Lytic transglycosylases in macromolecular transport systems of Gram-negative bacteria

Multi-resistance to antibiotics in Gram-negative bacteria has been reported in several studies, which make more effective methods of controlling and eliminating these bacteria necessary. To overcome multiresistant profiles, we used OMVs (Outer Membrane Vesicles) as carriers of levofloxacin to encapsulate and transport the drug from the extracellular medium into the cell, overcoming resistance barriers and inhibiting cell reproduction machinery. Prepackaged formulations in this manner were quite effective and, in some cases, totally inhibited bacterial growth by making the drug efficient again.

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

10.1101/748145 ◽

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.

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Bacterial Adaptor Membrane Fusion Proteins and the Structurally Dissimilar Outer Membrane Auxiliary Proteins Have Exchanged Central Domains inα-Proteobacteria

International Journal of Microbiology ◽

10.1155/2010/589391 ◽

2010 ◽

Vol 2010 ◽

pp. 1-5 ◽

Cited By ~ 3

Author(s):

Anthony Y. Xiao ◽

Jing Wang ◽

Milton H. Saier

Keyword(s):

Membrane Fusion ◽

Outer Membrane ◽

Fusion Proteins ◽

Transport Systems ◽

Gram Negative Bacteria ◽

Gram Negative ◽

Transporter Protein ◽

Functional Implications ◽

Membrane Fusion Proteins ◽

Helical Region

Transport systems frequently include auxiliary proteins that perform subfunctions within the transporter protein complex. Two such proteins found in Gram-negative bacteria are the Membrane Fusion Proteins (MFPs) and the Outer Membrane Auxiliary (OMA) proteins. We here demonstrate that OMAs present inα-proteobacteria (but not in other bacterial types) contain a longα-helical region that is homologous to corresponding regions in the MFPs. The results suggest that during their evolution, OMAs, specifically fromα-proteobacteria, exchanged their ownα-helical domain for one derived from an MFP. The structural and functional implications of these findings are discussed.

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

mBio ◽

10.1128/mbio.00194-19 ◽

2019 ◽

Vol 10 (2) ◽

Cited By ~ 4

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.

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Structural studies of proteins involved in the activity of novel antibiotics

Acta Crystallographica Section A Foundations and Advances ◽

10.1107/s2053273314092894 ◽

2014 ◽

Vol 70 (a1) ◽

pp. C710-C710

Author(s):

Lucile Moynie ◽

Luana Ferrara ◽

Antoni Tortajada ◽

James Naismith

Keyword(s):

Iron Acquisition ◽

Three Dimensional ◽

Multidrug Resistant ◽

New Drugs ◽

Transport Systems ◽

Resistant Bacteria ◽

Gram Negative Bacteria ◽

Structural Studies ◽

Gram Negative ◽

Novel Antibiotics

The emergence of multidrug-resistant Gram-negative bacteria such as P. aeruginosa has become a growing challenge for developing new drugs. One of the strategies recently used to improve the efficacy of the antibiotics is to increase their uptake by exploiting bacterial transport systems such as the siderophore-mediated iron acquisition system. BAL30072, a new monosulfactam conjugated to a siderophore moiety has been shown to have potent activity against many Gram-negative bacteria [1]. Several proteins affecting susceptibility to this antibiotic have been identified in Pseudomonas aeruginosa [2]. As part of the Translocation project (Innovative Medicines Initiative), we have undertaken structural studies of these proteins and have solved the three dimensional structures of two of these targets. The structure of PiuA, a TonB-dependent siderophore transporter involved in the uptake of siderophore antibiotics across the outer membrane [2,3] has been solved to a resolution of 1.9 Å. The structure of PiuC, an Fe(II)/α-ketoglutarate-dependent dioxygenase, has been solved to a resolution of 2.6 Å. The structural and biochemical studies of these proteins will help us to understand the mode of action of these novel antibiotics and subsequently help to design new drugs acting against multidrug-resistant bacteria.

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An exposed outer membrane haemin-binding protein facilitates haemin transport by a TonB-dependent receptor in Riemerella anatipestifer

Applied and Environmental Microbiology ◽

10.1128/aem.00367-21 ◽

2021 ◽

Author(s):

Mafeng Liu ◽

Siqi Liu ◽

Mi Huang ◽

Yaling Wang ◽

Mengying Wang ◽

...

Keyword(s):

