Isolation and Physical Characterization of Bioactive Lipopolysaccharides (Lipid A) From Nontoxic E. coli of Human Origin

2018 ◽  
pp. 223-268
Author(s):  
Henrich H. Paradies ◽  
Kurt Zimmermann
Keyword(s):  
Lipid A ◽  
Physical Characterization ◽  
Human Origin ◽  
E Coli

scholarly journals Antibacterial Activities and Characterization of Novel Inhibitors of LpxC

2002 ◽  
Vol 46 (6) ◽  
pp. 1793-1799 ◽  
Author(s):  
John M. Clements ◽  
Fanny Coignard ◽  
Ian Johnson ◽  
Stephen Chandler ◽  
Shilpa Palan ◽  
...  
Keyword(s):  
Burkholderia Cepacia ◽  
Lipid A ◽  
Antibacterial Activities ◽  
Vitro Assay ◽  
Gram Negative ◽  
E Coli ◽  
Antibacterial Target ◽  
Derivatives Of

ABSTRACT Lipid A is the hydrophobic anchor of lipopolysaccharide (LPS) and forms the major lipid component of the outer monolayer of the outer membrane of gram-negative bacteria. Lipid A is required for bacterial growth and virulence, and inhibition of its biosynthesis is lethal to bacteria. UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase (LpxC) is a metalloenzyme that catalyzes the second step in the biosynthesis of lipid A. Inhibitors of LpxC have previously been shown to have antibiotic activities. We have screened a metalloenzyme inhibitor library for antibacterial activities against an Escherichia coli strain with reduced LpxC activity. From this screen, a series of sulfonamide derivatives of the α-(R)-amino hydroxamic acids, exemplified by BB-78484 and BB-78485, have been identified as having potent inhibitory activities against LpxC in an in vitro assay. Leads from this series showed gram-negative selective activities against members of the Enterobacteriaceae, Serratia marcescens, Morganella morganii, Haemophilus influenzae, Moraxella catarrhalis, and Burkholderia cepacia. BB-78484 was bactericidal against E. coli, achieving 3-log killing in 4 h at a concentration 4 times above the MIC, as would be predicted for an inhibitor of lipid A biosynthesis. E. coli mutants with decreased susceptibility to BB-78484 were selected. Analysis of these mutants revealed that resistance arose as a consequence of mutations in the fabZ or lpxC genes. These data confirm the antibacterial target of BB-78484 and BB-78485 and validate LpxC as a target for gram-negative selective antibacterials.

scholarly journals Identification and Characterization of Two Klebsiella pneumoniaelpxL Lipid A Late Acyltransferases and Their Role in Virulence

2017 ◽  
Vol 85 (9) ◽  
Author(s):  
Grant Mills ◽  
Amy Dumigan ◽  
Timothy Kidd ◽  
Laura Hobley ◽  
José A. Bengoechea
Keyword(s):  
Lipid A ◽  
Galleria Mellonella ◽  
Inflammatory Responses ◽  
Two Component System ◽  
Content Type ◽  
E Coli ◽  
Wide Range ◽  
Candidate Target ◽  
Tract Infections

ABSTRACT Klebsiella pneumoniae causes a wide range of infections, from urinary tract infections to pneumonia. The lipopolysaccharide is a virulence factor of this pathogen, although there are gaps in our understanding of its biosynthesis. Here we report on the characterization of K. pneumoniae lpxL, which encodes one of the enzymes responsible for the late secondary acylation of immature lipid A molecules. Analysis of the available K. pneumoniae genomes revealed that this pathogen's genome encodes two orthologues of Escherichia coli LpxL. Using genetic methods and mass spectrometry, we demonstrate that LpxL1 catalyzes the addition of laureate and LpxL2 catalyzes the addition of myristate. Both enzymes acylated E. coli lipid A, whereas only LpxL2 mediated K. pneumoniae lipid A acylation. We show that LpxL1 is negatively regulated by the two-component system PhoPQ. The lipid A produced by the lpxL2 mutant lacked the 2-hydroxymyristate, palmitate, and 4-aminoarabinose decorations found in the lipid A synthesized by the wild type. The lack of 2-hydroxymyristate was expected since LpxO modifies the myristate transferred by LpxL2 to the lipid A. The absence of the other two decorations is most likely caused by the downregulation of phoPQ and pmrAB expression. LpxL2-dependent lipid A acylation protects Klebsiella from polymyxins, mediates resistance to phagocytosis, limits the activation of inflammatory responses by macrophages, and is required for pathogen survival in the wax moth (Galleria mellonella). Our findings indicate that the LpxL2 contribution to virulence is dependent on LpxO-mediated hydroxylation of the LpxL2-transferred myristate. Our studies suggest that LpxL2 might be a candidate target in the development of anti-K. pneumoniae drugs.

