complex that may form pores in the Gram-negative bacterial outer membrane. In vitro experiments defined the antimicrobial action of Hill_BB_C10074 against P. aeruginosa and transmission electron microscopy and electrochemical impedance spectroscopy confirmed the outer membrane disruptive power of Hill_BB_C10074 which was greater than the clinically relevant antibiotic, polymyxin B.ConclusionsCombining predictive tools with in vitro approaches, we have characterised Hill_BB_C10074 as an important insect antimicrobial peptide and promising candidate for the future development of clinical antimicrobials.

Transformable nano-antibiotics for mechanotherapy and immune activation against drug-resistant Gram-negative bacteria.

The dearth of antibiotic candidates against Gram-negative bacteria and the rise of antibiotic resistance create a global health concern. The challenge lies in the unique Gram-negative bacterial outer membrane that provides the impermeable barrier for antibiotics and sequesters antigen presentation. We designed a transformable nano-antibiotics (TNA) that can transform from nontoxic nanoparticles to bactericidal nanofibrils with reasonable rigidity (Young's modulus, 21.6 ± 5.9 MPa) after targeting β-barrel assembly machine A (BamA) and lipid polysaccharides (LPSs) of Gram-negative bacteria. After morphological transformation, the TNA can penetrate and damage the bacterial envelope, disrupt electron transport and multiple conserved biosynthetic and metabolic pathways, burst bacterial antigen release from the outer membrane, and subsequently activate the innate and adaptive immunity. TNA kills Gram-negative bacteria in vitro and in vivo with undetectable resistance through multiple bactericidal modes of action. TNA treatment-induced vaccination results in rapid and long-lasting immune responses, protecting against lethal reinfections.

PepDRED: De Novo Peptide Design with Strong Binding Affinity for Target Protein.

De novo design of peptides that bind specifically to functional proteins is beneficial for diagnostics and therapeutics. However, complex permutations and combinations of amino acids pose significant challenges to the rational design of peptides with desirable stability and affinity. Herein, we develop a computational-based evolution method, namely, peptidomimetics-driven recognition elements design (PepDRED), to derive hemoglobin-inspired peptidomimetics. PepDRED mimics the natural evolutionism pipeline to generate stable apovariant (AVs) structures for wild-type counterparts via automated point mutations and validates their efficiency through free binding energy analysis and per residue energy decomposition analysis. For application demonstration, we applied PepDRED to design de novo peptides to bind FhuA, a typical TonB-dependent transporter (TBDT). TBDTs are Gram-negative bacterial outer membrane proteins responsible for iron transport and vital for bacterial resistance. PepDRED generated a pool of AVs and proceeded to reach an optimized peptide, AV440, with a remarkable binding affinity of -21 kcal/mol. AV440 is ∼2.5-fold stronger than the existing FhuA inhibitor Microcin J25. Network energy analysis further unveils that incorporating methionine (M42) in the N-terminal region significantly enhances inter-residue contacts and binding affinity. PepDRED offers a prompt and efficient in silico approach to develop potent peptide candidates for target proteins.

Mass-produced gram-negative bacterial outer membrane vesicles activate cancer antigen-specific stem-like CD8+ T cells which enables an effective combination immunotherapy with anti-PD-1.

Despite the capability of extracellular vesicles (EVs) derived from Gram-negative and Gram-positive bacteria to induce potent anti-tumour responses, large-scale production of bacterial EVs remains as a hurdle for their development as novel cancer immunotherapeutic agents. Here, we developed manufacturing processes for mass production of Escherichia coli EVs, namely, outer membrane vesicles (OMVs). By combining metal precipitation and size-exclusion chromatography, we isolated 357mg in total protein amount of E. coli OMVs, which was equivalent to 3.93 × 1015 particles (1.10 × 1010 particles/μg in total protein amounts of OMVs) from 160 L of the conditioned medium. We show that these mass-produced E. coli OMVs led to complete remission of two mouse syngeneic tumour models. Further analysis of tumour microenvironment in neoantigen-expressing tumour models revealed that E. coli OMV treatment causes increased infiltration and activation of CD8+ T cells, especially those of cancer antigen-specific CD8+ T cells with high expression of TCF-1 and PD-1. Furthermore, E. coli OMVs showed synergistic anti-tumour activity with anti-PD-1 antibody immunotherapy, inducing substantial tumour growth inhibition and infiltration of activated cancer antigen-specific stem-like CD8+ T cells into the tumour microenvironment. These data highlight the potent anti-tumour activities of mass-produced E. coli OMVs as a novel candidate for developing next-generation cancer immunotherapeutic agents.

The role and mechanisms of gram-negative bacterial outer membrane vesicles in inflammatory diseases.

