Academic literature on the topic 'Gram-negative inner membrane'

This perspective addresses recent advances in lipid transport across the Gram-negative inner and outer membranes. While we include a summary of previously existing literature regarding this topic, we focus on the maintenance of lipid asymmetry (Mla) pathway. Discovered in 2009 by the Silhavy group [J. C. Malinverni, T. J. Silhavy, Proc. Natl. Acad. Sci. U.S.A. 106, 8009–8014 (2009)], Mla has become increasingly appreciated for its role in bacterial cell envelope physiology. Through the work of many, we have gained an increasingly mechanistic understanding of the function of Mla via genetic, biochemical, and structural methods. Despite this, there is a degree of controversy surrounding the directionality in which Mla transports lipids. While the initial discovery and subsequent studies have posited that it mediated retrograde lipid transport (removing glycerophospholipids from the outer membrane and returning them to the inner membrane), others have asserted the opposite. This Perspective aims to lay out the evidence in an unbiased, yet critical, manner for Mla-mediated transport in addition to postulation of mechanisms for anterograde lipid transport from the inner to outer membranes.

ABSTRACT Ceragenins are cholic acid-derived antimicrobial agents that mimic the activity of endogenous antimicrobial peptides. Ceragenins target bacterial membranes, yet the consequences of these interactions have not been fully elucidated. The role of the outer membrane in allowing access of the ceragenins to the cytoplasmic membrane of Gram-negative bacteria was studied using the ML-35p mutant strain of Escherichia coli that has been engineered to allow independent monitoring of small-molecule flux across the inner and outer membranes. The ceragenins CSA-8, CSA-13, and CSA-54 permeabilize the outer membrane of this bacterium, suggesting that the outer membrane does not play a major role in preventing the access of these agents to the cytoplasmic membrane. However, only the most potent of these ceragenins, CSA-13, was able to permeabilize the inner membrane. Interestingly, neither CSA-8 nor CSA-54 caused inner membrane permeabilization over a 30-min period, even at concentrations well above those required for bacterial toxicity. To further assess the role of membrane interactions, we measured membrane depolarization in Gram-positive bacteria with different membrane lipid compositions, as well as in Gram-negative bacteria. We found greatly increased membrane depolarization at the minimal bactericidal concentration of the ceragenins for bacterial species containing a high concentration of phosphatidylethanolamine or uncharged lipids in their cytoplasmic membranes. Although membrane lipid composition affected bactericidal efficiency, membrane depolarization was sufficient to cause lethality, providing that agents could access the cytoplasmic membrane. Consequently, we propose that in targeting bacterial cytoplasmic membranes, focus be placed on membrane depolarization as an indicator of potency.

Gram-negative bacteria balance synthesis of the outer membrane (OM), cell wall, and cytoplasmic contents during growth via unknown mechanisms. Here, we show that a dominant mutation (designatedmlaA*, maintenance of lipid asymmetry) that alters MlaA, a lipoprotein that removes phospholipids from the outer leaflet of the OM ofEscherichia coli, increases OM permeability, lipopolysaccharide levels, drug sensitivity, and cell death in stationary phase. Surprisingly, single-cell imaging revealed that death occurs after protracted loss of OM material through vesiculation and blebbing at cell-division sites and compensatory shrinkage of the inner membrane, eventually resulting in rupture and slow leakage of cytoplasmic contents. The death ofmlaA*cells was linked to fatty acid depletion and was not affected by membrane depolarization, suggesting that lipids flow from the inner membrane to the OM in an energy-independent manner. Suppressor analysis suggested that the dominantmlaA*mutation activates phospholipase A, resulting in increased levels of lipopolysaccharide and OM vesiculation that ultimately undermine the integrity of the cell envelope by depleting the inner membrane of phospholipids. This novel cell-death pathway suggests that balanced synthesis across both membranes is key to the mechanical integrity of the Gram-negative cell envelope.

