scholarly journals MlaFEDB displays flippase activity to promote phospholipid transport towards the outer membrane of Gram-negative bacteria

2020 ◽  
Author(s):  
Gareth W. Hughes ◽  
Pooja Sridhar ◽  
Stephanie A. Nestorow ◽  
Peter J. Wotherspoon ◽  
Benjamin F. Cooper ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Membrane Trafficking ◽  
Atp Hydrolysis ◽  
Gram Negative Bacteria ◽  
Inner Membrane ◽  
Membrane Protein Complex ◽  
Gram Negative ◽  
Inner Membrane Protein ◽  
Phospholipid Transport

AbstractMlaFEDB is a Gram-negative inner membrane protein complex involved in the inter membrane trafficking of phospholipids. Originally proposed to transport phospholipids in a retrograde direction, recent evidence suggests MlaFEDB may actually export phospholipids from the inner membrane to the periplasmic carrier protein, MlaC, potentially suggesting a role in either anterograde trafficking of phospholipids to the outer membrane or bidirectional phospholipid movement. MlaFEDB is part of the ABC transporter superfamily of proteins and has been shown to hydrolyse ATP through the cytoplasmic facing MlaF component. However, the movement of PLs from FEDB to MlaC has been shown to occur in an ATP independent fashion hence the role of ATP hydrolysis within this complex remains unclear. In this study we sought to elucidate the role of ATP and provide evidence to suggest MlaFEDB has flippase activity, utilising ATP hydrolysis to translocate phospholipids from the outer to the inner leaflet of the IM. We also show that in the absence of ATP MlaFEDB mediates the loading of MlaC with phospholipids directly from the inner leaflet only. Our data provides a novel role for MlaFEDB and presents a link between Mla driven phospholipid transport and ATP hydrolysis.

scholarly journals MsbA Is Not Required for Phospholipid Transport in Neisseria meningitidis

2005 ◽  
Vol 280 (43) ◽  
pp. 35961-35966 ◽  
Author(s):  
Boris Tefsen ◽  
Martine P. Bos ◽  
Frank Beckers ◽  
Jan Tommassen ◽  
Hans de Cock
Keyword(s):  
Neisseria Meningitidis ◽  
Outer Membrane ◽  
Gram Negative Bacteria ◽  
Inner Membrane ◽  
Gram Negative ◽  
Outer Leaflet ◽  
Growth Phenotype ◽  
Inner Membrane Protein ◽  
Phospholipid Transport

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.

scholarly journals The essential inner membrane protein YejM is a metalloenzyme

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Uma Gabale ◽  
Perla Arianna Peña Palomino ◽  
HyunAh Kim ◽  
Wenya Chen ◽  
Susanne Ressl
Keyword(s):  
Antibiotic Resistance ◽  
Membrane Protein ◽  
Outer Membrane ◽  
Critical Role ◽  
Membrane Properties ◽  
Gram Negative Bacteria ◽  
Inner Membrane ◽  
Gram Negative ◽  
Periplasmic Domain ◽  
Inner Membrane Protein

Abstract Recent recurrent outbreaks of Gram-negative bacteria show the critical need to target essential bacterial mechanisms to fight the increase of antibiotic resistance. Pathogenic Gram-negative bacteria have developed several strategies to protect themselves against the host immune response and antibiotics. One such strategy is to remodel the outer membrane where several genes are involved. yejM was discovered as an essential gene in E. coli and S. typhimurium that plays a critical role in their virulence by changing the outer membrane permeability. How the inner membrane protein YejM with its periplasmic domain changes membrane properties remains unknown. Despite overwhelming structural similarity between the periplasmic domains of two YejM homologues with hydrolases like arylsulfatases, no enzymatic activity has been previously reported for YejM. Our studies reveal an intact active site with bound metal ions in the structure of YejM periplasmic domain. Furthermore, we show that YejM has a phosphatase activity that is dependent on the presence of magnesium ions and is linked to its function of regulating outer membrane properties. Understanding the molecular mechanism by which YejM is involved in outer membrane remodeling will help to identify a new drug target in the fight against the increased antibiotic resistance.

scholarly journals Depolarization, Bacterial Membrane Composition, and the Antimicrobial Action of Ceragenins

