Journal articles: 'Gram-negative inner membrane'

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Powers, Matthew J., and M. Stephen Trent. "Intermembrane transport: Glycerophospholipid homeostasis of the Gram-negative cell envelope." Proceedings of the National Academy of Sciences 116, no. 35 (August 16, 2019): 17147–55. http://dx.doi.org/10.1073/pnas.1902026116.

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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.

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Orlando, Benjamin, Yanyan Li, and Maofu Liao. "Snapshots of Endotoxin Extraction from the Gram-negative Inner Membrane." Microscopy and Microanalysis 26, S2 (July 30, 2020): 2520. http://dx.doi.org/10.1017/s1431927620021893.

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Epand, Raquel F., Jake E. Pollard, Jonathan O. Wright, Paul B. Savage, and Richard M. Epand. "Depolarization, Bacterial Membrane Composition, and the Antimicrobial Action of Ceragenins." Antimicrobial Agents and Chemotherapy 54, no. 9 (June 28, 2010): 3708–13. http://dx.doi.org/10.1128/aac.00380-10.

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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.

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Sutterlin, Holly A., Handuo Shi, Kerrie L. May, Amanda Miguel, Somya Khare, Kerwyn Casey Huang, and Thomas J. Silhavy. "Disruption of lipid homeostasis in the Gram-negative cell envelope activates a novel cell death pathway." Proceedings of the National Academy of Sciences 113, no. 11 (February 29, 2016): E1565—E1574. http://dx.doi.org/10.1073/pnas.1601375113.

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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.

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Li, Xiangyuan, Lei Fu, Shan Zhang, Yipeng Wang, and Lianghui Gao. "How Alligator Immune Peptides Kill Gram-Negative Bacteria: A Lipid-Scrambling, Squeezing, and Extracting Mechanism Revealed by Theoretical Simulations." International Journal of Molecular Sciences 24, no. 13 (June 30, 2023): 10962. http://dx.doi.org/10.3390/ijms241310962.

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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.

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Pérez-Cruz, Carla, Lidia Delgado, Carmen López-Iglesias, and Elena Mercade. "Outer-Inner Membrane Vesicles Naturally Secreted by Gram-Negative Pathogenic Bacteria." PLOS ONE 10, no. 1 (January 12, 2015): e0116896. http://dx.doi.org/10.1371/journal.pone.0116896.

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Raina, Satish. "Lipopolysaccharides: Regulated Biosynthesis and Structural Diversity." International Journal of Molecular Sciences 24, no. 8 (April 19, 2023): 7498. http://dx.doi.org/10.3390/ijms24087498.

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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 [...]

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Makowski, Marcin, Mário R. Felício, Isabel C. M. Fensterseifer, Octávio L. Franco, Nuno C. Santos, and Sónia Gonçalves. "EcDBS1R4, an Antimicrobial Peptide Effective against Escherichia coli with In Vitro Fusogenic Ability." International Journal of Molecular Sciences 21, no. 23 (November 30, 2020): 9104. http://dx.doi.org/10.3390/ijms21239104.

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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.

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Banack, Trevor, Peter D. Kim, and William Firshein. "TrfA-Dependent Inner Membrane-Associated Plasmid RK2 DNA Synthesis and Association of TrfA with Membranes of Different Gram-Negative Hosts." Journal of Bacteriology 182, no. 16 (August 15, 2000): 4380–83. http://dx.doi.org/10.1128/jb.182.16.4380-4383.2000.

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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.

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Clausell, Adrià, Maria Garcia-Subirats, Montserrat Pujol, M. Antonia Busquets, Francesc Rabanal, and Yolanda Cajal. "Gram-Negative Outer and Inner Membrane Models: Insertion of Cyclic Cationic Lipopeptides." Journal of Physical Chemistry B 111, no. 3 (January 2007): 551–63. http://dx.doi.org/10.1021/jp064757+.

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Jordan, L. D., Y. Zhou, C. R. Smallwood, Y. Lill, K. Ritchie, W. T. Yip, S. M. Newton, and P. E. Klebba. "Energy-dependent motion of TonB in the Gram-negative bacterial inner membrane." Proceedings of the National Academy of Sciences 110, no. 28 (June 24, 2013): 11553–58. http://dx.doi.org/10.1073/pnas.1304243110.

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Marx, Lisa, Enrico Semeraro, Karl Lohner, and Georg Pabst. "Structural Properties of Inner and Outer Membrane Mimics of Gram-Negative Bacteria." Biophysical Journal 116, no. 3 (February 2019): 87a. http://dx.doi.org/10.1016/j.bpj.2018.11.512.

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Clifton, Luke A., Maximilian W. A. Skoda, Emma L. Daulton, Arwel V. Hughes, Anton P. Le Brun, Jeremy H. Lakey, and Stephen A. Holt. "Asymmetric phospholipid: lipopolysaccharide bilayers; a Gram-negative bacterial outer membrane mimic." Journal of The Royal Society Interface 10, no. 89 (December 6, 2013): 20130810. http://dx.doi.org/10.1098/rsif.2013.0810.

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The Gram-negative bacterial outer membrane (OM) is a complex and highly asymmetric biological barrier but the small size of bacteria has hindered advances in in vivo examination of membrane dynamics. Thus, model OMs, amenable to physical study, are important sources of data. Here, we present data from asymmetric bilayers which emulate the OM and are formed by a simple two-step approach. The bilayers were deposited on an SiO 2 surface by Langmuir–Blodgett deposition of phosphatidylcholine as the inner leaflet and, via Langmuir–Schaefer deposition, an outer leaflet of either Lipid A or Escherichia coli rough lipopolysaccharides (LPS). The membranes were examined using neutron reflectometry (NR) to examine the coverage and mixing of lipids between the bilayer leaflets. NR data showed that in all cases, the initial deposition asymmetry was mostly maintained for more than 16 h. This stability enabled the sizes of the headgroups and bilayer roughness of 1,2-dipalmitoyl- sn -glycero-3-phosphocholine and Lipid A, Rc-LPS and Ra-LPS to be clearly resolved. The results show that rough LPS can be manipulated like phospholipids and used to fabricate advanced asymmetric bacterial membrane models using well-known bilayer deposition techniques. Such models will enable OM dynamics and interactions to be studied under in vivo -like conditions.

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Cochrane, Stephen A., Brandon Findlay, Alireza Bakhtiary, Jeella Z. Acedo, Eva M. Rodriguez-Lopez, Pascal Mercier, and John C. Vederas. "Antimicrobial lipopeptide tridecaptin A1selectively binds to Gram-negative lipid II." Proceedings of the National Academy of Sciences 113, no. 41 (September 29, 2016): 11561–66. http://dx.doi.org/10.1073/pnas.1608623113.

