scholarly journals Asymmetric phospholipid: lipopolysaccharide bilayers; a Gram-negative bacterial outer membrane mimic

2013 ◽  
Vol 10 (89) ◽  
pp. 20130810 ◽  
Author(s):  
Luke A. Clifton ◽  
Maximilian W. A. Skoda ◽  
Emma L. Daulton ◽  
Arwel V. Hughes ◽  
Anton P. Le Brun ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Lipid A ◽  
Membrane Dynamics ◽  
Gram Negative ◽  
Outer Leaflet ◽  
Physical Study ◽  
Bacterial Outer Membrane ◽  
Membrane Models ◽  
Initial Deposition

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.

scholarly journals Effect of Divalent Cation Removal on the Structure of Gram-Negative Bacterial Outer Membrane Models

Langmuir ◽  
2014 ◽  
Vol 31 (1) ◽  
pp. 404-412 ◽  
Author(s):  
Luke A. Clifton ◽  
Maximilian W. A. Skoda ◽  
Anton P. Le Brun ◽  
Filip Ciesielski ◽  
Ivan Kuzmenko ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Divalent Cation ◽  
Gram Negative ◽  
Bacterial Outer Membrane ◽  
Membrane Models

scholarly journals Mutation and Suppressor Analysis of the Essential Lipopolysaccharide Transport Protein LptA Reveals Strategies To Overcome Severe Outer Membrane Permeability Defects in Escherichia coli

2017 ◽  
Vol 200 (2) ◽  
Author(s):  
Federica A. Falchi ◽  
Elisa A. Maccagni ◽  
Simone Puccio ◽  
Clelia Peano ◽  
Cristina De Castro ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Lipid A ◽  
Antibiotic Sensitivity ◽  
Major Constituent ◽  
Permeability Barrier ◽  
Gram Negative Bacteria ◽  
Inner Membrane ◽  
Gram Negative ◽  
Outer Leaflet ◽  
Increased Sensitivity

ABSTRACTIn Gram-negative bacteria, lipopolysaccharide (LPS) contributes to the robust permeability barrier of the outer membrane (OM), preventing the entry of toxic molecules, such as detergents and antibiotics. LPS is transported from the inner membrane (IM) to the OM by the Lpt multiprotein machinery. Defects in LPS transport compromise LPS assembly at the OM and result in increased antibiotic sensitivity. LptA is a key component of the Lpt machine that interacts with the IM protein LptC and chaperones LPS through the periplasm. We report here the construction oflptA41, a quadruple mutant in four conserved amino acids potentially involved in LPS or LptC binding. Although viable, the mutant displays increased sensitivity to several antibiotics (bacitracin, rifampin, and novobiocin) and the detergent SDS, suggesting thatlptA41affects LPS transport. Indeed,lptA41is defective in Lpt complex assembly, and its lipid A carries modifications diagnostic of LPS transport defects. We also selected and characterized two phenotypic bacitracin-resistant suppressors oflptA41. One mutant, in which only bacitracin sensitivity is suppressed, harbors a small in-frame deletion inmlaA, which codes for an OM lipoprotein involved in maintaining OM asymmetry by reducing accumulation of phospholipids in the outer leaflet. The other mutant, in which bacitracin, rifampin, and SDS sensitivity is suppressed, harbors an additional amino acid substitution in LptA41 and a nonsense mutation inopgH, encoding a glycosyltransferase involved in periplasmic membrane-derived oligosaccharide synthesis. Characterization of the suppressor mutants highlights different strategies adopted by the cell to overcome OM defects caused by impaired LPS transport.IMPORTANCELipopolysaccharide (LPS) is the major constituent of the outer membrane (OM) of most Gram-negative bacteria, forming a barrier against antibiotics. LPS is synthesized at the inner membrane (IM), transported across the periplasm, and assembled at the OM by the multiprotein Lpt complex. LptA is the periplasmic component of the Lpt complex, which bridges IM and OM and ferries LPS across the periplasm. How the cell coordinates the processes involved in OM biogenesis is not completely understood. We generated a mutant partially defective inlptAthat exhibited increased sensitivity to antibiotics and selected for suppressors of the mutant. The analysis of two independent suppressors revealed different strategies adopted by the cell to overcome defects in LPS biogenesis.

scholarly journals Structure-guided enzymology of the lipid A acyltransferase LpxM reveals a dual activity mechanism

2016 ◽  
Vol 113 (41) ◽  
pp. E6064-E6071 ◽  
Author(s):  
Dustin Dovala ◽  
Christopher M. Rath ◽  
Qijun Hu ◽  
William S. Sawyer ◽  
Steven Shia ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Lipid A ◽  
Structural Information ◽  
Mechanistic Model ◽  
Permeability Barrier ◽  
Gram Negative Bacteria ◽  
Label Free ◽  
Gram Negative ◽  
Outer Leaflet ◽  
Domains Of Life