Outer membrane vesicles (OMVs) are spherical, bilayered, and nanosized membrane vesicles that are secreted from gram-negative bacteria. OMVs play a pivotal role in delivering lipopolysaccharide, proteins and other virulence factors to target cells. Multiple studies have found that OMVs participate in various inflammatory diseases, including periodontal disease, gastrointestinal inflammation, pulmonary inflammation and sepsis, by triggering pattern recognition receptors, activating inflammasomes and inducing mitochondrial dysfunction. OMVs also affect inflammation in distant organs or tissues via long-distance cargo transport in various diseases, including atherosclerosis and Alzheimer's disease. In this review, we primarily summarize the role of OMVs in inflammatory diseases, describe the mechanism through which OMVs participate in inflammatory signal cascades, and discuss the effects of OMVs on pathogenic processes in distant organs or tissues with the aim of providing novel insights into the role and mechanism of OMVs in inflammatory diseases and the prevention and treatment of OMV-mediated inflammatory diseases.

Skewing of UV-induced cell death pathways using TLR4 ligands

Abstract Toll-like receptor 4 (TLR4) is a canonical innate immune receptor for the Gram-negative bacterial outer membrane component lipopolysaccharide (LPS). TLR4 deficiency has been shown to contribute to UV-induced carcinogenesis in epidermal cells. UV-induced cellular damage results in apoptosis which is characterized by a non-inflammatory cell death through the cleavage and activation of caspase-3. Utilizing bacterial enzyme combinational chemistry (BECC) we generated hypoacylated lipid A moieties from Escherichia coli, Pseudomonas aeruginosa, and Salmonella typhimurium which have moderately attenuated immune-stimulatory capacity. Continuously exposing TLR4 to these innate immune ligands leads to tolerization and decreased expression of the receptor in mammalian cells. We hypothesize that skewing cell death away from apoptosis using lipid A mimetics to lower TLR4 expression prior to UV exposure will reduce carcinogenic potential. To analyze the roll of TLR4 in UV-induced carcinogenesis, wildtype and TLR4-deficient bone marrow-derived macrophage (BMDM) cell lines were tolerized with hexa-acylated or penta-acylated lipid A from multiple Gram-negative species and exposed to either 10 or 25 mJ/cm 2doses of UVB radiation. UV-irradiated BMDM cells show evidence of apoptosis including cleavage of DNA creating a ladder, whereas UV-irradiated BMDM cells tolerized with lipid A mimetics show a lack of apoptosis. The data suggests that tolerization of TLR4 using canonical ligands may be a useful tool in promoting DNA repair and maintaining immune responses following UV-induced damage. The research was supported by a Fisher College of Science and Mathematics Undergraduate Research Grant.

Insights into the Binding Mode of Lipid A to the Anti-lipopolysaccharide Factor ALFPm3 from Penaeus monodon: An In Silico Study through MD Simulations

The globally expanding threat of antibiotic resistance calls for the development of new strategies for abating Gram-negative bacterial infections. The use of extracorporeal blood cleansing devices with affinity sorbents to selectively capture bacterial lipopolysaccharide (LPS), which is the major constituent of Gram-negative bacterial outer membranes and the responsible agent for eliciting an exacerbated innate immune response in the host during infection, has received outstanding interest. For that purpose, molecules that bind tightly to LPS are required to functionalize the affinity sorbents. Particularly, anti-LPS factors (ALFs) are promising LPS-sequestrating molecules. Hence, in this work, molecular dynamics (MD) simulations are used to investigate the interaction mechanism and binding pose of the ALF isoform 3 from Penaeus monodon (ALFPm3), which is referred to as "AL3" for the sake of simplicity, and lipid A (LA, the component of LPS that represents its endotoxic principle). We concluded that hydrophobic interactions are responsible for AL3-LA binding and that LA binds to AL3 within the protein cavity, where it buries its aliphatic tails, whereas the negatively charged phosphate groups are exposed to the medium. AL3 residues that are key for its interaction with LA were identified, and their conservation in other ALFs (specifically Lys39 and Tyr49) was also analyzed. Additionally, based on the MD-derived results, we provide a picture of the possible AL3-LA interaction mechanism. Finally, an in vitro validation of the in silico predictions was performed. Overall, the insights gained from this work can guide the design of novel therapeutics for treating sepsis, since they may be significantly valuable for designing LPS-sequestrating molecules that could functionalize affinity sorbents to be used for extracorporeal blood detoxification.