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Tridecaptin A1(TriA1) is a nonribosomal lipopeptide with selective antimicrobial activity against Gram-negative bacteria. Here we show that TriA1exerts its bactericidal effect by binding to the bacterial cell-wall precursor lipid II on the inner membrane, disrupting the proton motive force. Biochemical and biophysical assays show that binding to the Gram-negative variant of lipid II is required for membrane disruption and that only the proton gradient is dispersed. The NMR solution structure of TriA1in dodecylphosphocholine micelles with lipid II has been determined, and molecular modeling was used to provide a structural model of the TriA1–lipid II complex. These results suggest that TriA1kills Gram-negative bacteria by a mechanism of action using a lipid-II–binding motif.

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Dombach, Jamie L., Joaquin L. J. Quintana, Toni A. Nagy, Chun Wan, Amy L. Crooks, Haijia Yu, Chih-Chia Su, Edward W. Yu, Jingshi Shen, and Corrella S. Detweiler. "A small molecule that mitigates bacterial infection disrupts Gram-negative cell membranes and is inhibited by cholesterol and neutral lipids." PLOS Pathogens 16, no. 12 (December 8, 2020): e1009119. http://dx.doi.org/10.1371/journal.ppat.1009119.

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Infections caused by Gram-negative bacteria are difficult to fight because these pathogens exclude or expel many clinical antibiotics and host defense molecules. However, mammals have evolved a substantial immune arsenal that weakens pathogen defenses, suggesting the feasibility of developing therapies that work in concert with innate immunity to kill Gram-negative bacteria. Using chemical genetics, we recently identified a small molecule, JD1, that kills Salmonella enterica serovar Typhimurium (S. Typhimurium) residing within macrophages. JD1 is not antibacterial in standard microbiological media, but rapidly inhibits growth and curtails bacterial survival under broth conditions that compromise the outer membrane or reduce efflux pump activity. Using a combination of cellular indicators and super resolution microscopy, we found that JD1 damaged bacterial cytoplasmic membranes by increasing fluidity, disrupting barrier function, and causing the formation of membrane distortions. We quantified macrophage cell membrane integrity and mitochondrial membrane potential and found that disruption of eukaryotic cell membranes required approximately 30-fold more JD1 than was needed to kill bacteria in macrophages. Moreover, JD1 preferentially damaged liposomes with compositions similar to E. coli inner membranes versus mammalian cell membranes. Cholesterol, a component of mammalian cell membranes, was protective in the presence of neutral lipids. In mice, intraperitoneal administration of JD1 reduced tissue colonization by S. Typhimurium. These observations indicate that during infection, JD1 gains access to and disrupts the cytoplasmic membrane of Gram-negative bacteria, and that neutral lipids and cholesterol protect mammalian membranes from JD1-mediated damage. Thus, it may be possible to develop therapeutics that exploit host innate immunity to gain access to Gram-negative bacteria and then preferentially damage the bacterial cell membrane over host membranes.

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Vetterli, Stefan U., Katja Zerbe, Maik Müller, Matthias Urfer, Milon Mondal, Shuang-Yan Wang, Kerstin Moehle, et al. "Thanatin targets the intermembrane protein complex required for lipopolysaccharide transport inEscherichia coli." Science Advances 4, no. 11 (November 2018): eaau2634. http://dx.doi.org/10.1126/sciadv.aau2634.

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With the increasing resistance of many Gram-negative bacteria to existing classes of antibiotics, identifying new paradigms in antimicrobial discovery is an important research priority. Of special interest are the proteins required for the biogenesis of the asymmetric Gram-negative bacterial outer membrane (OM). Seven Lpt proteins (LptA to LptG) associate in most Gram-negative bacteria to form a macromolecular complex spanning the entire envelope, which transports lipopolysaccharide (LPS) molecules from their site of assembly at the inner membrane to the cell surface, powered by adenosine 5′-triphosphate hydrolysis in the cytoplasm. The periplasmic protein LptA comprises the protein bridge across the periplasm, which connects LptB2FGC at the inner membrane to LptD/E anchored in the OM. We show here that the naturally occurring, insect-derived antimicrobial peptide thanatin targets LptA and LptD in the network of periplasmic protein-protein interactions required to assemble the Lpt complex, leading to the inhibition of LPS transport and OM biogenesis inEscherichia coli.

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Zaknoon, Fadia, Keren Goldberg, Hadar Sarig, Raquel F. Epand, Richard M. Epand, and Amram Mor. "Antibacterial Properties of an Oligo-Acyl-Lysyl Hexamer Targeting Gram-Negative Species." Antimicrobial Agents and Chemotherapy 56, no. 9 (July 2, 2012): 4827–32. http://dx.doi.org/10.1128/aac.00511-12.

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ABSTRACTToward developing new tools for fighting resistance to antibiotics, we investigated the antibacterial properties of a new decanoyl-based oligo-acyl-lysyl (OAK) hexamer, aminododecanoyl-lysyl-[aminodecanoyl-lysyl]5(α12-5α10). The OAK exhibited preferential activity against Gram-negative bacteria (GNB), as determined using 36 strains, including diverse species, with an MIC90of 6.2 μM. The OAK's bactericidal mode of action was associated with rapid membrane depolarization and cell permeabilization, suggesting that the inner membrane was the primary target, whereas the observed binding affinity to lipoteichoic acid suggested that inefficacy against Gram-positive species resulted from a cell wall interaction preventing α12-5α10from reaching internal targets. Interestingly, perturbation of the inner membrane structure and function was preserved at sub-MIC values. This prompted us to assess the OAK's effect on the proton motive force-dependent efflux pump AcrAB-TolC, implicated in the low sensitivity of GNB to various antibiotics, including erythromycin. We found that under sub-MIC conditions, wild-typeEscherichia coliwas significantly more sensitive to erythromycin (the MIC dropped by >10-fold), unlike itsacr-deletion mutant. Collectively, the data suggest a useful approach for treating GNB infections through overcoming antibiotic efflux.

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Weerakoon, Dhanushka, Jan K. Marzinek, Peter J. Bond, Conrado Pedebos, and Syma Khalid. "Interactions of polymyxin B1 with the gram-negative inner membrane: A simulation study." Biophysical Journal 122, no. 3 (February 2023): 371a. http://dx.doi.org/10.1016/j.bpj.2022.11.2043.

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Dong, Haohao, Xiaodi Tang, Zhengyu Zhang, and Changjiang Dong. "Structural insight into lipopolysaccharide transport from the Gram-negative bacterial inner membrane to the outer membrane." Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids 1862, no. 11 (November 2017): 1461–67. http://dx.doi.org/10.1016/j.bbalip.2017.08.003.

