scholarly journals Structure of LetB reveals a tunnel for lipid transport across the bacterial envelope

2019 ◽  
Author(s):  
Georgia L. Isom ◽  
Nicolas Coudray ◽  
Mark R. MacRae ◽  
Collin T. McManus ◽  
Damian C. Ekiert ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Lipid Transport ◽  
Transport Systems ◽  
Permeability Barrier ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
E Coli ◽  
Outer Membranes ◽  
Hydrophobic Tunnel ◽  
Outer Membrane Permeability

Gram-negative bacteria are surrounded by an outer membrane composed of phospholipids and lipopolysaccharide (LPS), which acts as a barrier to the environment and contributes to antibiotic resistance. While mechanisms of LPS transport have been well characterised, systems that translocate phospholipids across the periplasm, such as MCE (Mammalian Cell Entry) transport systems, are less well understood. Here we show that E. coli MCE protein LetB (formerly YebT), forms a ∼0.6 megadalton complex in the periplasm. Our cryo-EM structure reveals that LetB consists of a stack of seven modular rings, creating a long hydrophobic tunnel through the centre of the complex. LetB is sufficiently large to span the gap between the inner and outer membranes, and mutations that shorten the tunnel abolish function. Lipids bind inside the tunnel, suggesting that it functions as a pathway for lipid transport. Cryo-EM structures in the open and closed states reveal a dynamic tunnel lining, with implications for gating or substrate translocation. Together, our results support a model in which LetB establishes a physical link between the bacterial inner and outer membranes, and creates a hydrophobic pathway for the translocation of lipids across the periplasm, to maintain the integrity of the outer membrane permeability barrier.

scholarly journals Correct Sorting of Lipoproteins into the Inner and Outer Membranes ofPseudomonas aeruginosaby theEscherichia coliLolCDE Transport System

mBio ◽  
2019 ◽  
Vol 10 (2) ◽  
Author(s):  
Christian Lorenz ◽  
Thomas J. Dougherty ◽  
Stephen Lory
Keyword(s):  
Escherichia Coli ◽  
Pseudomonas Aeruginosa ◽  
Outer Membrane ◽  
Transport Systems ◽  
Gram Negative Bacteria ◽  
Inner Membrane ◽  
Gram Negative ◽  
Content Type ◽  
E Coli ◽  
Outer Membranes

ABSTRACTBiogenesis of the outer membrane of Gram-negative bacteria depends on dedicated macromolecular transport systems. The LolABCDE proteins make up the machinery for lipoprotein trafficking from the inner membrane (IM) across the periplasm to the outer membrane (OM). The Lol apparatus is additionally responsible for differentiating OM lipoproteins from those for the IM. InEnterobacteriaceae, a default sorting mechanism has been proposed whereby an aspartic acid at position +2 of the mature lipoproteins prevents Lol recognition and leads to their IM retention. In other bacteria, the conservation of sequences immediately following the acylated cysteine is variable. Here we show that inPseudomonas aeruginosa, the three essential Lol proteins (LolCDE) can be replaced with those fromEscherichia coli. TheP. aeruginosalipoproteins MexA, OprM, PscJ, and FlgH, with different sequences at their N termini, were correctly sorted by either theE. coliorP. aeruginosaLolCDE. We further demonstrate that an inhibitor ofE. coliLolCDE is active againstP. aeruginosaonly when expressing theE. coliorthologues. Our work shows that Lol proteins recognize a wide range of signals, consisting of an acylated cysteine and a specific conformation of the adjacent domain, determining IM retention or transport to the OM.IMPORTANCEGram-negative bacteria build their outer membranes (OM) from components that are initially located in the inner membrane (IM). A fraction of lipoproteins is transferred to the OM by the transport machinery consisting of LolABCDE proteins. Our work demonstrates that the LolCDE complexes of the transport pathways ofEscherichia coliandPseudomonas aeruginosaare interchangeable, with theE. coliorthologues correctly sorting theP. aeruginosalipoproteins while retaining their sensitivity to a small-molecule inhibitor. These findings question the nature of IM retention signals, identified inE. colias aspartate at position +2 of mature lipoproteins. We propose an alternative model for the sorting of IM and OM lipoproteins based on their relative affinities for the IM and the ability of the promiscuous sorting machinery to deliver lipoproteins to their functional sites in the OM.

