scholarly journals Thanatin targets the intermembrane protein complex required for lipopolysaccharide transport inEscherichia coli

2018 ◽  
Vol 4 (11) ◽  
pp. eaau2634 ◽  
Author(s):  
Stefan U. Vetterli ◽  
Katja Zerbe ◽  
Maik Müller ◽  
Matthias Urfer ◽  
Milon Mondal ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Macromolecular Complex ◽  
Gram Negative Bacteria ◽  
Periplasmic Protein ◽  
Protein Protein Interactions ◽  
Inner Membrane ◽  
Gram Negative ◽  
Research Priority ◽  
Naturally Occurring ◽  
Bacterial Outer Membrane

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.

scholarly journals Activity-Related Conformational Changes in d,d-Carboxypeptidases Revealed by In Vivo Periplasmic Förster Resonance Energy Transfer Assay in Escherichia coli

mBio ◽  
2017 ◽  
Vol 8 (5) ◽  
Author(s):  
Nils Y. Meiresonne ◽  
René van der Ploeg ◽  
Mark A. Hink ◽  
Tanneke den Blaauwen
Keyword(s):  
Protein Interactions ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Gram Negative Bacteria ◽  
Periplasmic Protein ◽  
Periplasmic Space ◽  
Gram Negative ◽  
Conformation Changes ◽  
Antibiotic Screening

ABSTRACT One of the mechanisms of β-lactam antibiotic resistance requires the activity of d,d-carboxypeptidases (d,d-CPases) involved in peptidoglycan (PG) synthesis, making them putative targets for new antibiotic development. The activity of PG-synthesizing enzymes is often correlated with their association with other proteins. The PG layer is maintained in the periplasm between the two membranes of the Gram-negative cell envelope. Because no methods existed to detect in vivo interactions in this compartment, we have developed and validated a Förster resonance energy transfer assay. Using the fluorescent-protein donor-acceptor pair mNeonGreen-mCherry, periplasmic protein interactions were detected in fixed and in living bacteria, in single samples or in plate reader 96-well format. We show that the d,d-CPases PBP5, PBP6a, and PBP6b of Escherichia coli change dimer conformation between resting and active states. Complementation studies and changes in localization suggest that these d,d-CPases are not redundant but that their balanced activity is required for robust PG synthesis. IMPORTANCE The periplasmic space between the outer and the inner membrane of Gram-negative bacteria contains many essential regulatory, transport, and cell wall-synthesizing and -hydrolyzing proteins. To date, no assay is available to determine protein interactions in this compartment. We have developed a periplasmic protein interaction assay for living and fixed bacteria in single samples or 96-well-plate format. Using this assay, we were able to demonstrate conformation changes related to the activity of proteins that could not have been detected by any other living-cell method available. The assay uniquely expands our toolbox for antibiotic screening and mode-of-action studies. IMPORTANCE The periplasmic space between the outer and the inner membrane of Gram-negative bacteria contains many essential regulatory, transport, and cell wall-synthesizing and -hydrolyzing proteins. To date, no assay is available to determine protein interactions in this compartment. We have developed a periplasmic protein interaction assay for living and fixed bacteria in single samples or 96-well-plate format. Using this assay, we were able to demonstrate conformation changes related to the activity of proteins that could not have been detected by any other living-cell method available. The assay uniquely expands our toolbox for antibiotic screening and mode-of-action studies.

A General System for Studying Protein−Protein Interactions in Gram-Negative Bacteria

2008 ◽  
Vol 7 (8) ◽  
pp. 3319-3328 ◽  
Author(s):  
Dale A. Pelletier ◽  
Gregory B. Hurst ◽  
Linda J. Foote ◽  
Patricia K. Lankford ◽  
Catherine K. McKeown ◽  
...  
Keyword(s):  
Protein Interactions ◽  
General System ◽  
Gram Negative Bacteria ◽  
Protein Protein Interactions ◽  
Gram Negative

scholarly journals On the path to uncover the bacterial type II secretion system

2012 ◽  
Vol 367 (1592) ◽  
pp. 1059-1072 ◽  
Author(s):  
Badreddine Douzi ◽  
Alain Filloux ◽  
Romé Voulhoux
Keyword(s):  
Protein Interactions ◽  
Secretion System ◽  
Target Cells ◽  
Type Ii Secretion ◽  
Gram Negative Bacteria ◽  
Type Ii ◽  
Protein Protein Interactions ◽  
Gram Negative ◽  
Extracellular Milieu ◽  
Type Ii Secretion System