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McLeod, Sarah M., Paul R. Fleming, Kathleen MacCormack, Robert E. McLaughlin, James D. Whiteaker, Shin-ichiro Narita, Makiko Mori, Hajime Tokuda, and Alita A. Miller. "Small-Molecule Inhibitors of Gram-Negative Lipoprotein Trafficking Discovered by Phenotypic Screening." Journal of Bacteriology 197, no. 6 (January 12, 2015): 1075–82. http://dx.doi.org/10.1128/jb.02352-14.

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In Gram-negative bacteria, lipoproteins are transported to the outer membrane by the Lol system. In this process, lipoproteins are released from the inner membrane by the ABC transporter LolCDE and passed to LolA, a diffusible periplasmic molecular chaperone. Lipoproteins are then transferred to the outer membrane receptor protein, LolB, for insertion in the outer membrane. Here we describe the discovery and characterization of novel pyridineimidazole compounds that inhibit this process.Escherichia colimutants resistant to the pyridineimidazoles show no cross-resistance to other classes of antibiotics and map to either the LolC or LolE protein of the LolCDE transporter complex. The pyridineimidazoles were shown to inhibit the LolA-dependent release of the lipoprotein Lpp fromE. colispheroplasts. These results combined with bacterial cytological profiling are consistent with LolCDE-mediated disruption of lipoprotein targeting to the outer membrane as the mode of action of these pyridineimidazoles. The pyridineimidazoles are the first reported inhibitors of the LolCDE complex, a target which has never been exploited for therapeutic intervention. These compounds open the door to further interrogation of the outer membrane lipoprotein transport pathway as a target for antimicrobial therapy.

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Klebba, Phillip E. "ROSET Model of TonB Action in Gram-Negative Bacterial Iron Acquisition." Journal of Bacteriology 198, no. 7 (January 19, 2016): 1013–21. http://dx.doi.org/10.1128/jb.00823-15.

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Therotationalsurveillance andenergytransfer (ROSET) model of TonB action suggests a mechanism by which the electrochemical proton gradient across the Gram-negative bacterial inner membrane (IM) promotes the transport of iron through ligand-gated porins (LGP) in the outer membrane (OM). TonB associates with the IM by an N-terminal hydrophobic helix that forms a complex with ExbBD. It also contains a central extended length of rigid polypeptide that spans the periplasm and a dimericC-terminal-ββαβ-domain (CTD) with LysM motifs that binds the peptidoglycan (PG) layer beneath the OM bilayer. The TonB CTD forms a dimer with affinity for both PG- and TonB-independent OM proteins (e.g., OmpA), localizing it near the periplasmic interface of the OM bilayer. Porins and other OM proteins associate with PG, and this general affinity allows the TonB CTD dimer to survey the periplasmic surface of the OM bilayer. Energized rotational motion of the TonB N terminus in the fluid IM bilayer promotes the lateral movement of the TonB-ExbBD complex in the IM and of the TonB CTD dimer across the inner surface of the OM. When it encounters an accessible TonB box of a (ligand-bound) LGP, the monomeric form of the CTD binds and recruits it into a 4-stranded β-sheet. Because the CTD is rotating, this binding reaction transfers kinetic energy, created by the electrochemical proton gradient across the IM, through the periplasm to the OM protein. The equilibration of the TonB C terminus between the dimeric and monomeric forms that engage in different binding reactions allows the identification of iron-loaded LGP and then the internalization of iron through their trans-outer membrane β-barrels. Hence, the ROSET model postulates a mechanism for the transfer of energy from the IM to the OM, triggering iron uptake.

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Py, Béatrice, Laurent Loiseau, and Frédéric Barras. "An inner membrane platform in the type II secretion machinery of Gram‐negative bacteria." EMBO reports 2, no. 3 (March 2001): 244–48. http://dx.doi.org/10.1093/embo-reports/kve042.

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Simpson, Brent W., Janine M. May, David J. Sherman, Daniel Kahne, and Natividad Ruiz. "Lipopolysaccharide transport to the cell surface: biosynthesis and extraction from the inner membrane." Philosophical Transactions of the Royal Society B: Biological Sciences 370, no. 1679 (October 5, 2015): 20150029. http://dx.doi.org/10.1098/rstb.2015.0029.

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The cell surface of most Gram-negative bacteria is covered with lipopolysaccharide (LPS). The network of charges and sugars provided by the dense packing of LPS molecules in the outer leaflet of the outer membrane interferes with the entry of hydrophobic compounds into the cell, including many antibiotics. In addition, LPS can be recognized by the immune system and plays a crucial role in many interactions between bacteria and their animal hosts. LPS is synthesized in the inner membrane of Gram-negative bacteria, so it must be transported across their cell envelope to assemble at the cell surface. Over the past two decades, much of the research on LPS biogenesis has focused on the discovery and understanding of Lpt, a multi-protein complex that spans the cell envelope and functions to transport LPS from the inner membrane to the outer membrane. This paper focuses on the early steps of the transport of LPS by the Lpt machinery: the extraction of LPS from the inner membrane. The accompanying paper (May JM, Sherman DJ, Simpson BW, Ruiz N, Kahne D. 2015 Phil. Trans. R. Soc. B 370 , 20150027. ( doi:10.1098/rstb.2015.0027 )) describes the subsequent steps as LPS travels through the periplasm and the outer membrane to its final destination at the cell surface.

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Josts, Inokentijs, Katharina Veith, Vincent Normant, Isabelle J. Schalk, and Henning Tidow. "Structural insights into a novel family of integral membrane siderophore reductases." Proceedings of the National Academy of Sciences 118, no. 34 (August 20, 2021): e2101952118. http://dx.doi.org/10.1073/pnas.2101952118.

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Gram-negative bacteria take up the essential ion Fe3+ as ferric-siderophore complexes through their outer membrane using TonB-dependent transporters. However, the subsequent route through the inner membrane differs across many bacterial species and siderophore chemistries and is not understood in detail. Here, we report the crystal structure of the inner membrane protein FoxB (from Pseudomonas aeruginosa) that is involved in Fe-siderophore uptake. The structure revealed a fold with two tightly bound heme molecules. In combination with in vitro reduction assays and in vivo iron uptake studies, these results establish FoxB as an inner membrane reductase involved in the release of iron from ferrioxamine during Fe-siderophore uptake.

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Pérez-Cruz, Carla, Ornella Carrión, Lidia Delgado, Gemma Martinez, Carmen López-Iglesias, and Elena Mercade. "New Type of Outer Membrane Vesicle Produced by the Gram-Negative Bacterium Shewanella vesiculosa M7T: Implications for DNA Content." Applied and Environmental Microbiology 79, no. 6 (January 11, 2013): 1874–81. http://dx.doi.org/10.1128/aem.03657-12.