scholarly journals Phenotypic and multi-omics characterization of Escherichia coli K-12 adapted to quaternary ammonium compounds identifies lipid A and cell envelope alterations regulated by mar-sox-rob and stress inducible pathways

2020 ◽  
Author(s):  
Kari A.C. Green ◽  
Branden S.J. Gregorchuk ◽  
Shelby L. Reimer ◽  
Nicola H. Cartwright ◽  
Daniel R. Beniac ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Quaternary Ammonium ◽  
Lipid A ◽  
Quaternary Ammonium Compounds ◽  
Cross Resistance ◽  
E Coli ◽  
Lipid A Biosynthesis ◽  
K 12 ◽  
Ammonium Compounds

AbstractQuaternary ammonium compounds (QACs) benzalkonium (BZK) and cetrimide (CET) are common disinfectants used to inhibit or eradicate Gram-negative bacteria in clinical and agricultural products. QAC tolerance in Escherichia coli and other Enterobacterales species can confer cross-resistance to various clinically used antibiotics, making it important to understand mechanisms of QAC tolerance in greater depth. QAC adaptation by E. coli is hypothesized to alter MarRAB regulated genes that converge on the outer membrane, specifically, lipid A biosynthesis and transport genes, porins, and efflux pump systems. To test this, we performed a ‘multi’-omics and phenotypic characterization of E. coli K-12 adapted to BZK and CET, to assess how QACs alter cell growth, genomics, and proteomics. E. coli adapted to either BZK and CET resulted in strains with stable QAC tolerance when either drug was omitted, elongated and narrower cell morphologies by scanning electron microscopy, and reduced growth fitness when compared to un-adapted E. coli. Antimicrobial susceptibility testing revealed that QAC adaptation increased E. coli tolerance by ≥4-fold to BZK, CET, and other QACs but no antibiotic cross-resistance. Single nucleotide variants identified by whole genome sequencing and differentially accumulated proteins by liquid chromatography-mass spectrometry identified alterations to various QAC-adapted E. coli genes and proteins belonging to: lipid A biosynthesis and transport (lpxLM, msbA, mla), the mar-sox-rob regulatory pathway (marR, rob), DNA/protein translation (gyrA, rpsA, rpoB, rapA). These alterations validate the hypothesis that mar-sox-rob network plays a role in QAC tolerance and identifies additional stress inducible genetic and protein QAC tolerant biomarkers.ImportanceBacterial tolerance mechanisms associated with disinfectant QAC adaptation is hypothesized to overlap with the mar-sox-rob multiple antimicrobial resistance pathway but has not been directly shown. Here, we generate QAC tolerant E. coli strains and identify phenotypic changes associated with protein and genetic alterations caused by prolonged QAC exposure. We identified genes that overlap with known antibiotic resistance mechanisms as well as distinct genes and proteins specific to QAC adaptation that are useful for future bacterial disinfectant tolerance mechanism studies. However, these altered genes and proteins implicate MarR and Rob pathways specifically in QAC tolerance but, surprisingly, the involvement of mar-sox-rob pathways did not increase antibiotic cross-resistance. Many altered genes we identified were essential genes in lipid A biosynthesis/transport, DNA and RNA transcription, and protein regulation systems potentially explaining why only QAC cross-tolerance was observed and why we observed greater cell fitness costs despite MarR and Rob pathway involvement.

scholarly journals Extraction of ADP-Heptose and Kdo2-Lipid A from E. coli Deficient in the Heptosyltransferase I Gene

2021 ◽  
Vol 11 (18) ◽  
pp. 8314
Author(s):  
Jozafina Milicaj ◽  
Colleen D. Castro ◽  
Nadiya Jaunbocus ◽  
Erika A. Taylor
Keyword(s):  
Bacterial Infections ◽  
Visual Cues ◽  
Large Scale ◽  
Lipid A ◽  
Biosynthetic Enzymes ◽  
Bacterial Cells ◽  
Site Specific ◽  
E Coli ◽  
Key Steps

