scholarly journals Structure of bacterial phospholipid transporter MlaFEDB with substrate bound

2020 ◽  
Author(s):  
Nicolas Coudray ◽  
Georgia L. Isom ◽  
Mark R. MacRae ◽  
Mariyah N. Saiduddin ◽  
Gira Bhabha ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Cell Envelope ◽  
Sequence Similarity ◽  
Substrate Binding ◽  
Membrane Integrity ◽  
Binding Pocket ◽  
Pathogen Virulence ◽  
Phospholipid Transport ◽  
Distinct Features

In double-membraned bacteria, phospholipids must be transported across the cell envelope to maintain the outer membrane barrier, which plays a key role in antibiotic resistance and pathogen virulence. The Mla system has been implicated in phospholipid trafficking and outer membrane integrity, and includes an ABC transporter complex, MlaFEDB. The transmembrane subunit, MlaE, has minimal sequence similarity to other ABC transporters, and the structure of the entire inner membrane MlaFEDB complex remains unknown. Here we report the cryo-EM structure of the MlaFEDB complex at 3.05 Å resolution. Our structure reveals that while MlaE has many distinct features, it is distantly related to the LPS and MacAB transporters, as well as the eukaryotic ABCA/ABCG families. MlaE adopts an outward-open conformation, resulting in a continuous pathway for phospholipid transport from the MlaE substrate-binding site to the pore formed by the ring of MlaD. Unexpectedly, two phospholipids are bound in the substrate-binding pocket of MlaFEDB, raising the possibility that multiple lipid substrates may be translocated each transport cycle. Site-specific crosslinking confirms that lipids bind in this pocket in vivo. Our structure provides mechanistic insight into substrate recognition and transport by the MlaFEDB complex.

scholarly journals Structure of bacterial phospholipid transporter MlaFEDB with substrate bound

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Nicolas Coudray ◽  
Georgia L Isom ◽  
Mark R MacRae ◽  
Mariyah N Saiduddin ◽  
Gira Bhabha ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Cell Envelope ◽  
Sequence Similarity ◽  
Substrate Recognition ◽  
Membrane Integrity ◽  
Transport Pathway ◽  
Mechanistic Insight ◽  
Membrane Barrier ◽  
Phospholipid Transport ◽  
Insight Into

In double-membraned bacteria, phospholipid transport across the cell envelope is critical to maintain the outer membrane barrier, which plays a key role in virulence and antibiotic resistance. An MCE transport system called Mla has been implicated in phospholipid trafficking and outer membrane integrity, and includes an ABC transporter, MlaFEDB. The transmembrane subunit, MlaE, has minimal sequence similarity to other transporters, and the structure of the entire inner-membrane MlaFEDB complex remains unknown. Here, we report the cryo-EM structure of MlaFEDB at 3.05 Å resolution, revealing distant relationships to the LPS and MacAB transporters, as well as the eukaryotic ABCA/ABCG families. A continuous transport pathway extends from the MlaE substrate-binding site, through the channel of MlaD, and into the periplasm. Unexpectedly, two phospholipids are bound to MlaFEDB, suggesting that multiple lipid substrates may be transported each cycle. Our structure provides mechanistic insight into substrate recognition and transport by MlaFEDB.

scholarly journals Cell Envelope Perturbation Induces Oxidative Stress and Changes in Iron Homeostasis in Vibrio cholerae

2009 ◽  
Vol 191 (17) ◽  
pp. 5398-5408 ◽  
Author(s):  
Aleksandra E. Sikora ◽  
Sinem Beyhan ◽  
Michael Bagdasarian ◽  
Fitnat H. Yildiz ◽  
Maria Sandkvist
Keyword(s):  
Oxidative Stress ◽  
Gene Expression ◽  
Outer Membrane ◽  
Vibrio Cholerae ◽  
Iron Homeostasis ◽  
Cell Envelope ◽  
Membrane Integrity ◽  
Genetic Alterations ◽  
Iron Storage ◽  
Study Gene Expression