2010 ◽  
Vol 54 (9) ◽  
pp. 3708-3713 ◽  
Author(s):  
Raquel F. Epand ◽  
Jake E. Pollard ◽  
Jonathan O. Wright ◽  
Paul B. Savage ◽  
Richard M. Epand
Keyword(s):  
Outer Membrane ◽  
Cytoplasmic Membrane ◽  
Bacterial Species ◽  
Membrane Depolarization ◽  
Membrane Lipid ◽  
Gram Negative Bacteria ◽  
Inner Membrane ◽  
Gram Negative ◽  
Cytoplasmic Membranes

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

scholarly journals Structural insights into a novel family of integral membrane siderophore reductases

2021 ◽  
Author(s):  
Inokentijs Josts ◽  
Katharina Veith ◽  
Vincent Normant ◽  
Isabelle J. Schalk ◽  
Henning Tidow
Keyword(s):  
Crystal Structure ◽  
Membrane Protein ◽  
Outer Membrane ◽  
Bacterial Species ◽  
Gram Negative Bacteria ◽  
Inner Membrane ◽  
Gram Negative ◽  
Functional Studies ◽  
Inner Membrane Protein ◽  
Structural Insights

AbstractGram-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 P. aeruginosa) that is involved in Fe-siderophore uptake. The structure revealed a novel fold with two tightly-bound heme molecules. In combination with functional studies these results establish FoxB as an inner membrane reductase involved in the release of iron from ferrioxamine during Fe-siderophore uptake.

scholarly journals The inner membrane protein YhdP modulates the rate of anterograde phospholipid flow in Escherichia coli

2020 ◽  
Author(s):  
Jacqueline Grimm ◽  
Handuo Shi ◽  
Wei Wang ◽  
Angela M. Mitchell ◽  
Ned S. Wingreen ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Outer Membrane ◽  
Permeability Barrier ◽  
Gram Negative Bacteria ◽  
High Flux ◽  
Inner Membrane ◽  
Gram Negative ◽  
Selective Permeability ◽  
Delayed Cell Death ◽  
Inner Membrane Protein

AbstractThe outer membrane (OM) of Gram-negative bacteria is a selective permeability barrier that allows uptake of nutrients while simultaneously protecting the cell from harmful compounds. The basic pathways and molecular machinery responsible for transporting lipopolysaccharides (LPS), lipoproteins, and β-barrel proteins to the OM have been identified, but very little is known about phospholipid (PL) transport. To identify genes capable of affecting PL transport, we screened for genetic interactions with mlaA*, a mutant in which anterograde PL transport causes the inner membrane (IM) to shrink and eventually rupture; characterization of mlaA*-mediated lysis suggested that PL transport can occur via a high-flux, diffusive flow mechanism. We found that YhdP, an IM protein involved in maintaining the OM permeability barrier, modulates the rate of PL transport during mlaA*-mediated lysis. Deletion of yhdP from mlaA* reduced the rate of IM transport to the OM by 50%, slowing shrinkage of the IM and delaying lysis. As a result, the weakened OM of ΔydhP cells was further compromised and ruptured before the IM during mlaA*-mediated death. These findings demonstrate the existence of a high-flux, diffusive pathway for PL flow in Escherichia coli that is modulated by YhdP.Significance StatementThe outer membrane (OM) of Gram-negative bacteria serves as a barrier that protects cells from harmful chemical compounds, including many antibiotics. Understanding how bacteria build this barrier is an important step in engineering strategies to circumvent it. A long-standing mystery in the field is how phospholipids (PLs) are transported from the inner membrane (IM) to the OM. We previously discovered that a mutation in the gene mlaA causes rapid flow of PLs to the OM, eventually resulting in IM rupture. Here, we found that deletion of the gene yhdP delayed cell death in the mlaA mutant by slowing flow of PLs to the OM. These findings reveal a high-flux, diffusive pathway for PL transport in Gram-negative bacteria modulated by YhdP.