Gram-negative bacteria have evolved several secretory pathways to release enzymes or toxins into the surrounding environment or into the target cells. The type II secretion system (T2SS) is conserved in Gram-negative bacteria and involves a set of 12 to 16 different proteins. Components of the T2SS are located in both the inner and outer membranes where they assemble into a supramolecular complex spanning the bacterial envelope, also called the secreton. The T2SS substrates transiently go through the periplasm before they are translocated across the outer membrane and exposed to the extracellular milieu. The T2SS is unique in its ability to promote secretion of large and sometimes multimeric proteins that are folded in the periplasm. The present review describes recently identified protein–protein interactions together with structural and functional advances in the field that have contributed to improve our understanding on how the type II secretion apparatus assembles and on the role played by individual proteins of this highly sophisticated system.

Polyamines: Naturally occurring small molecule modulators of electrostatic protein–protein interactions

2010 ◽  
Vol 104 (2) ◽  
pp. 118-125 ◽  
Author(s):  
Anja Berwanger ◽  
Susanne Eyrisch ◽  
Inge Schuster ◽  
Volkhard Helms ◽  
Rita Bernhardt
Keyword(s):  
Small Molecule ◽  
Protein Interactions ◽  
Protein Protein Interactions ◽  
Naturally Occurring ◽  
Small Molecule Modulators

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.

scholarly journals YejM Modulates Activity of the YciM/FtsH Protease Complex To Prevent Lethal Accumulation of Lipopolysaccharide

mBio ◽  
2020 ◽  
Vol 11 (2) ◽  
Author(s):  
Randi L. Guest ◽  
Daniel Samé Guerra ◽  
Maria Wissler ◽  
Jacqueline Grimm ◽  
Thomas J. Silhavy
Keyword(s):  
Escherichia Coli ◽  
Outer Membrane ◽  
Gram Negative Bacteria ◽  
Inner Membrane ◽  
Gram Negative ◽  
Content Type ◽  
Essential Protein ◽  
Ftsh Protease ◽  
Acyl Chains ◽  
Lipid Balance

ABSTRACT Lipopolysaccharide (LPS) is an essential glycolipid present in the outer membrane (OM) of many Gram-negative bacteria. Balanced biosynthesis of LPS is critical for cell viability; too little LPS weakens the OM, while too much LPS is lethal. In Escherichia coli, this balance is maintained by the YciM/FtsH protease complex, which adjusts LPS levels by degrading the LPS biosynthesis enzyme LpxC. Here, we provide evidence that activity of the YciM/FtsH protease complex is inhibited by the essential protein YejM. Using strains in which LpxC activity is reduced, we show that yciM is epistatic to yejM, demonstrating that YejM acts upstream of YciM to prevent toxic overproduction of LPS. Previous studies have shown that this toxicity can be suppressed by deleting lpp, which codes for a highly abundant OM lipoprotein. It was assumed that deletion of lpp restores lipid balance by increasing the number of acyl chains available for glycerophospholipid biosynthesis. We show that this is not the case. Rather, our data suggest that preventing attachment of lpp to the peptidoglycan sacculus allows excess LPS to be shed in vesicles. We propose that this loss of OM material allows continued transport of LPS to the OM, thus preventing lethal accumulation of LPS within the inner membrane. Overall, our data justify the commitment of three essential inner membrane proteins to avoid toxic over- or underproduction of LPS. IMPORTANCE Gram-negative bacteria are encapsulated by an outer membrane (OM) that is impermeable to large and hydrophobic molecules. As such, these bacteria are intrinsically resistant to several clinically relevant antibiotics. To better understand how the OM is established or maintained, we sought to clarify the function of the essential protein YejM in Escherichia coli. Here, we show that YejM inhibits activity of the YciM/FtsH protease complex, which regulates synthesis of the essential OM glycolipid lipopolysaccharide (LPS). Our data suggest that disrupting proper communication between LPS synthesis and transport to the OM leads to accumulation of LPS within the inner membrane (IM). The lethality associated with this event can be suppressed by increasing OM vesiculation. Our research has identified a completely novel signaling pathway that we propose coordinates LPS synthesis and transport.

scholarly journals Non-naturally Occurring Helical Molecules Can Interfere with p53–MDM2 and p53–MDMX Protein–Protein Interactions

2019 ◽  
Vol 67 (10) ◽  
pp. 1139-1143 ◽  
Author(s):  
Aoze Su ◽  
Siyuan Wang ◽  
Akane Sada ◽  
Yuko Otani ◽  
Luhan Zhai ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Protein Protein Interactions ◽  
Naturally Occurring ◽  
Helical Molecules

scholarly journals Dynamic interaction of fluoroquinolones with magnesium ions monitored using bacterial outer membrane nanopores