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ABSTRACTOuter membrane vesicles (OMVs) from Gram-negative bacteria are known to be involved in lateral DNA transfer, but the presence of DNA in these vesicles has remained difficult to explain. An ultrastructural study of the Antarctic psychrotolerant bacteriumShewanella vesiculosaM7Thas revealed that this Gram-negative bacterium naturally releases conventional one-bilayer OMVs through a process in which the outer membrane is exfoliated and only the periplasm is entrapped, together with a more complex type of OMV, previously undescribed, which on formation drag along inner membrane and cytoplasmic content and can therefore also entrap DNA. These vesicles, with a double-bilayer structure and containing electron-dense material, were visualized by transmission electron microscopy (TEM) after high-pressure freezing and freeze-substitution (HPF-FS), and their DNA content was fluorometrically quantified as 1.8 ± 0.24 ng DNA/μg OMV protein. The new double-bilayer OMVs were estimated by cryo-TEM to represent 0.1% of total vesicles. The presence of DNA inside the vesicles was confirmed by gold DNA immunolabeling with a specific monoclonal IgM against double-stranded DNA. In addition, a proteomic study of purified membrane vesicles confirmed the presence of plasma membrane and cytoplasmic proteins in OMVs from this strain. Our data demonstrate the existence of a previously unobserved type of double-bilayer OMV in the Gram-negative bacteriumShewanella vesiculosaM7Tthat can incorporate DNA, for which we propose the name outer-inner membrane vesicle (O-IMV).

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Douglass, Martin V., François Cléon, and M. Stephen Trent. "Cardiolipin aids in lipopolysaccharide transport to the gram-negative outer membrane." Proceedings of the National Academy of Sciences 118, no. 15 (April 8, 2021): e2018329118. http://dx.doi.org/10.1073/pnas.2018329118.

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In Escherichia coli, cardiolipin (CL) is the least abundant of the three major glycerophospholipids in the gram-negative cell envelope. However, E. coli harbors three distinct enzymes that synthesize CL: ClsA, ClsB, and ClsC. This redundancy suggests that CL is essential for bacterial fitness, yet CL-deficient bacteria are viable. Although multiple CL–protein interactions have been identified, the role of CL still remains unclear. To identify genes that impact fitness in the absence of CL, we analyzed high-density transposon (Tn) mutant libraries in combinatorial CL synthase mutant backgrounds. We found LpxM, which is the last enzyme in lipid A biosynthesis, the membrane anchor of lipopolysaccharide (LPS), to be critical for viability in the absence of clsA. Here, we demonstrate that CL produced by ClsA enhances LPS transport. Suppressors of clsA and lpxM essentiality were identified in msbA, a gene that encodes the indispensable LPS ABC transporter. Depletion of ClsA in ∆lpxM mutants increased accumulation of LPS in the inner membrane, demonstrating that the synthetic lethal phenotype arises from improper LPS transport. Additionally, overexpression of ClsA alleviated ΔlpxM defects associated with impaired outer membrane asymmetry. Mutations that lower LPS levels, such as a YejM truncation or alteration in the fatty acid pool, were sufficient in overcoming the synthetically lethal ΔclsA ΔlpxM phenotype. Our results support a model in which CL aids in the transportation of LPS, a unique glycolipid, and adds to the growing repertoire of CL–protein interactions important for bacterial transport systems.

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Huntley, Jason F., Patrick G. Conley, Kayla E. Hagman, and Michael V. Norgard. "Characterization of Francisella tularensis Outer Membrane Proteins." Journal of Bacteriology 189, no. 2 (November 17, 2006): 561–74. http://dx.doi.org/10.1128/jb.01505-06.

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ABSTRACT Francisella tularensis is a gram-negative coccobacillus that is capable of causing severe, fatal disease in a number of mammalian species, including humans. Little is known about the proteins that are surface exposed on the outer membrane (OM) of F. tularensis, yet identification of such proteins is potentially fundamental to understanding the initial infection process, intracellular survival, virulence, immune evasion and, ultimately, vaccine development. To facilitate the identification of putative F. tularensis outer membrane proteins (OMPs), the genomes of both the type A strain (Schu S4) and type B strain (LVS) were subjected to six bioinformatic analyses for OMP signatures. Compilation of the bioinformatic predictions highlighted 16 putative OMPs, which were cloned and expressed for the generation of polyclonal antisera. Total membranes were extracted from both Schu S4 and LVS by spheroplasting and osmotic lysis, followed by sucrose density gradient centrifugation, which separated OMs from cytoplasmic (inner) membrane and other cellular compartments. Validation of OM separation and enrichment was confirmed by probing sucrose gradient fractions with antibodies to putative OMPs and inner membrane proteins. F. tularensis OMs typically migrated in sucrose gradients between densities of 1.17 and 1.20 g/ml, which differed from densities typically observed for other gram-negative bacteria (1.21 to 1.24 g/ml). Finally, the identities of immunogenic proteins were determined by separation on two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and mass spectrometric analysis. This is the first report of a direct method for F. tularensis OM isolation that, in combination with computational predictions, offers a more comprehensive approach for the characterization of F. tularensis OMPs.

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Maktabi, Sepehr, Jeffrey W. Schertzer, and Paul R. Chiarot. "Dewetting-induced formation and mechanical properties of synthetic bacterial outer membrane models (GUVs) with controlled inner-leaflet lipid composition." Soft Matter 15, no. 19 (2019): 3938–48. http://dx.doi.org/10.1039/c9sm00223e.

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Kim, Jin-Young, Seong-Cheol Park, Moon-Young Yoon, Kyung-Soo Hahm, and Yoonkyung Park. "C-terminal amidation of PMAP-23: translocation to the inner membrane of Gram-negative bacteria." Amino Acids 40, no. 1 (May 30, 2010): 183–95. http://dx.doi.org/10.1007/s00726-010-0632-1.

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Ma, Miao, Margaux Lustig, Michèle Salem, Dominique Mengin-Lecreulx, Gilles Phan, and Isabelle Broutin. "MexAB-OprM Efflux Pump Interaction with the Peptidoglycan of Escherichia coli and Pseudomonas aeruginosa." International Journal of Molecular Sciences 22, no. 10 (May 18, 2021): 5328. http://dx.doi.org/10.3390/ijms22105328.

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One of the major families of membrane proteins found in prokaryote genome corresponds to the transporters. Among them, the resistance-nodulation-cell division (RND) transporters are highly studied, as being responsible for one of the most problematic mechanisms used by bacteria to resist to antibiotics, i.e., the active efflux of drugs. In Gram-negative bacteria, these proteins are inserted in the inner membrane and form a tripartite assembly with an outer membrane factor and a periplasmic linker in order to cross the two membranes to expulse molecules outside of the cell. A lot of information has been collected to understand the functional mechanism of these pumps, especially with AcrAB-TolC from Escherichia coli, but one missing piece from all the suggested models is the role of peptidoglycan in the assembly. Here, by pull-down experiments with purified peptidoglycans, we precise the MexAB-OprM interaction with the peptidoglycan from Escherichia coli and Pseudomonas aeruginosa, highlighting a role of the peptidoglycan in stabilizing the MexA-OprM complex and also differences between the two Gram-negative bacteria peptidoglycans.