Alligator sinensis cathelicidins (As-CATHs) are antimicrobial peptides extracted from alligators that enable alligators to cope with diseases caused by bacterial infections. This study assessed the damaging effects of sequence-truncated and residue-substituted variants of As-CATH4, AS4-1, AS4-5, and AS4-9 (with decreasing charges but increasing hydrophobicity) on the membranes of Gram-negative bacteria at the molecular level by using coarse-grained molecular dynamics simulations. The simulations predicted that all the variants disrupt the structures of the inner membrane of Gram-negative bacteria, with AS4-9 having the highest antibacterial activity that is able to squeeze the membrane and extract lipids from the membrane. However, none of them can disrupt the structure of asymmetric outer membrane of Gram-negative bacteria, which is composed of lipopolysaccharides in the outer leaflet and phospholipids in the inner leaflet. Nonetheless, the adsorption of AS4-9 induces lipid scrambling in the membrane by lowering the free energy of a phospholipid flipping from the inner leaflet up to the outer leaflet. Upon binding onto the lipid-scrambled outer membrane, AS4-9s are predicted to squeeze and extract phospholipids from the membrane, AS4-5s have a weak pull-out effect, and AS4-1s mainly stay free in water without any lipid-extracting function. These findings provide inspiration for the development of potent therapeutic agents targeting bacteria.

The cell envelope of Gram-negative bacteria contains two distinct membranes, an inner (IM) and an outer (OM) membrane, separated by the periplasm, a hydrophilic compartment that includes a thin layer of peptidoglycan [...]

Discovering antibiotic molecules able to hold the growing spread of antimicrobial resistance is one of the most urgent endeavors that public health must tackle. The case of Gram-negative bacterial pathogens is of special concern, as they are intrinsically resistant to many antibiotics, due to an outer membrane that constitutes an effective permeability barrier. Antimicrobial peptides (AMPs) have been pointed out as potential alternatives to conventional antibiotics, as their main mechanism of action is membrane disruption, arguably less prone to elicit resistance in pathogens. Here, we investigate the in vitro activity and selectivity of EcDBS1R4, a bioinspired AMP. To this purpose, we have used bacterial cells and model membrane systems mimicking both the inner and the outer membranes of Escherichia coli, and a variety of optical spectroscopic methodologies. EcDBS1R4 is effective against the Gram-negative E. coli, ineffective against the Gram-positive Staphylococcus aureus and noncytotoxic for human cells. EcDBS1R4 does not form stable pores in E. coli, as the peptide does not dissipate its membrane potential, suggesting an unusual mechanism of action. Interestingly, EcDBS1R4 promotes a hemi-fusion of vesicles mimicking the inner membrane of E. coli. This fusogenic ability of EcDBS1R4 requires the presence of phospholipids with a negative curvature and a negative charge. This finding suggests that EcDBS1R4 promotes a large lipid spatial reorganization able to reshape membrane curvature, with interesting biological implications herein discussed.

ABSTRACT TrfA, the replication initiator protein of broad-host-range plasmid RK2, was tested for its ability to bind to the membrane of four different gram-negative hosts in addition to Escherichia coli: Pseudomonas aeruginosa, Pseudomonas putida, Salmonella enterica serovar Typhimurium, andRhodobacter sphaeroides. Cells harboring TrfA-encoding plasmids were fractionated into soluble, inner membrane, and outer membrane fractions. The fractions were subjected to Western blotting, and the blots were probed with antibody to the TrfA proteins. TrfA was found to fractionate with the cell membranes of all species tested. When the two membrane fractions of these species were tested for their ability to synthesize plasmid DNA endogenously (i.e., without added template or enzymes), only the inner membrane fraction was capable of extensive synthesis that was inhibited by anti-TrfA antibody in a manner similar to that of the original host species, E. coli. In addition, although DNA synthesis did occur in the outer membrane fraction, it was much less extensive than that exhibited by the inner membrane fraction and only slightly affected by anti-TrfA antibody. Plasmid DNA synthesized by the inner membrane fraction of one representative species, P. aeruginosa, was characteristic of supercoil and intermediate forms of the plasmid. Extensive DNA synthesis was observed in the soluble fraction of another representative species, R. sphaeroides, but it was completely unaffected by anti-TrfA antibody, suggesting that such synthesis was due to repair and/or nonspecific chain extension of plasmid DNA fragments.