2020 ◽  
Vol 11 (38) ◽  
pp. 10344-10353
Author(s):  
Jiajun Wang ◽  
Jigneshkumar Dahyabhai Prajapati ◽  
Ulrich Kleinekathöfer ◽  
Mathias Winterhalter
Keyword(s):  
Outer Membrane ◽  
Divalent Cations ◽  
Dynamic Interaction ◽  
Gram Negative Bacteria ◽  
Magnesium Ions ◽  
Gram Negative ◽  
Bacterial Outer Membrane

Divalent cations alter the translocation of antibiotic molecules through the Gram-negative bacteria outer membrane nanopores.

Epitope mapping of monoclonal antibodies specific for the directly cross-linkedmeso-diaminopimelic acid peptidoglycan found in the anaerobic beer spoilage bacteriumPectinatus cerevisiiphilus

1999 ◽  
Vol 45 (9) ◽  
pp. 779-785 ◽  
Author(s):  
Barry Ziola ◽  
Sheryl L Gares ◽  
Brandene Lorrain ◽  
Lori Gee ◽  
W M Ingledew ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Outer Membrane ◽  
Epitope Mapping ◽  
Sodium Dodecyl ◽  
Diaminopimelic Acid ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Spoilage Bacteria ◽  
Beer Spoilage ◽  
Bacterial Outer Membrane

Nineteen monoclonal antibodies (Mabs) were isolated based on reactivity with disrupted Pectinatus cerevisiiphilus cells. All of the Mabs reacted with cells from which the outer membrane had been stripped by incubation with sodium dodecyl sulphate, suggesting the peptidoglycan (PG) layer was involved in binding. Mab reactivity with purified PG confirmed this. Epitope mapping revealed the Mabs in total recognize four binding sites on the PG. Mabs specific for each of the four sites also bound strongly to disrupted Pectinatus frisingensis, Selenomonas lacticifix, Zymophilus paucivorans, and Zymophilus raffinosivorans cells, but weakly to disrupted Megasphaera cerevisiae cells. No antibody reactivity was seen with disrupted cells of 11 other species of Gram-negative bacteria. These results confirm that a common PG structure is used by several species of anaerobic Gram-negative beer spoilage bacteria. These results also indicate that PG-specific Mabs can be used to rapidly detect a range of anaerobic Gram-negative beer spoilage bacteria, provided the bacterial outer membrane is first removed to allow antibody binding.Key words: beer spoilage, epitope mapping, monoclonal antibodies, Pectinatus, peptidoglycan.

scholarly journals The Type IV Pilus Assembly Complex: Biogenic Interactions among the Bundle-Forming Pilus Proteins of Enteropathogenic Escherichia coli

2002 ◽  
Vol 184 (13) ◽  
pp. 3457-3465 ◽  
Author(s):  
Sandra W. Ramer ◽  
Gary K. Schoolnik ◽  
Cheng-Yen Wu ◽  
Jaiweon Hwang ◽  
Sarah A. Schmidt ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Outer Membrane ◽  
Protein Interactions ◽  
Protein Protein Interactions ◽  
Cell Compartment ◽  
Inner Membrane ◽  
Outer Membranes ◽  
Type Iv ◽  
Pilin Subunit ◽  
Membrane Compartments

ABSTRACT Production of type IV bundle-forming pili (BFP) by enteropathogenic Escherichia coli (EPEC) requires the protein products of 12 genes of the 14-gene bfp operon. Antisera against each of these proteins were used to demonstrate that in-frame deletion of individual genes within the operon reduces the abundance of other bfp operon-encoded proteins. This result was demonstrated not to be due to downstream polar effects of the mutations but rather was taken as evidence for protein-protein interactions and their role in the stabilization of the BFP assembly complex. These data, combined with the results of cell compartment localization studies, suggest that pilus formation requires the presence of a topographically discrete assembly complex that is composed of BFP proteins in stoichiometric amounts. The assembly complex appears to consist of an inner membrane component containing three processed, pilin-like proteins, BfpI, -J, and -K, that localize with BfpE, -L, and -A (the major pilin subunit); an outer membrane, secretin-like component, BfpB and -G; and a periplasmic component composed of BfpU. Of these, only BfpL consistently localizes with both the inner and outer membranes and thus, together with BfpU, may articulate between the Bfp proteins in the inner membrane and outer membrane compartments.

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