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Berger, Carolin, Guillaume P. Robin, Ulla Bonas, and Ralf Koebnik. "Membrane topology of conserved components of the type III secretion system from the plant pathogen Xanthomonas campestris pv. vesicatoria." Microbiology 156, no. 7 (July 1, 2010): 1963–74. http://dx.doi.org/10.1099/mic.0.039248-0.

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Type III secretion (T3S) systems play key roles in the assembly of flagella and the translocation of bacterial effector proteins into eukaryotic host cells. Eleven proteins which are conserved among Gram-negative plant and animal pathogenic bacteria have been proposed to build up the basal structure of the T3S system, which spans both inner and outer bacterial membranes. We studied six conserved proteins, termed Hrc, predicted to reside in the inner membrane of the plant pathogen Xanthomonas campestris pv. vesicatoria. The membrane topology of HrcD, HrcR, HrcS, HrcT, HrcU and HrcV was studied by translational fusions to a dual alkaline phosphatase–β-galactosidase reporter protein. Two proteins, HrcU and HrcV, were found to have the same membrane topology as the Yersinia homologues YscU and YscV. For HrcR, the membrane topology differed from the model for the homologue from Yersinia, YscR. For our data on three other protein families, exemplified by HrcD, HrcS and HrcT, we derived the first topology models. Our results provide what is believed to be the first complete model of the inner membrane topology of any bacterial T3S system and will aid in elucidating the architecture of T3S systems by ultrastructural analysis.

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Zhang, Ge, Vadim Baidin, Karanbir S. Pahil, Eileen Moison, David Tomasek, Nitya S. Ramadoss, Arnab K. Chatterjee, et al. "Cell-based screen for discovering lipopolysaccharide biogenesis inhibitors." Proceedings of the National Academy of Sciences 115, no. 26 (May 7, 2018): 6834–39. http://dx.doi.org/10.1073/pnas.1804670115.

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New drugs are needed to treat gram-negative bacterial infections. These bacteria are protected by an outer membrane which prevents many antibiotics from reaching their cellular targets. The outer leaflet of the outer membrane contains LPS, which is responsible for creating this permeability barrier. Interfering with LPS biogenesis affects bacterial viability. We developed a cell-based screen that identifies inhibitors of LPS biosynthesis and transport by exploiting the nonessentiality of this pathway inAcinetobacter. We used this screen to find an inhibitor of MsbA, an ATP-dependent flippase that translocates LPS across the inner membrane. Treatment with the inhibitor caused mislocalization of LPS to the cell interior. The discovery of an MsbA inhibitor, which is universally conserved in all gram-negative bacteria, validates MsbA as an antibacterial target. Because our cell-based screen reports on the function of the entire LPS biogenesis pathway, it could be used to identify compounds that inhibit other targets in the pathway, which can provide insights into vulnerabilities of the gram-negative cell envelope.

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Li, Shiqi, Ruohua Ren, Letian Lyu, Jiangning Song, Yajun Wang, Tsung-Wu Lin, Anton Le Brun, Hsien-Yi Hsu, and Hsin-Hui Shen. "Solid and Liquid Surface-Supported Bacterial Membrane Mimetics as a Platform for the Functional and Structural Studies of Antimicrobials." Membranes 12, no. 10 (September 20, 2022): 906. http://dx.doi.org/10.3390/membranes12100906.

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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.

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Segovia, Roser, Judith Solé, Ana Maria Marqués, Yolanda Cajal, and Francesc Rabanal. "Unveiling the Membrane and Cell Wall Action of Antimicrobial Cyclic Lipopeptides: Modulation of the Spectrum of Activity." Pharmaceutics 13, no. 12 (December 17, 2021): 2180. http://dx.doi.org/10.3390/pharmaceutics13122180.

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Antibiotic resistance is a major public health challenge, and Gram-negative multidrug-resistant bacteria are particularly dangerous. The threat of running out of active molecules is accelerated by the extensive use of antibiotics in the context of the COVID-19 pandemic, and new antibiotics are urgently needed. Colistin and polymyxin B are natural antibiotics considered as last resort drugs for multi-resistant infections, but their use is limited because of neuro- and nephrotoxicity. We previously reported a series of synthetic analogues inspired in natural polymyxins with a flexible scaffold that allows multiple modifications to improve activity and reduce toxicity. In this work, we focus on modifications in the hydrophobic domains, describing analogues that broaden or narrow the spectrum of activity including both Gram-positive and Gram-negative bacteria, with MICs in the low µM range and low hemolytic activity. Using biophysical methods, we explore the interaction of the new molecules with model membranes that mimic the bacterial inner and outer membranes, finding a selective effect on anionic membranes and a mechanism of action based on the alteration of membrane function. Transmission electron microscopy observation confirms that polymyxin analogues kill microbial cells primarily by damaging membrane integrity. Redistribution of the hydrophobicity within the polymyxin molecule seems a plausible approach for the design and development of safer and more selective antibiotics.

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Nguyen, Hang Thi, Lisa A. O’Donovan, Henrietta Venter, Cecilia C. Russell, Adam McCluskey, Stephen W. Page, Darren J. Trott, and Abiodun D. Ogunniyi. "Comparison of Two Transmission Electron Microscopy Methods to Visualize Drug-Induced Alterations of Gram-Negative Bacterial Morphology." Antibiotics 10, no. 3 (March 17, 2021): 307. http://dx.doi.org/10.3390/antibiotics10030307.

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In this study, we optimized and compared different transmission electron microscopy (TEM) methods to visualize changes to Gram-negative bacterial morphology induced by treatment with a robenidine analogue (NCL195) and colistin combination. Aldehyde-fixed bacterial cells (untreated, treated with colistin or NCL195 + colistin) were prepared using conventional TEM methods and compared with ultrathin Tokuyasu cryo-sections. The results of this study indicate superiority of ultrathin cryo-sections in visualizing the membrane ultrastructure of Escherichia coli and Pseudomonas aeruginosa, with a clear delineation of the outer and inner membrane as well as the peptidoglycan layer. We suggest that the use of ultrathin cryo-sectioning can be used to better visualize and understand drug interaction mechanisms on the bacterial cell membrane.