The bacterial cell envelopes possess a complex multilayered architecture exhibiting unique properties evolved to regulate interactions with the external environment and molecules with antimicrobial properties. A molecular understanding of the interactions of external molecules with the bacterial envelope will aid in the development of novel antibacterial formulations. Using detailed molecular models of the bacterial cell envelope, we have carried out molecular dynamics simulations and free energy computations to gain insights into the interactions of membrane targeting antibacterials and surfactants. We have elucidated the molecular basis for the interaction and transport of antibacterial therapeutics with both the Gram-negative inner membrane (IM) and outer membrane (OM) as well as the periplasmic peptidoglycan cell wall. Free-energy computations reveal the presence of a barrier in the core-saccharide region of the OM for the translocation of thymol a naturally occurring antibacterial while the external O-antigen region is easily traversed. In contrast, thymol spontaneously inserts into the IM and lipid diffusivities show a distinct increase in the presence of thymol. The all-atom simulations for these asymmetric bacterial membranes are challenging due to the large number of atoms involved in these models. Therefore, coarse-grained MARTINI models are preferred for these systems. We have compared the all-atom (CHARMM36) and coarse-grained (MARTINI) models of the IM, periplasmic peptidoglycan (PGN) and OM. The structural and barrier properties of the membrane were contrasted. Our results indicate that the MARTINI models accurately capture insertion free energies for small molecules and other structural properties with all-atom models of both the IM and PGN layer. We have also illustrated the lipid composition effects on the IM properties and affirmed the need to employ accurate all atom models for these membranes. Surfactants, with their ability to solubilize lipids, are another class of widely used antibacterial agents. We observe that the PGN layer does not offer a barrier to isolated surfactant molecules, however the passage of surfactant aggregates were restricted. We also observed greater changes to the IM structural and mechanical properties in the presence of laurate and rationalize differences in the efficacy of these surfactants with different aggregation properties, chain lengths, and electrostatic interaction with PGN. In the last part of the thesis, we have assessed the ability of ve coarse-grained MARTINI 1, 2-dipalmitoylsn- glycero-3-phosphocholine (DPPC) membranes to capture the ripple phase in membranes. Our study illustrates that the presence of the partly interdigitated ripple-like states are a strong function of system-size and occur as kinetically trapped structures in smaller lipid patches. The present MARTINI force elds will require additional re-parametrization to capture the ripple phase. Coupled with free energy computations our in silico study reveals lea etresolved insertion properties in bacterial membranes allowing one to assess the ability of naturally occurring small molecules such as thymol and surfactants to penetrate various membrane components. Molecular insights gained from our study can potentially be used in the design of novel antibacterial formulations to improve the efficacy of therapeutics and disinfectants to eventually combat the rise of resistant bacterial strains.

Gonorrhoea is the term used to describe the clinical manifestations of infection with the bacterium Neisseria gonorrhoeae. Neisseria gonorrhoeae is a Gram-negative diplococcus which usually infects the columnar epithelium of mucous membranes, including the lower male and female genital tracts, the rectum, the pharynx, and the conjunctivae. Transmission is by direct exposure of a mucous membrane to infected secretions, classically via sexual contact. Those who are most at risk of infection include younger age groups (15–29 years), inner-city residents, ethnic minority groups, and men who have sex with men. This chapter discusses the etiology, symptoms, demographics, natural history, complications, demographics, diagnosis, prognosis, and treatment of gonorrhoea.