ABSTRACT The Vibrio cholerae type II secretion (T2S) machinery is a multiprotein complex that spans the cell envelope. When the T2S system is inactivated, cholera toxin and other exoproteins accumulate in the periplasmic compartment. Additionally, loss of secretion via the T2S system leads to a reduced growth rate, compromised outer membrane integrity, and induction of the extracytoplasmic stress factor RpoE (A. E. Sikora, S. R. Lybarger, and M. Sandkvist, J. Bacteriol. 189:8484-8495, 2007). In this study, gene expression profiling reveals that inactivation of the T2S system alters the expression of genes encoding cell envelope components and proteins involved in central metabolism, chemotaxis, motility, oxidative stress, and iron storage and acquisition. Consistent with the gene expression data, molecular and biochemical analyses indicate that the T2S mutants suffer from internal oxidative stress and increased levels of intracellular ferrous iron. By using a tolA mutant of V. cholerae that shares a similar compromised membrane phenotype but maintains a functional T2S machinery, we show that the formation of radical oxygen species, induction of oxidative stress, and changes in iron physiology are likely general responses to cell envelope damage and are not unique to T2S mutants. Finally, we demonstrate that disruption of the V. cholerae cell envelope by chemical treatment with polymyxin B similarly results in induction of the RpoE-mediated stress response, increased sensitivity to oxidants, and a change in iron metabolism. We propose that many types of extracytoplasmic stresses, caused either by genetic alterations of outer membrane constituents or by chemical or physical damage to the cell envelope, induce common signaling pathways that ultimately lead to internal oxidative stress and misregulation of iron homeostasis.

scholarly journals Peptidoglycan Association of Murein Lipoprotein Is Required for KpsD-Dependent Group 2 Capsular Polysaccharide Expression and Serum Resistance in a UropathogenicEscherichia coliIsolate

mBio ◽  
2017 ◽  
Vol 8 (3) ◽  
Author(s):  
Jingyu Diao ◽  
Catrien Bouwman ◽  
Donghong Yan ◽  
Jing Kang ◽  
Anand K. Katakam ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Capsular Polysaccharide ◽  
Cell Envelope ◽  
Bloodstream Infections ◽  
Serum Resistance ◽  
E Coli ◽  
Serum Killing ◽  
Group 2

ABSTRACTMurein lipoprotein (Lpp) and peptidoglycan-associated lipoprotein (Pal) are major outer membrane lipoproteins inEscherichia coli. Their roles in cell-envelope integrity have been documented inE. colilaboratory strains, and while Lpp has been linked to serum resistancein vitro, the underlying mechanism has not been established. Here,lppandpalmutants of uropathogenicE. colistrain CFT073 showed reduced survival in a mouse bacteremia model, but only thelppmutant was sensitive to serum killingin vitro. The peptidoglycan-bound Lpp form was specifically required for preventing complement-mediated bacterial lysisin vitroand complement-mediated clearancein vivo. Compared to the wild-type strain, thelppmutant had impaired K2 capsular polysaccharide production and was unable to respond to exposure to serum by elevating capsular polysaccharide amounts. These properties correlated with altered cellular distribution of KpsD, the predicted outer membrane translocon for “group 2” capsular polysaccharides. We identified a novel Lpp-dependent association between functional KpsD and peptidoglycan, highlighting important interplay between cell envelope components required for resistance to complement-mediated lysis in uropathogenicE. coliisolates.IMPORTANCEUropathogenicE. coli(UPEC) isolates represent a significant cause of nosocomial urinary tract and bloodstream infections. Many UPEC isolates are resistant to serum killing. Here, we show that a major cell-envelope lipoprotein (murein lipoprotein) is required for serum resistancein vitroand for complement-mediated bacterial clearancein vivo. This is mediated, in part, through a novel mechanism by which murein lipoprotein affects the proper assembly of a key component of the machinery involved in production of “group 2” capsules. The absence of murein lipoprotein results in impaired production of the capsule layer, a known participant in complement resistance. These results demonstrate an important role for murein lipoprotein in complex interactions between different outer membrane biogenesis pathways and further highlight the importance of lipoprotein assembly and transport in bacterial pathogenesis.