Gram-negative bacteria possess a characteristic outer membrane, of which the lipid A constituent elicits a strong host immune response through the Toll-like receptor 4 complex, and acts as a component of the permeability barrier to prevent uptake of bactericidal compounds. Lipid A species comprise the bulk of the outer leaflet of the outer membrane and are produced through a multistep biosynthetic pathway conserved in most Gram-negative bacteria. The final steps in this pathway involve the secondary acylation of lipid A precursors. These are catalyzed by members of a superfamily of enzymes known as lysophospholipid acyltransferases (LPLATs), which are present in all domains of life and play important roles in diverse biological processes. To date, characterization of this clinically important class of enzymes has been limited by a lack of structural information and the availability of only low-throughput biochemical assays. In this work, we present the structure of the bacterial LPLAT protein LpxM, and we describe a high-throughput, label-free mass spectrometric assay to characterize acyltransferase enzymatic activity. Using our structure and assay, we identify an LPLAT thioesterase activity, and we provide experimental evidence to support an ordered-binding and “reset” mechanistic model for LpxM function. This work enables the interrogation of other bacterial acyltransferases’ structure–mechanism relationships, and the assay described herein provides a foundation for quantitatively characterizing the enzymology of any number of clinically relevant LPLAT proteins.

scholarly journals Dewetting-induced formation and mechanical properties of synthetic bacterial outer membrane models (GUVs) with controlled inner-leaflet lipid composition

Soft Matter ◽  
2019 ◽  
Vol 15 (19) ◽  
pp. 3938-3948 ◽  
Author(s):  
Sepehr Maktabi ◽  
Jeffrey W. Schertzer ◽  
Paul R. Chiarot
Keyword(s):  
Mechanical Properties ◽  
Outer Membrane ◽  
Lipid Composition ◽  
Membrane Lipid ◽  
Giant Unilamellar Vesicles ◽  
Gram Negative ◽  
Membrane Lipid Composition ◽  
Bacterial Outer Membrane ◽  
Membrane Models ◽  
Microfluidic Technique

We report on a microfluidic technique for fabricating monodisperse asymmetric giant unilamellar vesicles (GUVs) possessing the Gram-negative bacterial outer membrane lipid composition.

scholarly journals Interplay ofKlebsiella pneumoniaefabZandlpxCMutations Leads to LpxC Inhibitor-Dependent Growth Resulting from Loss of Membrane Homeostasis

mSphere ◽  
2018 ◽  
Vol 3 (5) ◽  
Author(s):  
Mina Mostafavi ◽  
Lisha Wang ◽  
Lili Xie ◽  
Kenneth T. Takeoka ◽  
Daryl L. Richie ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Lipid A ◽  
Gram Negative Bacteria ◽  
Resistance Mutations ◽  
Cellular Mechanisms ◽  
Gram Negative ◽  
Content Type ◽  
Outer Leaflet ◽  
Chemical Inhibition ◽  
Dependent Growth

ABSTRACTTight coordination of inner and outer membrane biosynthesis is very important in Gram-negative bacteria. Biosynthesis of the lipid A moiety of lipopolysaccharide, which comprises the outer leaflet of the outer membrane has garnered interest for Gram-negative antibacterial discovery. In particular, several potent inhibitors of LpxC (the first committed step of the lipid A pathway) are described. Here we show that serial passaging ofKlebsiella pneumoniaein increasing levels of an LpxC inhibitor yielded mutants that grew only in the presence of the inhibitor. These strains had mutations infabZandlpxCoccurring together (encoding either FabZR121L/LpxCV37Gor FabZF51L/LpxCV37G).K. pneumoniaemutants having only LpxCV37Gor LpxCV37Aor various FabZ mutations alone were less susceptible to the LpxC inhibitor and did not require LpxC inhibition for growth. Western blotting revealed that LpxCV37Gaccumulated to high levels, and electron microscopy of cells harboring FabZR121L/LpxCV37Gindicated an extreme accumulation of membrane in the periplasm when cells were subcultured without LpxC inhibitor. Significant accumulation of detergent-like lipid A pathway intermediates that occur downstream of LpxC (e.g., lipid X and disaccharide monophosphate [DSMP]) was also seen. Taken together, our results suggest that redirection of lipid A pathway substrate by less active FabZ variants, combined with increased activity from LpxCV37Gwas overdriving the lipid A pathway, necessitating LpxC chemical inhibition, since native cellular maintenance of membrane homeostasis was no longer functioning.IMPORTANCEEmergence of antibiotic resistance has prompted efforts to identify and optimize novel inhibitors of antibacterial targets such as LpxC. This enzyme catalyzes the first committed step of lipid A synthesis, which is necessary to generate lipopolysaccharide and ultimately the Gram-negative protective outer membrane. Investigation of this pathway and its interrelationship with inner membrane (phospholipid) biosynthesis or other pathways is therefore highly important to the fundamental understanding of Gram-negative bacteria and by extension to antibiotic discovery. Here we exploited the availability of a novel LpxC inhibitor to engender the generation ofK. pneumoniaeresistant mutants whose growth depends on chemical inhibition of LpxC. Inhibitor dependency resulted from the interaction of different resistance mutations and was based on loss of normal cellular mechanisms required to establish membrane homeostasis. This study provides new insights into the importance of this process inK. pneumoniaeand how it may be linked to novel biosynthetic pathway inhibitors.