scholarly journals DpaA detaches Braun’s lipoprotein from peptidoglycan

2021 ◽  
Author(s):  
Matthias Winkle ◽  
Víctor M. Hernández-Rocamora ◽  
Karthik Pullela ◽  
Emily C. A. Goodall ◽  
Alessandra M. Martorana ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Cell Envelope ◽  
Stress Conditions ◽  
Permeability Barrier ◽  
Gram Negative Bacteria ◽  
Sequence Motifs ◽  
Gram Negative ◽  
E Coli ◽  
Impermeable Barrier ◽  
Unique Cell

ABSTRACTGram-negative bacteria have a unique cell envelope with a lipopolysaccharide-containing outer membrane that is tightly connected to a thin layer of peptidoglycan. The tight connection between the outer membrane and peptidoglycan is needed to maintain the outer membrane as an impermeable barrier for many toxic molecules and antibiotics. Enterobacteriaceae such as Escherichia coli covalently attach the abundant outer membrane-anchored lipoprotein Lpp (Braun’s lipoprotein) to tripeptides in peptidoglycan, mediated by the transpeptidases LdtA, LdtB and LdtC. LdtD and LdtE are members of the same family of LD-transpeptidases but they catalyse a different reaction, the formation of 3-3 cross-links in the peptidoglycan. The function of the sixth homologue in E. coli, LdtF remains unclear, although it has been shown to become essential in cells with inhibited LPS export to the outer membrane. We now show that LdtF hydrolyses the Lpp-peptidoglycan linkage, detaching Lpp from peptidoglycan, and have renamed LdtF to peptidoglycan meso-diaminopimelic acid protein amidase A (DpaA). We show that the detachment of Lpp from peptidoglycan is beneficial for the cell under certain stress conditions and that the deletion of dpaA allows frequent transposon inactivation in the lapB (yciM) gene, whose product down-regulates lipopolysaccharide biosynthesis. DpaA-like proteins have characteristic sequence motifs and are present in many Gram-negative bacteria of which some have no Lpp, raising the possibility that DpaA has other substrates in these species. Overall, our data show that the Lpp-peptidoglycan linkage in E. coli is more dynamic than previously appreciated.IMPORTANCEGram-negative bacteria have a complex cell envelope with two membranes and a periplasm containing the peptidoglycan layer. The outer membrane is firmly connected to the peptidoglycan by highly abundant proteins. The outer membrane-anchored Braun’s lipoprotein (Lpp) is the most abundant protein in E. coli and about one third of the Lpp molecules become covalently attached to tripeptides in peptidoglycan. The attachment of Lpp to peptidoglycan stabilizes the cell envelope and is crucial for the outer membrane to function as a permeability barrier for a range of toxic molecules and antibiotics. So far the attachment of Lpp to peptidoglycan has been considered to be irreversible. We have now identified an amidase, DpaA, which is capable of detaching Lpp from PG and we show that the detachment of Lpp is important under certain stress conditions. DpaA-like proteins are present in many Gram-negative bacteria and may have different substrates in these species.

scholarly journals DpaA Detaches Braun’s Lipoprotein from Peptidoglycan

mBio ◽  
2021 ◽  
Vol 12 (3) ◽  
Author(s):  
Matthias Winkle ◽  
Víctor M. Hernández-Rocamora ◽  
Karthik Pullela ◽  
Emily C. A. Goodall ◽  
Alessandra M. Martorana ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Cell Envelope ◽  
Stress Conditions ◽  
Permeability Barrier ◽  
Gram Negative Bacteria ◽  
Sequence Motifs ◽  
Gram Negative ◽  
Content Type ◽  
E Coli ◽  
Impermeable Barrier