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36

Xu, Yongbin, Arne Moeller, So-Young Jun, Minho Le, Bo-Young Yoon, Jin-Sik Kim, Kangseok Lee, and Nam-Chul Ha. "Assembly and Channel Opening of Outer Membrane Protein in Tripartite Drug Efflux Pumps of Gram-negative Bacteria." Journal of Biological Chemistry 287, no. 15 (February 3, 2012): 11740–50. http://dx.doi.org/10.1074/jbc.m111.329375.

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Gram-negative bacteria are capable of expelling diverse xenobiotic substances from within the cell by use of three-component efflux pumps in which the energy-activated inner membrane transporter is connected to the outer membrane channel protein via the membrane fusion protein. In this work, we describe the crystal structure of the membrane fusion protein MexA from the Pseudomonas aeruginosa MexAB-OprM pump in the hexameric ring arrangement. Electron microscopy study on the chimeric complex of MexA and the outer membrane protein OprM reveals that MexA makes a tip-to-tip interaction with OprM, which suggests a docking model for MexA and OprM. This docking model agrees well with genetic results and depicts detailed interactions. Opening of the OprM channel is accompanied by the simultaneous exposure of a protein structure resembling a six-bladed cogwheel, which intermeshes with the complementary cogwheel structure in the MexA hexamer. Taken together, we suggest an assembly and channel opening model for the MexAB-OprM pump. This study provides a better understanding of multidrug resistance in Gram-negative bacteria.

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Gawarzewski, Iris, Sander H. J. Smits, Lutz Schmitt, and Joachim Jose. "Structural comparison of the transport units of type V secretion systems." Biological Chemistry 394, no. 11 (November 1, 2013): 1385–98. http://dx.doi.org/10.1515/hsz-2013-0162.

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Abstract Pathogenic gram-negative bacteria have evolved several secretion mechanisms to translocate adhesins, enzymes, toxins, and other virulence factors across the inner and outer membranes. Currently, eight different secretion systems, type I–type VIII (T1SS–T8SS) plus the chaperone-usher (CU) pathway, have been identified, which act in one-step or two-step mechanisms to traverse both membrane barriers. The type V secretion system (T5SS) is dependent first on the Sec translocon within the inner membrane. The periplasmic intermediates are then secreted through aqueous pores formed by β-barrels in the outer membrane. Until now, transport across the outer membrane has not been understood on a molecular level. With respect to special characteristics revealed by crystal structure analysis, bioinformatic and biochemical data, five subgroups of T5SS were defined. Here, we compare the transport moieties of members of four subgroups based on X-ray crystal structures. For the fifth subgroup, which was identified only recently, no structures have thus far been reported. We also discuss different models for the translocation process across the outer membrane with respect to recent findings.

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Samantarrai, Devyani, Annapoorni Lakshman Sagar, Ramurthy Gudla, and Dayananda Siddavattam. "TonB-Dependent Transporters in Sphingomonads: Unraveling Their Distribution and Function in Environmental Adaptation." Microorganisms 8, no. 3 (March 3, 2020): 359. http://dx.doi.org/10.3390/microorganisms8030359.

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TonB-dependent transport system plays a critical role in the transport of nutrients across the energy-deprived outer membrane of Gram-negative bacteria. It contains a specialized outer membrane TonB-dependent transporter (TBDT) and energy generating (ExbB/ExbD) and transducing (TonB) inner membrane multi-protein complex, called TonB complex. Very few TonB complex protein-coding sequences exist in the genomes of Gram-negative bacteria. Interestingly, the TBDT coding alleles are phenomenally high, especially in the genomes of bacteria surviving in complex and stressful environments. Sphingomonads are known to survive in highly polluted environments using rare, recalcitrant, and toxic substances as their sole source of carbon. Naturally, they also contain a huge number of TBDTs in the outer membrane. Out of them, only a few align with the well-characterized TBDTs. The functions of the remaining TBDTs are not known. Predictions made based on genome context and expression pattern suggest their involvement in the transport of xenobiotic compounds across the outer membrane.

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Bootsma, Hester J., Piet C. Aerts, George Posthuma, Theo Harmsen, Jan Verhoef, Hans van Dijk, and Frits R. Mooi. "Moraxella (Branhamella)catarrhalis BRO β-Lactamase: a Lipoprotein of Gram-Positive Origin?" Journal of Bacteriology 181, no. 16 (August 15, 1999): 5090–93. http://dx.doi.org/10.1128/jb.181.16.5090-5093.1999.

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ABSTRACT In the past 20 years, BRO β-lactamase-producing Moraxella catarrhalis strains have emerged. We show that BRO is expressed as a 33-kDa lipoprotein associated with the inner leaflet of the outer membrane. To our knowledge, this is the first description of a lipidated β-lactamase in a gram-negative species.

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Sperandeo, Paola, Rachele Cescutti, Riccardo Villa, Cristiano Di Benedetto, Daniela Candia, Gianni Dehò, and Alessandra Polissi. "Characterization of lptA and lptB, Two Essential Genes Implicated in Lipopolysaccharide Transport to the Outer Membrane of Escherichia coli." Journal of Bacteriology 189, no. 1 (October 20, 2006): 244–53. http://dx.doi.org/10.1128/jb.01126-06.

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ABSTRACT The outer membrane (OM) of gram-negative bacteria is an asymmetric lipid bilayer that protects the cell from toxic molecules. Lipopolysaccharide (LPS) is an essential component of the OM in most gram-negative bacteria, and its structure and biosynthesis are well known. Nevertheless, the mechanisms of transport and assembly of this molecule in the OM are poorly understood. To date, the only proteins implicated in LPS transport are MsbA, responsible for LPS flipping across the inner membrane, and the Imp/RlpB complex, involved in LPS targeting to the OM. Here, we present evidence that two Escherichia coli essential genes, yhbN and yhbG, now renamed lptA and lptB, respectively, participate in LPS biogenesis. We show that mutants depleted of LptA and/or LptB not only produce an anomalous LPS form, but also are defective in LPS transport to the OM and accumulate de novo-synthesized LPS in a novel membrane fraction of intermediate density between the inner membrane (IM) and the OM. In addition, we show that LptA is located in the periplasm and that expression of the lptA-lptB operon is controlled by the extracytoplasmic σ factor RpoE. Based on these data, we propose that LptA and LptB are implicated in the transport of LPS from the IM to the OM of E. coli.

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Aronova, N. V., N. V. Pavlovich, M. V. Tsimbalistova, S. N. Golovin, and A. S. Anisimova. "The Role of Outer Membrane Vesicles of Agents of Particularly Dangerous Infections in the Pathogenesis and Immunogenesis of Infectious Process." Problems of Particularly Dangerous Infections, no. 4 (January 24, 2022): 6–15. http://dx.doi.org/10.21055/0370-1069-2021-4-6-15.