scholarly journals The outer membrane proteins OmpA, FhuA, OmpF, EstA, BtuB and OmpX have unique lipopolysaccharide fingerprints

2018 ◽  
Author(s):  
Jonathan Shearer ◽  
Damien Jefferies ◽  
Syma Khalid
Keyword(s):  
Outer Membrane ◽  
Outer Membrane Proteins ◽  
Coarse Grain ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Unique Pattern ◽  
E Coli ◽  
Outer Leaflet ◽  
Wide Range

AbstractThe outer membrane of Gram-negative bacteria has a highly complex asymmetrical architecture, containing a mixture of phospholipids in the inner leaflet and in the outer leaflet they contain almost exclusively lipopolysaccharide (LPS) molecules. In E. coli, the outer membrane contains a wide range proteins with a beta barrel architecture, that vary in size from the smallest having eight strands to larger barrels composed of twenty-two strands. Here we report coarse-grain molecular dynamics simulations of six proteins from the E. coli outer membrane OmpA, OmpX, BtuB, FhuA, OmpF and EstA in a range of membrane environments, which are representative of the in vivo for different strains of E. coli. We show that each protein has a unique pattern of interaction with the surrounding membrane, which is influenced by the composition of the protein, the level of LPS in the outer leaflet and the differing mobilities of the lipids in the two leaflets of the membrane. Overall we present analyses from over 200 microseconds of simulation for each protein.Author summaryWe present data from over 200 microseconds of coarse-grain simulations that show the complexities of protein-lipid interactions within the outer membranes of Gram-negative bacteria. We show that the slow movement of lipolysaccharide molecules necessitate simulations of over 30 microsecond duration to achieve converged properties such as protein tilt angle. Each of the six proteins studied here shows a unique pattern of interactions with the outer membrane and thus constitute a ‘fingerprint’ or ‘signature’.

scholarly journals RickettsiaLipid A Biosynthesis Utilizes the Late Acyltransferase LpxJ for Secondary Fatty Acid Addition

2018 ◽  
Vol 200 (19) ◽  
Author(s):  
Mark L. Guillotte ◽  
Joseph J. Gillespie ◽  
Courtney E. Chandler ◽  
M. Sayeedur Rahman ◽  
Robert K. Ernst ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Outer Membrane ◽  
Lipid A ◽  
Spotted Fever ◽  
Acyl Chain ◽  
Membrane Dynamics ◽  
Rickettsia Typhi ◽  
Gram Negative ◽  
Content Type ◽  
Host Pathogen

ABSTRACTMembers of theRickettsiagenus are obligate intracellular, Gram-negative coccobacilli that infect mammalian and arthropod hosts. Several rickettsial species are human pathogens and are transmitted by blood-feeding arthropods. In Gram-negative parasites, the outer membrane (OM) sits at the nexus of the host-pathogen interaction and is rich in lipopolysaccharide (LPS). The lipid A component of LPS anchors the molecule to the bacterial surface and is an endotoxic agonist of Toll-like receptor 4 (TLR4). Despite the apparent importance of lipid A in maintaining OM integrity, as well as its inflammatory potential during infection, this molecule is poorly characterized inRickettsiapathogens. In this work, we have identified and characterized new members of the recently discovered LpxJ family of lipid A acyltransferases in bothRickettsia typhiandRickettsia rickettsii, the etiological agents of murine typhus and Rocky Mountain spotted fever, respectively. Our results demonstrate that these enzymes catalyze the addition of a secondary acyl chain (C14/C16) to the 3′-linked primary acyl chain of the lipid A moiety in the final steps of the Raetz pathway of lipid A biosynthesis. Since lipid A architecture is fundamental to bacterial OM integrity, we believe that rickettsial LpxJ may be important in maintaining membrane dynamics to facilitate molecular interactions at the host-pathogen interface that are required for adhesion and invasion of mammalian cells. This work contributes to our understanding of rickettsial outer membrane physiology and sets a foundation for further exploration of the envelope and its role in pathogenesis.IMPORTANCELipopolysaccharide (LPS) triggers an inflammatory response through the TLR4-MD2 receptor complex and inflammatory caspases, a process mediated by the lipid A moiety of LPS. Species ofRickettsiadirectly engage both extracellular and intracellular immunosurveillance, yet little is known about rickettsial lipid A. Here, we demonstrate that the alternative lipid A acyltransferase, LpxJ, fromRickettsia typhiandR. rickettsiicatalyzes the addition of C16fatty acid chains into the lipid A 3′-linked primary acyl chain, accounting for major structural differences relative to the highly inflammatory lipid A ofEscherichia coli.