scholarly journals Appropriate Regulation of the σE-Dependent Envelope Stress Response Is Necessary To Maintain Cell Envelope Integrity and Stationary-Phase Survival in Escherichia coli

2017 ◽  
Vol 199 (12) ◽  
Author(s):  
Hervé Nicoloff ◽  
Saumya Gopalkrishnan ◽  
Sarah E. Ades
Keyword(s):  
Escherichia Coli ◽  
Stationary Phase ◽  
Outer Membrane ◽  
Stress Responses ◽  
Cell Envelope ◽  
Membrane Integrity ◽  
Point Mutations ◽  
Content Type ◽  
Envelope Stress ◽  
Outer Membrane Porins

ABSTRACT The alternative sigma factor σE is a key component of the Escherichia coli response to cell envelope stress and is required for viability even in the absence of stress. The activity of σE increases during entry into stationary phase, suggesting an important role for σE when nutrients are limiting. Elevated σE activity has been proposed to activate a pathway leading to the lysis of nonculturable cells that accumulate during early stationary phase. To better understand σE-directed cell lysis and the role of σE in stationary phase, we investigated the effects of elevated σE activity in cultures grown for 10 days. We demonstrate that high σE activity is lethal for all cells in stationary phase, not only those that are nonculturable. Spontaneous mutants with reduced σE activity, due primarily to point mutations in the region of σE that binds the −35 promoter motif, arise and take over cultures within 5 to 6 days after entry into stationary phase. High σE activity leads to large reductions in the levels of outer membrane porins and increased membrane permeability, indicating membrane defects. These defects can be counteracted and stationary-phase lethality delayed significantly by stabilizing membranes with Mg2+ and buffering the growth medium or by deleting the σE-dependent small RNAs (sRNAs) MicA, RybB, and MicL, which inhibit the expression of porins and Lpp. Expression of these sRNAs also reverses the loss of viability following depletion of σE activity. Our results demonstrate that appropriate regulation of σE activity, ensuring that it is neither too high nor too low, is critical for envelope integrity and cell viability. IMPORTANCE The Gram-negative cell envelope and cytoplasm differ significantly, and separate responses have evolved to combat stress in each compartment. An array of cell envelope stress responses exist, each of which is focused on different parts of the envelope. The σE response is conserved in many enterobacteria and is tuned to monitor pathways for the maturation and delivery of outer membrane porins, lipoproteins, and lipopolysaccharide to the outer membrane. The activity of σE is tightly regulated to match the production of σE regulon members to the needs of the cell. In E. coli, loss of σE results in lethality. Here we demonstrate that excessive σE activity is also lethal and results in decreased membrane integrity, the very phenotype the system is designed to prevent.

scholarly journals Evidence for phospholipid export from the gram-negative inner membrane: time to rethink the Mla pathway?

2018 ◽  
Author(s):  
Gareth W. Hughes ◽  
Stephen C. L. Hall ◽  
Claire S. Laxton ◽  
Pooja Sridhar ◽  
Amirul H. Mahadi ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Outer Membrane ◽  
Binding Pocket ◽  
Periplasmic Protein ◽  
Inner Membrane ◽  
Gram Negative ◽  
Membrane Atpase ◽  
Phospholipid Transport ◽  
First Time ◽  
Β Sheet

AbstractThe Mla pathway is believed to be involved in maintaining the asymmetrical Gram-negative outer membrane via retrograde phospholipid transport. The pathway is composed of 3 components: the outer membrane MlaA-OmpC/F complex, a soluble periplasmic protein, MlaC, and the inner membrane ATPase, MlaFEDB complex. Here we solve the crystal structure of MlaC in its phospholipid free closed apo conformation, revealing a novel pivoting β-sheet mechanism which functions to open and close the phospholipid-binding pocket. Using the apo form of MlaC we provide evidence that the Mla pathway functions in an anterograde rather than a retrograde direction by showing the inner membrane MlaFEDB machinery exports phospholipids and transfers them to MlaC in the periplasm. We confirm that the lipid export process occurs through the MlaD component of the MlaFEDB complex. This lipid export process is shown to be independent of ATP. Our data provides, for the first time, evidence of an apparatus for lipid export to the outer membrane.