LPS-aggregating proteins GBP1 and GBP2 are each sufficient to enhance caspase-4 activation both in cellulo and in vitro

The gamma-interferon (IFNγ)-inducible guanylate-binding proteins (GBPs) promote host defense against gram-negative cytosolic bacteria in part through the induction of an inflammatory cell death pathway called pyroptosis. To activate pyroptosis, GBPs facilitate sensing of the gram-negative bacterial outer membrane component lipopolysaccharide (LPS) by the noncanonical caspase-4 inflammasome. There are seven human GBP paralogs, and it is unclear how each GBP contributes to LPS sensing and pyroptosis induction. GBP1 forms a multimeric microcapsule on the surface of cytosolic bacteria through direct interactions with LPS. The GBP1 microcapsule recruits caspase-4 to bacteria, a process deemed essential for caspase-4 activation. In contrast to GBP1, closely related paralog GBP2 is unable to bind bacteria on its own but requires GBP1 for direct bacterial binding. Unexpectedly, we find that GBP2 overexpression can restore gram-negative-induced pyroptosis in GBP1KO cells, without GBP2 binding to the bacterial surface. A mutant of GBP1 that lacks the triple arginine motif required for microcapsule formation also rescues pyroptosis in GBP1KO cells, showing that binding to bacteria is dispensable for GBPs to promote pyroptosis. Instead, we find that GBP2, like GBP1, directly binds and aggregates "free" LPS through protein polymerization. We demonstrate that supplementation of either recombinant polymerized GBP1 or GBP2 to an invitro reaction is sufficient to enhance LPS-induced caspase-4 activation. This provides a revised mechanistic framework for noncanonical inflammasome activation where GBP1 or GBP2 assembles cytosol-contaminating LPS into a protein-LPS interface for caspase-4 activation as part of a coordinated host response to gram-negative bacterial infections.

Bacterial Outer Membrane Vesicles Promote Lung Inflammatory Responses and Macrophage Activation via Multi-Signaling Pathways.

Emerging evidence suggests that Gram-negative bacteria release bacterial outer membrane vesicles (OMVs) and that these play an important role in the pathogenesis of bacterial infection-mediated inflammatory responses and organ damage. Despite the fact that scattered reports have shown that OMVs released from Gram-negative bacteria may function via the TLR2/4-signaling pathway or induce pyroptosis in macrophages, our study reveals a more complex role of OMVs in the development of inflammatory lung responses and macrophage pro-inflammatory activation. We first confirmed that various types of Gram-negative bacteria release similar OMVs which prompt pro-inflammatory activation in both bone marrow-derived macrophages and lung alveolar macrophages. We further demonstrated that mice treated with OMVs via intratracheal instillation developed significant inflammatory lung responses. Using mouse inflammation and autoimmune arrays, we identified multiple altered cytokine/chemokines in both bone marrow-derived macrophages and alveolar macrophages, suggesting that OMVs have a broader spectrum of function compared to LPS. Using TLR4 knock-out cells, we found that OMVs exert more robust effects on activating macrophages compared to LPS. We next examined multiple signaling pathways, including not only cell surface antigens, but also intracellular receptors. Our results confirmed that bacterial OMVs trigger both surface protein-mediated signaling and intracellular signaling pathways, such as the S100-A8 protein-mediated pathway. In summary, our studies confirm that bacterial OMVs strongly induced macrophage pro-inflammatory activation and inflammatory lung responses via multi-signaling pathways. Bacterial OMVs should be viewed as a repertoire of pathogen-associated molecular patterns (PAMPs), exerting more robust effects than Gram-negative bacteria-derived LPS.

Molecular simulation of lignin-related aromatic compound permeation through gram-negative bacterial outer membranes

Lignin, an abundant aromatic heteropolymer in secondary plant cell walls, is the single largest source of renewable aromatics in the biosphere. Leveraging this resource for renewable bioproducts through targeted microbial action depends on lignin fragment uptake by microbial hosts and subsequent enzymatic action to obtain the desired product. Recent computational work has emphasized that bacterial inner membranes are permeable to many aromatic compounds expected from lignin depolymerization processes. In this study, we expand on these findings through simulations for 42 lignin-related compounds across a gram-negative bacterial outer membrane model. Unbiased simulation trajectories indicate that spontaneous crossing for the full outer membrane is relatively rare at molecular simulation timescales, primarily due to preferential membrane partitioning and slow diffusion within the lipopolysaccharide layer within the outer membrane. Membrane partitioning and permeability coefficients were determined through replica exchange umbrella sampling simulations to overcome sampling limitations. We find that the glycosylated lipopolysaccharides found in the outer membrane increase the permeation barrier to many lignin-related compounds, particularly the most hydrophobic compounds. However, the effect is relatively modest; at industrially relevant concentrations, uncharged lignin-related compounds will readily diffuse across the outer membrane without the need for specific porins. Together, our results provide insight into the permeability of the bacterial outer membrane for assessing lignin fragment uptake and the future production of renewable bioproducts.