scholarly journals Current Progress in the Structural and Biochemical Characterization of Proteins Involved in the Assembly of Lipopolysaccharide

2018 ◽  
Vol 2018 ◽  
pp. 1-32 ◽  
Author(s):  
Thomas E. Bohl ◽  
Hideki Aihara
Keyword(s):  
Outer Membrane ◽  
Lipid A ◽  
Biochemical Characterization ◽  
Protein Structures ◽  
Transport Systems ◽  
Biosynthetic Enzymes ◽  
Human Pathogens ◽  
Core Oligosaccharide ◽  
Gram Negative ◽  
Outer Leaflet

The lipid component of the outer leaflet of the outer membrane of Gram-negative bacteria is primarily composed of the glycolipid lipopolysaccharide (LPS), which serves to form a protective barrier against hydrophobic toxins and many antibiotics. LPS is comprised of three regions: the lipid A membrane anchor, the nonrepeating core oligosaccharide, and the repeating O-antigen polysaccharide. The lipid A portion is also referred to as endotoxin as its overstimulation of the toll-like receptor 4 during systemic infection precipitates potentially fatal septic shock. Because of the importance of LPS for the viability and virulence of human pathogens, understanding how LPS is synthesized and transported to the outer leaflet of the outer membrane is important for developing novel antibiotics to combat resistant Gram-negative strains. The following review describes the current state of our understanding of the proteins responsible for the synthesis and transport of LPS with an emphasis on the contribution of protein structures to our understanding of their functions. Because the lipid A portion of LPS is relatively well conserved, a detailed description of the biosynthetic enzymes in the Raetz pathway of lipid A synthesis is provided. Conversely, less well-conserved biosynthetic enzymes later in LPS synthesis are described primarily to demonstrate conserved principles of LPS synthesis. Finally, the conserved LPS transport systems are described in detail.

scholarly journals Phospholipid retention in the absence of asymmetry strengthens the outer membrane permeability barrier to last-resort antibiotics

2018 ◽  
Vol 115 (36) ◽  
pp. E8518-E8527 ◽  
Author(s):  
Matthew J. Powers ◽  
M. Stephen Trent
Keyword(s):  
Membrane Permeability ◽  
Outer Membrane ◽  
Lipid A ◽  
Permeability Barrier ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Outer Leaflet ◽  
Two Factors ◽  
Evolution Experiment ◽  
Outer Membrane Permeability

The outer membrane of Gram-negative bacteria is a critical barrier that prevents entry of noxious compounds. Integral to this functionality is the presence of lipopolysaccharide (LPS) or lipooligosaccharide (LOS), a molecule that is located exclusively in the outer leaflet of the outer membrane. Its lipid anchor, lipid A, is a glycolipid whose hydrophobicity and net negative charge are primarily responsible for the robustness of the membrane. Because of this, lipid A is a hallmark of Gram-negative physiology and is generally essential for survival. Rare exceptions have been described, includingAcinetobacter baumannii, which can survive in the absence of lipid A, albeit with significant growth and membrane permeability defects. Here, we show by an evolution experiment that LOS-deficientA. baumanniican rapidly improve fitness over the course of only 120 generations. We identified two factors which negatively contribute to fitness in the absence of LOS, Mla and PldA. These proteins are involved in glycerophospholipid transport (Mla) and lipid degradation (PldA); both are active only on mislocalized, surface-exposed glycerophospholipids. Elimination of these two mechanisms was sufficient to cause a drastic fitness improvement in LOS-deficientA. baumannii. The LOS-deficient double mutant grows as robustly as LOS-positive wild-type bacteria while remaining resistant to the last-resort polymyxin antibiotics. These data provide strong biological evidence for the directionality of Mla-mediated glycerophospholipid transport in Gram-negative bacteria and furthers our knowledge of asymmetry-maintenance mechanisms in the context of the outer membrane barrier.

Sign in / Sign up

Export Citation Format

Share Document