ABSTRACT Gram-negative bacteria have a unique cell envelope with a lipopolysaccharide-containing outer membrane that is tightly connected to a thin layer of peptidoglycan. The tight connection between the outer membrane and peptidoglycan is needed to maintain the outer membrane as an impermeable barrier for many toxic molecules and antibiotics. Enterobacteriaceae such as Escherichia coli covalently attach the abundant outer membrane-anchored lipoprotein Lpp (Braun’s lipoprotein) to tripeptides in peptidoglycan, mediated by the transpeptidases LdtA, LdtB, and LdtC. LdtD and LdtE are members of the same family of ld-transpeptidases but they catalyze a different reaction, the formation of 3-3 cross-links in the peptidoglycan. The function of the sixth homologue in E. coli, LdtF, remains unclear, although it has been shown to become essential in cells with inhibited lipopolysaccharide export to the outer membrane. We now show that LdtF hydrolyzes the Lpp-peptidoglycan linkage, detaching Lpp from peptidoglycan, and have renamed LdtF to peptidoglycan meso-diaminopimelic acid protein amidase A (DpaA). We show that the detachment of Lpp from peptidoglycan is beneficial for the cell under certain stress conditions and that the deletion of dpaA allows frequent transposon inactivation in the lapB (yciM) gene, whose product downregulates lipopolysaccharide biosynthesis. DpaA-like proteins have characteristic sequence motifs and are present in many Gram-negative bacteria, of which some have no Lpp, raising the possibility that DpaA has other substrates in these species. Overall, our data show that the Lpp-peptidoglycan linkage in E. coli is more dynamic than previously appreciated. IMPORTANCE Gram-negative bacteria have a complex cell envelope with two membranes and a periplasm containing the peptidoglycan layer. The outer membrane is firmly connected to the peptidoglycan by highly abundant proteins. The outer membrane-anchored Braun’s lipoprotein (Lpp) is the most abundant protein in E. coli, and about one-third of the Lpp molecules become covalently attached to tripeptides in peptidoglycan. The attachment of Lpp to peptidoglycan stabilizes the cell envelope and is crucial for the outer membrane to function as a permeability barrier for a range of toxic molecules and antibiotics. So far, the attachment of Lpp to peptidoglycan has been considered to be irreversible. We have now identified an amidase, DpaA, which is capable of detaching Lpp from peptidoglycan, and we show that the detachment of Lpp is important under certain stress conditions. DpaA-like proteins are present in many Gram-negative bacteria and may have different substrates in these species.

scholarly journals Architectures of lipid transport systems for the bacterial outer membrane

2016 ◽  
Author(s):  
Damian C. Ekiert ◽  
Gira Bhabha ◽  
Garrett Greenan ◽  
Sergey Ovchinnikov ◽  
Jeffery S. Cox ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Lipid Binding ◽  
Cell Entry ◽  
Lipid Transport ◽  
Transport Systems ◽  
List Type ◽  
Central Channel ◽  
Membrane Protein Complex ◽  
E Coli ◽  
Outer Membranes

SUMMARYHow phospholipids are trafficked between the bacterial inner and outer membranes through the intervening hydrophilic space of the periplasm is not known. Here we report that members of the mammalian cell entry (MCE) protein family form structurally diverse hexameric rings and barrels with a central channel capable of mediating lipid transport. TheE. coliMCE protein, MlaD, forms a ring as part of a larger ABC transporter complex in the inner membrane, and employs a soluble lipid-binding protein to ferry lipids between MlaD and an outer membrane protein complex. In contrast, EM structures of two otherE. coliMCE proteins show that YebT forms an elongated tube consisting of seven stacked MCE rings, and PqiB adopts a syringe-like architecture. Both YebT and PqiB create channels of sufficient length to span the entire periplasmic space. This work reveals diverse architectures of highly conserved protein-based channels implicated in the transport of lipids between the inner and outer membranes of bacteria and some eukaryotic organelles.HIGHLIGHTSMCE proteins adopt diverse architectures for transporting lipids across the bacterial periplasmCryo-EM and X-ray structures reveal how the MlaFEDB complex, along with MlaC, might shuttle lipids across the periplasm3.9 Å cryo-EM structure of PqiB reveals a syringe-like architecture with a continuous central channelYebT forms a a segmented tube-like structure, and YebT and PqiB are poised to directly link the inner and outer membranes to facilitate lipid transport.