scholarly journals Restoring Balance to the Outer Membrane: YejM’s Role in LPS Regulation

mBio ◽  
2020 ◽  
Vol 11 (6) ◽  
pp. e02624-20
Author(s):  
Brent W. Simpson ◽  
Martin V. Douglass ◽  
M. Stephen Trent
Keyword(s):  
Membrane Protein ◽  
Cell Surface ◽  
Outer Membrane ◽  
Strong Evidence ◽  
Cell Envelope ◽  
Gram Negative Bacteria ◽  
Inner Membrane ◽  
Gram Negative ◽  
Multiple Functions ◽  
Inner Membrane Protein

ABSTRACTGram-negative bacteria produce an asymmetric outer membrane (OM) that is particularly impermeant to many antibiotics and characterized by lipopolysaccharide (LPS) exclusively at the cell surface. LPS biogenesis remains an ideal target for therapeutic intervention, as disruption could kill bacteria or increase sensitivity to existing antibiotics. While it has been known that LPS synthesis is regulated by proteolytic control of LpxC, the enzyme that catalyzes the first committed step of LPS synthesis, it remains unknown which signals direct this regulation. New details have been revealed during study of a cryptic essential inner membrane protein, YejM. Multiple functions have been proposed over the years for YejM, including a controversial hypothesis that it transports cardiolipin from the inner membrane to the OM. Strong evidence now indicates that YejM senses LPS in the periplasm and directs proteolytic regulation. Here, we discuss the standing literature of YejM and highlight exciting new insights into cell envelope maintenance.

scholarly journals New functional identity of the essential inner membrane protein YejM: the cardiolipin translocator is also a metalloenzyme

2020 ◽  
Author(s):  
Uma Gabale ◽  
Perla A. Peña Palomino ◽  
HyunAh Kim ◽  
Wenya Chen ◽  
Susanne Ressl
Keyword(s):  
Antibiotic Resistance ◽  
Membrane Protein ◽  
Outer Membrane ◽  
Essential Gene ◽  
Critical Role ◽  
Gram Negative Bacteria ◽  
Inner Membrane ◽  
Gram Negative ◽  
Periplasmic Domain ◽  
Inner Membrane Protein

AbstractRecent recurrent outbreaks of Gram-negative bacteria show the critical need to target essential bacterial mechanisms to fight the increase of antibiotic resistance. Pathogenic Gram-negative bacteria have developed several strategies to protect themselves against the host immune response and antibiotics. One strategy is to remodel the outer membrane where several genes are involved. yejM was discovered as an essential gene in E. coli and S. typhimurium that plays a critical role in their virulence by changing the outer membrane permeability by translocating and increasing the cardiolipin lipid concentration. How the inner membrane protein YejM with its periplasmic domain acts as a cardiolipin translocator remains unknown. Despite overwhelming structural similarity of the periplasmic domains of two YejM homologues with hydrolases like arylsulfatases, no enzymatic activity has been reported for YejM. Our studies reveal an intact active site with bound metal ions in the structure of YejM periplasmic domain. Furthermore, we show that YejM has a phosphatase activity that is dependent on the presence of magnesium ions and is linked to its cardiolipin translocation properties. Understanding the molecular mechanism by which YejM is involved in OM remodeling will help to identify a new drug target in the fight against the increased antibiotic resistance.

scholarly journals MexAB-OprM Efflux Pump Interaction with the Peptidoglycan of Escherichia coli and Pseudomonas aeruginosa

2021 ◽  
Vol 22 (10) ◽  
pp. 5328
Author(s):  
Miao Ma ◽  
Margaux Lustig ◽  
Michèle Salem ◽  
Dominique Mengin-Lecreulx ◽  
Gilles Phan ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Pseudomonas Aeruginosa ◽  
Cell Division ◽  
Membrane Proteins ◽  
Outer Membrane ◽  
Efflux Pump ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Active Efflux

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.

scholarly journals Structural insights into a novel family of integral membrane siderophore reductases

2021 ◽  
Vol 118 (34) ◽  
pp. e2101952118
Author(s):  
Inokentijs Josts ◽  
Katharina Veith ◽  
Vincent Normant ◽  
Isabelle J. Schalk ◽  
Henning Tidow
Keyword(s):  
Iron Uptake ◽  
Bacterial Species ◽  
Gram Negative Bacteria ◽  
Inner Membrane ◽  
Gram Negative ◽  
Inner Membrane Protein ◽  
Uptake Studies ◽  
Structural Insights

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.

Sign in / Sign up

Export Citation Format

Share Document