Solid and Liquid Surface-Supported Bacterial Membrane Mimetics as a Platform for the Functional and Structural Studies of Antimicrobials.

Increasing antibiotic resistance has provoked the urgent need to investigate the interactions of antimicrobials with bacterial membranes. The reasons for emerging antibiotic resistance and innovations in novel therapeutic approaches are highly relevant to the mechanistic interactions between antibiotics and membranes. Due to the dynamic nature, complex compositions, and small sizes of native bacterial membranes, bacterial membrane mimetics have been developed to allow for the in vitro examination of structures, properties, dynamics, and interactions. In this review, three types of model membranes are discussed: monolayers, supported lipid bilayers, and supported asymmetric bilayers; this review highlights their advantages and constraints. From monolayers to asymmetric bilayers, biomimetic bacterial membranes replicate various properties of real bacterial membranes. The typical synthetic methods for fabricating each model membrane are introduced. Depending on the properties of lipids and their biological relevance, various lipid compositions have been used to mimic bacterial membranes. For example, mixtures of phosphatidylethanolamines (PE), phosphatidylglycerols (PG), and cardiolipins (CL) at various molar ratios have been used, approaching actual lipid compositions of Gram-positive bacterial membranes and inner membranes of Gram-negative bacteria. Asymmetric lipid bilayers can be fabricated on solid supports to emulate Gram-negative bacterial outer membranes. To probe the properties of the model bacterial membranes and interactions with antimicrobials, three common characterization techniques, including quartz crystal microbalance with dissipation (QCM-D), surface plasmon resonance (SPR), and neutron reflectometry (NR) are detailed in this review article. Finally, we provide examples showing that the combination of bacterial membrane models and characterization techniques is capable of providing crucial information in the design of new antimicrobials that combat bacterial resistance.

Interaction between Antimicrobial Peptide Magainin 2 and Nonlipid Components in the Bacterial Outer Envelope.

Antimicrobial peptides (AMPs) offer advantages over conventional antibiotics; for example, bacteria develop more resistance to small-molecule antibiotics than to AMPs. The interaction of the AMPs with the lipopolysaccharide (LPS) layer of the Gram-negative bacteria cell envelope is not well understood. A MARTINI model was constructed of a Gram-negative bacterial outer membrane interacting with the AMP Magainin 2. In a 20 μs molecular dynamics (MD) simulation, the AMP diffused to the LPS layer of the cell envelope and remained there, suggesting interactions between the Magainin 2 and the LPS layer, causing the AMP to concentrate at that position. The free energy profile for the insertion of the Magainin 2 into the membrane was also calculated using umbrella sampling, which showed that the AMP positioned such that the cationic side chains of the AMP coordinated to the negatively charged phosphate groups of the LPS layer. These simulations indicate that the AMP Magainin 2 partition into the LPS layer of a bacterial membrane.

Microcins reveal natural mechanisms of bacterial manipulation to inform therapeutic development.

Microcins are an understudied and poorly characterized class of antimicrobial peptides. Despite the existence of only 15 examples, all identified from the Enterobacteriaceae, microcins display diversity in sequence, structure, target cell uptake, cytotoxic mechanism of action and target specificity. Collectively, these features describe some of the unique means nature has contrived for molecules to cross the 'impermeable' barrier of the Gram-negative bacterial outer membrane and inflict cytotoxic effects. Microcins appear to be widely dispersed among different species and in different environments, where they function in regulating microbial communities in diverse ways, including through competition. Growing evidence suggests that microcins may be adapted for therapeutic uses such as antimicrobial drugs, microbiome modulators or facilitators of peptide uptake into cells. Advancing our biological, ecological and biochemical understanding of the roles of microcins in bacterial interactions, and learning how to regulate and modify microcin activity, is essential to enable such therapeutic applications.

Remodelling of the Gram-negative bacterial Kdo2-lipid A and its functional implications.

The lipopolysaccharide (LPS) is a characteristic molecule of the outer leaflet of the Gram-negative bacterial outer membrane, which consists of lipid A, core oligosaccharide, and O antigen. The lipid A is embedded in outer membrane and provides an efficient permeability barrier, which is particularly important to reduce the permeability of antibiotics, toxic cationic metals, and antimicrobial peptides. LPS, an important modulator of innate immune responses ranging from localized inflammation to disseminated sepsis, displays a high level of structural and functional heterogeneity, which arise due to regulated differences in the acylation of the lipid A and the incorporation of non-stoichiometric modifications in lipid A and the core oligosaccharide. This review focuses on the current mechanistic understanding of the synthesis and assembly of the lipid A molecule and its most salient non-stoichiometric modifications.