scholarly journals Outer Membrane Disruption Overcomes Intrinsic, Acquired, and Spontaneous Antibiotic Resistance

mBio ◽  
2020 ◽  
Vol 11 (5) ◽  
Author(s):  
Craig R. MacNair ◽  
Eric D. Brown
Keyword(s):  
Antibiotic Resistance ◽  
Outer Membrane ◽  
Biofilm Formation ◽  
Antibiotic Use ◽  
Membrane Disruption ◽  
Permeability Barrier ◽  
Gram Negative Bacteria ◽  
Gram Positive ◽  
Gram Negative ◽  
Outer Membrane Permeability

ABSTRACT Disruption of the outer membrane (OM) barrier allows for the entry of otherwise inactive antimicrobials into Gram-negative pathogens. Numerous efforts to implement this approach have identified a large number of OM perturbants that sensitize Gram-negative bacteria to many clinically available Gram-positive active antibiotics. However, there is a dearth of investigation into the strengths and limitations of this therapeutic strategy, with an overwhelming focus on characterization of individual potentiator molecules. Herein, we look to explore the utility of exploiting OM perturbation to sensitize Gram-negative pathogens to otherwise inactive antimicrobials. We identify the ability of OM disruption to change the rules of Gram-negative entry, overcome preexisting and spontaneous resistance, and impact biofilm formation. Disruption of the OM expands the threshold of hydrophobicity compatible with Gram-negative activity to include hydrophobic molecules. We demonstrate that while resistance to Gram-positive active antibiotics is surprisingly common in Gram-negative pathogens, OM perturbation overcomes many antibiotic inactivation determinants. Further, we find that OM perturbation reduces the rate of spontaneous resistance to rifampicin and impairs biofilm formation. Together, these data suggest that OM disruption overcomes many of the traditional hurdles encountered during antibiotic treatment and is a high priority approach for further development. IMPORTANCE The spread of antibiotic resistance is an urgent threat to global health that necessitates new therapeutics. Treatments for Gram-negative pathogens are particularly challenging to identify due to the robust outer membrane permeability barrier in these organisms. Recent discovery efforts have attempted to overcome this hurdle by disrupting the outer membrane using chemical perturbants and have yielded several new peptides and small molecules that allow the entry of otherwise inactive antimicrobials. However, a comprehensive investigation into the strengths and limitations of outer membrane perturbants as antibiotic partners is currently lacking. Herein, we interrogate the interaction between outer membrane perturbation and several common impediments to effective antibiotic use. Interestingly, we discover that outer membrane disruption is able to overcome intrinsic, spontaneous, and acquired antibiotic resistance in Gram-negative bacteria, meriting increased attention toward this approach.

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.

scholarly journals “Asymmetry Is the Rhythmic Expression of Functional Design,” a Quotation from Jan Tschichold

2020 ◽  
Vol 202 (18) ◽  
Author(s):  
Joe Lutkenhaus
Keyword(s):  
Outer Membrane ◽  
Regulatory Role ◽  
Permeability Barrier ◽  
Gram Negative Bacteria ◽  
Functional Design ◽  
Lipid Asymmetry ◽  
Gram Negative ◽  
Outer Membranes ◽  
Rhythmic Expression

ABSTRACT The outer membranes of Gram-negative bacteria provide a permeability barrier to antibiotics and other harmful chemicals. The integrity of this barrier relies on the maintenance of the lipid asymmetry of the outer membrane, and studies of suppressors of a decades-old mutant reveal that YejM plays a key regulatory role and provide a model for the maintenance of this asymmetry.

scholarly journals Outer Membrane Lipid Secretion and the Innate Immune Response to Gram-Negative Bacteria

2020 ◽  
Vol 88 (7) ◽  
Author(s):  
Nicole P. Giordano ◽  
Melina B. Cian ◽  
Zachary D. Dalebroux
Keyword(s):  
Outer Membrane ◽  
Mammalian Cells ◽  
Lipid A ◽  
Membrane Lipid ◽  
Membrane Curvature ◽  
Host Immunity ◽  
Host Recognition ◽  
Permeability Barrier ◽  
Gram Negative Bacteria ◽  
Gram Negative