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The literature review is devoted to the modern concepts of the vesiculation phenomenon and its biological role in pathogenic bacteria – causative agents of particularly dangerous human infections. Data on the production, structure, composition, and functions of the outer membrane vesicles (OMV) of bacteria have been summarized. In recent years, the interest of researchers in the formation of spherical structures (so called bubbles or vesicles) from outer membrane of gram-negative bacteria has significantly increased. Such structures are surrounded by the double layer of a phospholipid membrane, the outer layer of which is enriched with lipopolysaccharide molecules. The inner space of vesicles could include various antigens, receptors, adhesins, toxins, enzymes, porins, etc. The formation of vesicles by the outer membranes of bacteria is recognized as a normal physiological manifestation of bacterial activity aimed at adaptation to environmental conditions. The investigation of the biological role of OMV showed their connection with the pathogenesis and immunogenesis of bacterial diseases. The review provides information on the peculiarity of induction, OMV composition and their participation in the processes of patho- and immunogenesis of severe infections caused by groups I–II PBA – the gram-negative causative agents of plague, tularemia, brucellosis, glanders, melioidosis, cholera, and formation of extracellular vesicles in a gram-positive anthrax pathogen. The particular attention is paid to the issue of developing safe and effective next-generation vaccine preparations based on bacterial vesicles.

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Chi, Ximin, Qiongxuan Fan, Yuanyuan Zhang, Ke Liang, Li Wan, Qiang Zhou, and Yanyan Li. "Structural mechanism of phospholipids translocation by MlaFEDB complex." Cell Research 30, no. 12 (September 3, 2020): 1127–35. http://dx.doi.org/10.1038/s41422-020-00404-6.

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AbstractIn Gram-negative bacteria, phospholipids are major components of the inner membrane and the inner leaflet of the outer membrane, playing an essential role in forming the unique dual-membrane barrier to exclude the entry of most antibiotics. Understanding the mechanisms of phospholipid translocation between the inner and outer membrane represents one of the major challenges surrounding bacterial phospholipid homeostasis. The conserved MlaFEDB complex in the inner membrane functions as an ABC transporter to drive the translocation of phospholipids between the inner membrane and the periplasmic protein MlaC. However, the mechanism of phospholipid translocation remains elusive. Here we determined three cryo-EM structures of MlaFEDB from Escherichia coli in its nucleotide-free and ATP-bound conformations, and performed extensive functional studies to verify and extend our findings from structural analyses. Our work reveals unique structural features of the entire MlaFEDB complex, six well-resolved phospholipids in three distinct cavities, and large-scale conformational changes upon ATP binding. Together, these findings define the cycle of structural rearrangement of MlaFEDB in action, and suggest that MlaFEDB uses an extrusion mechanism to extract and release phospholipids through the central translocation cavity.

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Long, Feng, Chih-Chia Su, Hsiang-Ting Lei, Jani Reddy Bolla, Sylvia V. Do, and Edward W. Yu. "Structure and mechanism of the tripartite CusCBA heavy-metal efflux complex." Philosophical Transactions of the Royal Society B: Biological Sciences 367, no. 1592 (April 19, 2012): 1047–58. http://dx.doi.org/10.1098/rstb.2011.0203.

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Gram-negative bacteria frequently expel toxic chemicals through tripartite efflux pumps that span both the inner and outer membranes. The three parts are the inner membrane, substrate-binding transporter (or pump); a periplasmic membrane fusion protein (MFP, or adaptor); and an outer membrane-anchored channel. The fusion protein connects the transporter to the channel within the periplasmic space. One such efflux system CusCBA is responsible for extruding biocidal Cu(I) and Ag(I) ions. We previously described the crystal structures of both the inner membrane transporter CusA and the MFP CusB of Escherichia coli . We also determined the co-crystal structure of the CusBA adaptor–transporter efflux complex, showing that the transporter CusA, which is present as a trimer, interacts with six CusB protomers and that the periplasmic domain of CusA is involved in these interactions. Here, we summarize the structural information of these efflux proteins, and present the accumulated evidence that this efflux system uses methionine residues to bind and export Cu(I) and Ag(I). Genetic and structural analyses suggest that the CusA pump is capable of picking up the metal ions from both the periplasm and the cytoplasm. We propose a stepwise shuttle mechanism for this pump to export metal ions from the cell.

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Lenders, Michael H. H., Sven Reimann, Sander H. J. Smits, and Lutz Schmitt. "Molecular insights into type I secretion systems." Biological Chemistry 394, no. 11 (November 1, 2013): 1371–84. http://dx.doi.org/10.1515/hsz-2013-0171.

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Abstract Type 1 secretion systems are one of the main machineries in Gram-negative bacteria involved in the secretion of a wide range of substrates from the cytoplasm across the inner and outer membrane in one step to the extracellular space. The range of substrates varies from small proteins up to large surface layer proteins of about 900 kDa. Most of the substrates have a non-cleavable C-terminal secretion signal and so-called GG repeats that are able to bind calcium ions. The translocator complex is composed of a trimeric outer membrane protein that provides a pore in the outer membrane. A multimeric membrane fusion protein spans the periplasm and forms a continuous channel connecting the outer membrane protein with a dimeric ATP-binding cassette transporter in the inner membrane. The ATP-binding cassette-transporter is thought to form a channel through the inner membrane and energizes the transport process. This review will provide a detailed view of the components of the translocator and will summarize structural as well as functional data.

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Awang, Tadsanee, Phoom Chairatana, Ranjit Vijayan, and Prapasiri Pongprayoon. "Evaluation of the Binding Mechanism of Human Defensin 5 in a Bacterial Membrane: A Simulation Study." International Journal of Molecular Sciences 22, no. 22 (November 17, 2021): 12401. http://dx.doi.org/10.3390/ijms222212401.

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Human α-defensin 5 (HD5) is a host-defense peptide exhibiting broad-spectrum antimicrobial activity. The lipopolysaccharide (LPS) layer on the Gram-negative bacterial membrane acts as a barrier to HD5 insertion. Therefore, the pore formation and binding mechanism remain unclear. Here, the binding mechanisms at five positions along the bacterial membrane axis were investigated using Molecular Dynamics. (MD) simulations. We found that HD5 initially placed at positions 1 to 3 moved up to the surface, while HD5 positioned at 4 and 5 remained within the membrane interacting with the middle and inner leaflet of the membrane, respectively. The arginines were key components for tighter binding with 3-deoxy-d-manno-octulosonic acid (KDO), phosphates of the outer and inner leaflets. KDO appeared to retard the HD5 penetration.

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Xiang, Quanju, Haiyan Wang, Zhongshan Wang, Yizheng Zhang, and Changjiang Dong. "Characterization of lipopolysaccharide transport protein complex." Open Life Sciences 9, no. 2 (February 1, 2014): 131–38. http://dx.doi.org/10.2478/s11535-013-0250-5.