Outer Membrane ◽

Binding Protein ◽

Bacterial Pathogen ◽

Essential Element ◽

Acid Sites ◽

Transport Systems ◽

Gram Negative Bacteria ◽

Gram Negative ◽

Riemerella Anatipestifer ◽

Iron Source

Iron is an essential element for the replication of most bacteria, including Riemerella anatipestifer (R. anatipestifer, RA), a gram-negative bacterial pathogen of ducks and other birds. R. anatipestifer utilizes haemoglobin-derived haemin as an iron source; however, the mechanism by which this bacterium acquires haemin from haemoglobin is largely unknown. Here, RhuA disruption was shown to impair iron utilization from duck haemoglobin in R. anatipestifer CH-1. Moreover, the putative lipoprotein RhuA was identified as a surface-exposed, outer membrane haemin-binding protein, but it could not extract haemin from duck haemoglobin. Mutagenesis studies showed that recombinant RhuAY144A, RhuAY177A and RhuAH149A lost haemin-binding ability, suggesting that amino acid sites tyrosine 144 (Y144), Y177 and histidine 149 (H149) are crucial for haemin binding. Furthermore, RhuR, the gene adjacent to RhuA, encodes a TonB2-dependent haemin transporter. The function of RhuA in duck haemoglobin utilization was abolished in the RhuR mutant strain, and recombinant RhuA was able to bind the cell surface of R. anatipestifer CH-1ΔRhuA rather than R. anatipestifer CH-1ΔRhuRΔRhuA, indicating that RhuA associates with RhuR to function. The sequence of the RhuR-RhuA haemin utilization locus exhibits no similarity with those of characterized haemin transport systems. Thus, this locus is a novel haemin uptake locus with homologues distributed mainly in the Bacteroidetes phylum. IMPORTANCE In vertebrates, haemin from haemoglobin is an important iron source for infectious bacteria. Many bacteria can obtain haemin from haemoglobin, but the mechanisms of haemin acquisition from haemoglobin differ among bacteria. Moreover, most studies have focused on the mechanism of haemin acquisition from mammalian haemoglobin. In this study, we found that the RhuR-RhuA locus of R. anatipestifer CH-1, a duck pathogen, is involved in haemin acquisition from duck haemoglobin via a unique pathway. RhuA was identified as an exposed outer membrane haemin-binding protein, and RhuR was identified as a TonB2-dependent haemin transporter. Moreover, the function of RhuA in haemoglobin utilization is RhuR dependent, not vice versa. The homologues of RhuR and RhuA are widely distributed in bacteria in marine environments, animals, and plants, representing a novel haemin transportation system of gram-negative bacteria. This study not only was important for understanding haemin uptake in R. anatipestifer but also enriched the knowledge about the haemin transportation pathway in gram-negative bacteria.

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Peptidoglycomics reveals compositional changes in peptidoglycan between biofilm- and planktonic-derived Pseudomonas aeruginosa

Journal of Biological Chemistry ◽

10.1074/jbc.ra119.010505 ◽

2019 ◽

Vol 295 (2) ◽

pp. 504-516

Author(s):

Erin M. Anderson ◽

David Sychantha ◽

Dyanne Brewer ◽

Anthony J. Clarke ◽

Jennifer Geddes-McAlister ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Growth Conditions ◽

Environmental Stressors ◽

Gram Negative Bacteria ◽

Physiological Changes ◽

Differential Analysis ◽

Gram Negative ◽

Lytic Transglycosylases ◽

Compositional Changes ◽

External Stimuli

Peptidoglycan (PG) is a critical component of the bacterial cell wall and is composed of a repeating β-1,4–linked disaccharide of N-acetylglucosamine and N-acetylmuramic acid appended with a highly conserved stem peptide. In Gram-negative bacteria, PG is assembled in the cytoplasm and exported into the periplasm where it undergoes considerable maturation, modification, or degradation depending on the growth phase or presence of environmental stressors. These modifications serve important functions in diverse processes, including PG turnover, cell elongation/division, and antibiotic resistance. Conventional methods for analyzing PG composition are complex and time-consuming. We present here a streamlined MS-based method that combines differential analysis with statistical 1D annotation approaches to quantitatively compare PGs produced in planktonic- and biofilm-cultured Pseudomonas aeruginosa. We identified a core assembly of PG that is present in high abundance and that does not significantly differ between the two growth states. We also identified an adaptive PG assembly that is present in smaller amounts and fluctuates considerably between growth states in response to physiological changes. Biofilm-derived adaptive PG exhibited significant changes compared with planktonic-derived PG, including amino acid substitutions of the stem peptide and modifications that indicate changes in the activity of amidases, deacetylases, and lytic transglycosylases. The results of this work also provide first evidence of de-N-acetylated muropeptides from P. aeruginosa. The method developed here offers a robust and reproducible workflow for accurately determining PG composition in samples that can be used to assess global PG fluctuations in response to changing growth conditions or external stimuli.

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Bacterial-Mucosal Cell Junctional Complexes

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100050731 ◽

1974 ◽

Vol 32 ◽

pp. 144-145

Author(s):

Roger C. Wagner

Keyword(s):

Intestinal Wall ◽

Rat Colon ◽

Mucosal Cell ◽

Gram Negative Bacteria ◽

Gram Negative ◽

Mucosal Cells ◽

Mucosal Lining ◽

Degenerative Alterations ◽

Junctional Complexes ◽

Intestinal Mucosal Cells

Bacteria exhibit the ability to adhere to the apical surfaces of intestinal mucosal cells. These attachments either precede invasion of the intestinal wall by the bacteria with accompanying inflammation and degeneration of the mucosa or represent permanent anchoring sites where the bacteria never totally penetrate the mucosal cells.Endemic gram negative bacteria were found attached to the surface of mucosal cells lining the walls of crypts in the rat colon. The bacteria did not intrude deeper than 0.5 urn into the mucosal cells and no degenerative alterations were detectable in the mucosal lining.

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Rapid demonstration of septic or infectious arthritis due to Gram-negative bacteria

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100123830 ◽

1992 ◽

Vol 50 (1) ◽

pp. 686-687

Author(s):

Jacob S. Hanker ◽

Paul R. Gross ◽

Beverly L. Giammara

Keyword(s):

Chronic Arthritis ◽

Blood Cultures ◽

Gram Negative Bacteria ◽

Infectious Arthritis ◽

Bacterial Arthritis ◽

Gram Positive ◽

Gram Negative ◽

Gram Positive Bacteria

Blood cultures are positive in approximately only 50 per cent of the patients with nongonococcal bacterial infectious arthritis and about 20 per cent of those with gonococcal arthritis. But the concept that gram-negative bacteria could be involved even in chronic arthritis is well-supported. Gram stains are more definitive in staphylococcal arthritis caused by gram-positive bacteria than in bacterial arthritis due to gram-negative bacteria. In the latter situation where gram-negative bacilli are the problem, Gram stains are helpful for 50% of the patients; they are only helpful for 25% of the patients, however, where gram-negative gonococci are the problem. In arthritis due to gram-positive Staphylococci. Gramstained smears are positive for 75% of the patients.

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