scholarly journals YraP Contributes to Cell Envelope Integrity and Virulence ofSalmonella entericaSerovar Typhimurium

2018 ◽  
Vol 86 (11) ◽  
Author(s):  
Faye C. Morris ◽  
Timothy J. Wells ◽  
Jack A. Bryant ◽  
Anna E. Schager ◽  
Yanina R. Sevastsyanovich ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Cell Envelope ◽  
Systemic Infection ◽  
Eukaryotic Cell ◽  
Infection Model ◽  
Content Type ◽  
Protein Levels ◽  
K 12

ABSTRACTMutations in σE-regulated lipoproteins have previously been shown to impact bacterial viability under conditions of stress and duringin vivoinfection. YraP is conserved across a number of Gram-negative pathogens, includingNeisseria meningitidis, where the homolog is a component of the Bexsero meningococcal group B vaccine. Investigations using laboratory-adaptedEscherichia coliK-12 have shown thatyraPmutants have elevated sensitivity to a range of compounds, including detergents and normally ineffective antibiotics. In this study, we investigate the role of the outer membrane lipoprotein YraP in the pathogenesis ofSalmonella entericaserovar Typhimurium. We show that mutations inS. TyphimuriumyraPresult in a defective outer membrane barrier with elevated sensitivity to a range of compounds. This defect is associated with attenuated virulence in an oral infection model and during the early stages of systemic infection. We show that this attenuation is not a result of defects in lipopolysaccharide and O-antigen synthesis, changes in outer membrane protein levels, or the ability to adhere to and invade eukaryotic cell linesin vitro.

scholarly journals Structural basis for VIPP1 oligomerization and maintenance of thylakoid membrane integrity

2020 ◽  
Author(s):  
Tilak Kumar Gupta ◽  
Sven Klumpe ◽  
Karin Gries ◽  
Steffen Heinz ◽  
Wojciech Wietrzynski ◽  
...  
Keyword(s):  
Membrane Integrity ◽  
Point Mutations ◽  
Binding Pocket ◽  
Thylakoid Membranes ◽  
Lipid Binding ◽  
Structural Basis ◽  
Amphipathic Helices ◽  
Cryo Electron Microscopy

AbstractVesicle-inducing protein in plastids (VIPP1) is essential for the biogenesis and maintenance of thylakoid membranes, which transform light into life. However, it is unknown how VIPP1 performs its vital membrane-shaping function. Here, we use cryo-electron microscopy to determine structures of cyanobacterial VIPP1 rings, revealing how VIPP1 monomers flex and interweave to form basket-like assemblies of different symmetries. Three VIPP1 monomers together coordinate a non-canonical nucleotide binding pocket that is required for VIPP1 oligomerization. Inside the ring’s lumen, amphipathic helices from each monomer align to form large hydrophobic columns, enabling VIPP1 to bind and curve membranes. In vivo point mutations in these hydrophobic surfaces cause extreme thylakoid swelling under high light, indicating an essential role of VIPP1 lipid binding in resisting stress-induced damage. Our study provides a structural basis for understanding how the oligomerization of VIPP1 drives the biogenesis of thylakoid membranes and protects these life-giving membranes from environmental stress.

scholarly journals Bacteriocin Release Protein Triggers Dimerization of Outer Membrane Phospholipase A In Vivo

1999 ◽  
Vol 181 (10) ◽  
pp. 3281-3283 ◽  
Author(s):  
Niek Dekker ◽  
Jan Tommassen ◽  
Hubertus M. Verheij
Keyword(s):  
Escherichia Coli ◽  
Outer Membrane ◽  
Cell Envelope ◽  
Lag Time ◽  
Phospholipase A ◽  
Cross Linking ◽  
Outer Membrane Phospholipase A ◽  
Bacteriocin Release Protein ◽  
Chemical Cross Linking