ABSTRACT The outer membrane (OM) of Gram-negative bacteria is an asymmetric lipid bilayer that consists of inner leaflet phospholipids and outer leaflet lipopolysaccharides (LPS). The asymmetric character and unique biochemistry of LPS molecules contribute to the OM’s ability to function as a molecular permeability barrier that protects the bacterium against hazards in the environment. Assembly and regulation of the OM have been extensively studied for understanding mechanisms of antibiotic resistance and bacterial defense against host immunity; however, there is little knowledge on how Gram-negative bacteria release their OMs into their environment to manipulate their hosts. Discoveries in bacterial lipid trafficking, OM lipid homeostasis, and host recognition of microbial patterns have shed new light on how microbes secrete OM vesicles (OMVs) to influence inflammation, cell death, and disease pathogenesis. Pathogens release OMVs that contain phospholipids, like cardiolipins, and components of LPS molecules, like lipid A endotoxins. These multiacylated lipid amphiphiles are molecular patterns that are differentially detected by host receptors like the Toll-like receptor 4/myeloid differentiation factor 2 complex (TLR4/MD-2), mouse caspase-11, and human caspases 4 and 5. We discuss how lipid ligands on OMVs engage these pattern recognition receptors on the membranes and in the cytosol of mammalian cells. We then detail how bacteria regulate OM lipid asymmetry, negative membrane curvature, and the phospholipid-to-LPS ratio to control OMV formation. The goal is to highlight intersections between OM lipid regulation and host immunity and to provide working models for how bacterial lipids influence vesicle formation.

scholarly journals A small-molecule inhibitor of BamA impervious to efflux and the outer membrane permeability barrier

2019 ◽  
Vol 116 (43) ◽  
pp. 21748-21757 ◽  
Author(s):  
Elizabeth M. Hart ◽  
Angela M. Mitchell ◽  
Anna Konovalova ◽  
Marcin Grabowicz ◽  
Jessica Sheng ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Small Molecule ◽  
Cytoplasmic Membrane ◽  
Multidrug Resistant ◽  
Resistant Bacteria ◽  
Permeability Barrier ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Bam Complex ◽  
Induced Aggregation

The development of new antimicrobial drugs is a priority to combat the increasing spread of multidrug-resistant bacteria. This development is especially problematic in gram-negative bacteria due to the outer membrane (OM) permeability barrier and multidrug efflux pumps. Therefore, we screened for compounds that target essential, nonredundant, surface-exposed processes in gram-negative bacteria. We identified a compound, MRL-494, that inhibits assembly of OM proteins (OMPs) by the β-barrel assembly machine (BAM complex). The BAM complex contains one essential surface-exposed protein, BamA. We constructed a bamA mutagenesis library, screened for resistance to MRL-494, and identified the mutation bamAE470K. BamAE470K restores OMP biogenesis in the presence of MRL-494. The mutant protein has both altered conformation and activity, suggesting it could either inhibit MRL-494 binding or allow BamA to function in the presence of MRL-494. By cellular thermal shift assay (CETSA), we determined that MRL-494 stabilizes BamA and BamAE470K from thermally induced aggregation, indicating direct or proximal binding to both BamA and BamAE470K. Thus, it is the altered activity of BamAE470K responsible for resistance to MRL-494. Strikingly, MRL-494 possesses a second mechanism of action that kills gram-positive organisms. In microbes lacking an OM, MRL-494 lethally disrupts the cytoplasmic membrane. We suggest that the compound cannot disrupt the cytoplasmic membrane of gram-negative bacteria because it cannot penetrate the OM. Instead, MRL-494 inhibits OMP biogenesis from outside the OM by targeting BamA. The identification of a small molecule that inhibits OMP biogenesis at the cell surface represents a distinct class of antibacterial agents.

scholarly journals A Peptidomimetic Antibiotic Targets Outer Membrane Proteins and Disrupts Selectively the Outer Membrane in Escherichia coli

2015 ◽  
Vol 291 (4) ◽  
pp. 1921-1932 ◽  
Author(s):  
Matthias Urfer ◽  
Jasmina Bogdanovic ◽  
Fabio Lo Monte ◽  
Kerstin Moehle ◽  
Katja Zerbe ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Outer Membrane ◽  
Outer Membrane Proteins ◽  
Permeability Barrier ◽  
Gram Negative ◽  
Fluorescence Studies ◽  
E Coli ◽  
Interaction Sites ◽  
External Stresses ◽  
Antibiotic Discovery

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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