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AbstractLipopolysaccharide (LPS) is an essential component of the outer membranes (OM) of most Gram-negative bacteria, which plays a crucial role in protection of the bacteria from toxic compounds and harsh conditions. The LPS is biosynthesized at the cytoplasmic side of inner membrane (IM), and then transported across the aqueous periplasmic compartment and assembled correctly at the outer membrane. This process is accomplished by seven LPS transport proteins (LptA-G), but the transport mechanism remains poorly understood. Here, we present findings by pull down assays in which the periplasmic component LptA interacts with both the IM complex LptBFGC and the OM complex LptDE in vitro, but not with complex LptBFG. Using purified Lpt proteins, we have successfully reconstituted the seven transport proteins as a complex in vitro. In addition, the LptC may play an essential role in regulating the conformation of LptBFG to secure the lipopolysaccharide from the inner membrane. Our results contribute to the understanding of lipopolysaccharide transport mechanism and will provide a platform to study the detailed mechanism of the LPS transport in vitro.

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Mensa, Bruk, Yong Ho Kim, Sungwook Choi, Richard Scott, Gregory A. Caputo, and William F. DeGrado. "Antibacterial Mechanism of Action of Arylamide Foldamers." Antimicrobial Agents and Chemotherapy 55, no. 11 (August 15, 2011): 5043–53. http://dx.doi.org/10.1128/aac.05009-11.

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ABSTRACTSmall arylamide foldamers designed to mimic the amphiphilic nature of antimicrobial peptides (AMPs) have shown potent bactericidal activity against both Gram-negative and Gram-positive strains without many of the drawbacks of natural AMPs. These foldamers were shown to cause large changes in the permeability of the outer membrane ofEscherichia coli. They cause more limited permeabilization of the inner membrane which reaches critical levels corresponding with the time required to bring about bacterial cell death. Transcriptional profiling ofE. colitreated with sublethal concentrations of the arylamides showed induction of genes related to membrane and oxidative stresses, with some overlap with the effects observed for polymyxin B. Protein secretion into the periplasm and the outer membrane is also compromised, possibly contributing to the lethality of the arylamide compounds. The induction of membrane stress response regulons such asrcscoupled with morphological changes at the membrane observed by electron microscopy suggests that the activity of the arylamides at the membrane represents a significant contribution to their mechanism of action.

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Urfer, Matthias, Jasmina Bogdanovic, Fabio Lo Monte, Kerstin Moehle, Katja Zerbe, Ulrich Omasits, Christian H. Ahrens, Gabriella Pessi, Leo Eberl, and John A. Robinson. "A Peptidomimetic Antibiotic Targets Outer Membrane Proteins and Disrupts Selectively the Outer Membrane in Escherichia coli." Journal of Biological Chemistry 291, no. 4 (December 1, 2015): 1921–32. http://dx.doi.org/10.1074/jbc.m115.691725.

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Increasing antibacterial resistance presents a major challenge in antibiotic discovery. One attractive target in Gram-negative bacteria is the unique asymmetric outer membrane (OM), which acts as a permeability barrier that protects the cell from external stresses, such as the presence of antibiotics. We describe a novel β-hairpin macrocyclic peptide JB-95 with potent antimicrobial activity against Escherichia coli. This peptide exhibits no cellular lytic activity, but electron microscopy and fluorescence studies reveal an ability to selectively disrupt the OM but not the inner membrane of E. coli. The selective targeting of the OM probably occurs through interactions of JB-95 with selected β-barrel OM proteins, including BamA and LptD as shown by photolabeling experiments. Membrane proteomic studies reveal rapid depletion of many β-barrel OM proteins from JB-95-treated E. coli, consistent with induction of a membrane stress response and/or direct inhibition of the Bam folding machine. The results suggest that lethal disruption of the OM by JB-95 occurs through a novel mechanism of action at key interaction sites within clusters of β-barrel proteins in the OM. These findings open new avenues for developing antibiotics that specifically target β-barrel proteins and the integrity of the Gram-negative OM.

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Tefsen, Boris, Martine P. Bos, Frank Beckers, Jan Tommassen, and Hans de Cock. "MsbA Is Not Required for Phospholipid Transport in Neisseria meningitidis." Journal of Biological Chemistry 280, no. 43 (August 25, 2005): 35961–66. http://dx.doi.org/10.1074/jbc.m509026200.

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The outer membrane of Gram-negative bacteria contains phospholipids and lipopolysaccharide (LPS) in the inner and outer leaflet, respectively. Little is known about the transport of the phospholipids from their site of synthesis to the outer membrane. The inner membrane protein MsbA of Escherichia coli, which is involved in the transport of LPS across the inner membrane, has been reported to be involved in phospholipid transport as well. Here, we have reported the construction and the characterization of a Neisseria meningitidis msbA mutant. The mutant was viable, and it showed a retarded growth phenotype and contained very low amounts of LPS. However, it produced an outer membrane, demonstrating that phospholipid transport was not affected by the mutation. Notably, higher amounts of phospholipids were produced in the msbA mutant than in its isogenic parental strain, provided that capsular biosynthesis was also disrupted. Although these results confirmed that MsbA functions in LPS transport, they also demonstrated that it is not required for phospholipid transport, at least not in N. meningitidis.

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Kim, Peter D., and William Firshein. "Isolation of an Inner Membrane-Derived Subfraction That Supports In Vitro Replication of a Mini-RK2 Plasmid inEscherichia coli." Journal of Bacteriology 182, no. 6 (March 15, 2000): 1757–60. http://dx.doi.org/10.1128/jb.182.6.1757-1760.2000.

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ABSTRACT Previous results have demonstrated that the inner, but not the outer, membrane fraction of Escherichia coli is the site of membrane-associated DNA replication of plasmid RK2, a broad-host-range plasmid capable of replication in a wide variety of gram-negative hosts (K. Michaels, J. Mei, and W. Firshein, Plasmid 32:19–31, 1994). To resolve the inner membrane replication site further, the procedure of Ishidate et al. (K. Ishidate, E. S. Creeger, J. Zrike, S. Deb, G. Glauner, T. J. MacAlister, and L. I. Rothfield, J. Biol. Chem. 261:428–443, 1986) was used to separate the inner membrane into a number of subfractions, of which only one, a small subfraction containing only 10% of the entire membrane, was found to synthesize DNA inhibited by antibody prepared against the plasmid-encoded initiation protein TrfA. This is the same subfraction that was also found to bind oriV and TrfA to the greatest extent in filter binding assays (J. Mei, S. Benashski, and W. Firshein, J. Bacteriol. 177:6766–6772, 1995).

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Journal articles on the topic 'Gram-negative inner membrane'

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