The enzymes involved in lipopolysaccharide (LPS) biosynthesis, including Heptosyltransferase I (HepI), are critical for maintaining the integrity of the bacterial cell wall, and therefore these LPS biosynthetic enzymes are validated targets for drug discovery to treat Gram-negative bacterial infections. Enzymes involved in the biosynthesis of lipopolysaccharides (LPSs) utilize substrates that are synthetically complex, with numerous stereocenters and site-specific glycosylation patterns. Due to the relatively complex substrate structures, characterization of these enzymes has necessitated strategies to generate bacterial cells with gene disruptions to enable the extraction of these substrates from large scale bacterial growths. Like many LPS biosynthetic enzymes, Heptosyltransferase I binds two substrates: the sugar acceptor substrate, Kdo2-Lipid A, and the sugar donor substrate, ADP-l-glycero-d-manno-heptose (ADPH). HepI characterization experiments require copious amounts of Kdo2-Lipid A and ADPH, and unsuccessful extractions of these two substrates can lead to serious delays in collection of data. While there are papers and theses with protocols for extraction of these substrates, they are often missing small details essential to the success of the extraction. Herein detailed protocols are given for extraction of ADPH and Kdo2-Lipid A (KLA) from E. coli, which have had proven success in the Taylor lab. Key steps in the extraction of ADPH are clearing the extract through ultracentrifugation and keeping all water that touches anything in the extraction, including filters, at a pH of 8.0. Key steps in the extraction of KLA are properly lysing the dried down cells before starting the extraction, maximizing yield by allowing precipitate to form overnight, appropriately washing the pellet with phenol and dissolving the KLA in 1% TEA using visual cues, rather than a specific volume. These protocols led to increased yield and a higher success rate of extractions thereby enabling the characterization of HepI.

Rapid, Refined, and Robust Method for Expression, Purification, and Characterization of Recombinant Human Amyloid-beta M1-42

2019 ◽  
Author(s):  
Priya Prakash ◽  
Travis Lantz ◽  
Krupal P. Jethava ◽  
Gaurav Chopra
Keyword(s):  
Amyloid Beta ◽  
Western Blots ◽  
Force Microscopy ◽  
E Coli ◽  
Atomic Force ◽  
Set Up ◽  
Short Time

Amyloid plaques found in the brains of Alzheimer’s disease (AD) patients primarily consists of amyloid beta 1-42 (Ab42). Commercially, Ab42 is synthetized using peptide synthesizers. We describe a robust methodology for expression of recombinant human Ab(M1-42) in Rosetta(DE3)pLysS and BL21(DE3)pLysS competent E. coli with refined and rapid analytical purification techniques. The peptide is isolated and purified from the transformed cells using an optimized set-up for reverse-phase HPLC protocol, using commonly available C18 columns, yielding high amounts of peptide (~15-20 mg per 1 L culture) in a short time. The recombinant Ab(M1-42) forms characteristic aggregates similar to synthetic Ab42 aggregates as verified by western blots and atomic force microscopy to warrant future biological use. Our rapid, refined, and robust technique to purify human Ab(M1-42) can be used to synthesize chemical probes for several downstream in vitro and in vivo assays to facilitate AD research.

Characterization of Healthy and Tumor Oral Cell Lines of Human Origin. The preliminary stage in the assessment of relevant chemical compounds with impact on dentistry

2018 ◽  
Vol 69 (10) ◽  
pp. 2889-2894
Author(s):  
Ion Virgil Corlan ◽  
Adelina Cheveresan ◽  
Delia Berceanu Vaduva ◽  
Cristian Nica ◽  
Alin Faur ◽  
...  
Keyword(s):  
Squamous Cell Carcinoma ◽  
Cell Carcinoma ◽  
Cell Lines ◽  
Squamous Cell ◽  
Chemical Compounds ◽  
Tongue Squamous Cell Carcinoma ◽  
Gingival Fibroblasts ◽  
Human Origin ◽  
Preliminary Stage

The present study was aimed to evaluate the confluence percentage of three oral cell lines, namely primary gingival keratinocytes (PGK), primary gingival fibroblasts (HGF) and tongue squamous cell carcinoma (SCC-4). All cells have been monitored at different passages for 21 days. Evaluation of confluence percentage reveals the fact that primary gingival keratinocytes and tongue squamous cell carcinoma at small passages requires a period of about two weeks to reach a confluence of approximately 80% while for the gingival fibroblasts a period of about three times smaller is satisfactory.

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