ABSTRACT Bacteriocin release protein is known to activate outer membrane phospholipase A (OMPLA), which results in the release of colicin fromEscherichia coli. In vivo chemical cross-linking experiments revealed that the activation coincides with dimerization of OMPLA. Permeabilization of the cell envelope and dimerization were characterized by a lag time of 2 h.

scholarly journals Mutational Analysis of the Escherichia coli K-12 TolA N-Terminal Region and Characterization of Its TolQ-Interacting Domain by Genetic Suppression

1998 ◽  
Vol 180 (24) ◽  
pp. 6433-6439 ◽  
Author(s):  
Pierre Germon ◽  
Thierry Clavel ◽  
Anne Vianney ◽  
Raymond Portalier ◽  
Jean Claude Lazzaroni
Keyword(s):  
Escherichia Coli ◽  
Outer Membrane ◽  
Cytoplasmic Membrane ◽  
Transmembrane Domain ◽  
Mutational Analysis ◽  
Cell Envelope ◽  
Transmembrane Helix ◽  
Membrane Integrity ◽  
Genetic Suppression ◽  
K 12

ABSTRACT The Tol-Pal proteins of Escherichia coli are involved in maintaining outer membrane integrity. They form two complexes in the cell envelope. Transmembrane domains of TolQ, TolR, and TolA interact in the cytoplasmic membrane, while TolB and Pal form a complex near the outer membrane. The N-terminal transmembrane domain of TolA anchors the protein to the cytoplasmic membrane and interacts with TolQ and TolR. Extensive mutagenesis of the N-terminal part of TolA was carried out to characterize the residues involved in such processes. Mutations affecting the function of TolA resulted in a lack or an alteration in TolA-TolQ or TolR-TolA interactions but did not affect the formation of TolQ-TolR complexes. Our results confirmed the importance of residues serine 18 and histidine 22, which are part of an SHLS motif highly conserved in the TolA and the related TonB proteins from different organisms. Genetic suppression experiments were performed to restore the functional activity of some tolA mutants. The suppressor mutations all affected the first transmembrane helix of TolQ. These results confirmed the essential role of the transmembrane domain of TolA in triggering interactions with TolQ and TolR.

scholarly journals The Mxi-Spa Type III Secretory Pathway ofShigella flexneri Requires an Outer Membrane Lipoprotein, MxiM, for Invasin Translocation

1999 ◽  
Vol 67 (4) ◽  
pp. 1982-1991 ◽  
Author(s):  
Raymond Schuch ◽  
Anthony T. Maurelli
Keyword(s):  
Outer Membrane ◽  
Type Iii Secretion ◽  
Secretory Pathway ◽  
Cell Envelope ◽  
Shigella Flexneri ◽  
Culture Cell ◽  
Tissue Culture Cell ◽  
Virulence Plasmid ◽  
Type Iii

ABSTRACT Invasion of epithelial cells by Shigella flexneri is mediated by a set of translocated bacterial invasins, the Ipa proteins, and its dedicated type III secretion system, called Mxi-Spa. We show here that mxiM, part of the mxi-spa locus in the S. flexneri virulence plasmid, encodes an indispensable type III secretion apparatus component, required for both Ipa translocation and tissue culture cell invasion. We demonstrated that mature MxiM, first identified as a putative lipoprotein, is lipidated in vivo. Consistent with features of known lipoproteins, MxiM (i) can be labeled with [3H]palmitate and [2-3H]glycerol, (ii) is associated with the cell envelope, (iii) is secreted independently of the type III pathway, and (iv) requires an intact lipoprotein modification and processing site for full activity. The lipidated form of MxiM was detected primarily in the outer membrane, where it establishes a peripheral association with the inner leaflet. Through analysis of subcellular Ipa distribution in a mxiM null mutant background, MxiM was found to be required for the assembly and/or function of outer, but not inner, membrane regions of Mxi-Spa. This function probably requires interactions with other Mxi-Spa subunits within the periplasmic space. We discuss implications of these findings with respect to the function of MxiM and the structure of Mxi-Spa as a whole.

Sign in / Sign up

Export Citation Format

Share Document