Structural characterization of phosphoethanolamine-modified lipid A from probiotic <i>Escherichia coli</i> strain Nissle 1917

Jo, Sung‐Hyun; Park, Han-Gyu; Song, Won-Suk; Kim, Seong–Min; Kim, Eun-Jung; Yang, Yung‐Hun; Kim, Jae‐Seok; Kim, Byung‐Gee; Kim, Yun‐Gon

doi:10.1039/c9ra02375e

Cited by 7 publications

(8 citation statements)

References 50 publications

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“…The main species was detected at m/z 1796.2 ( Figure 8 ) matching with the typical E. coli lipid A glucosamine disaccharide backbone carrying four 14:0 (3-OH), one 14:0, and one 12:0 acyl chains and decorated by two phosphates. Moreover, peaks with mass differences of 28 (-CH 2 CH 2 - unit) and 14 (-CH 2 - unit) amu (atomic mass unit), indicative of the occurrence of species differing in the length of the acyl chains, were also found, as previously reported for other E. coli strains [ 45 ]. Importantly, a peak at m/z 1876.2 corresponding to tris -phosphorylated lipid A species (i.e., carrying a pyrophosphate group) was observed in all the strains ( Figure 8 ).…”

Section: Resultssupporting

confidence: 78%

Overexpression of lpxT Gene in Escherichia coli Inhibits Cell Division and Causes Envelope Defects without Changing the Overall Phosphorylation Level of Lipid A

et al. 2020

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LpxT is an inner membrane protein that transfers a phosphate group from the essential lipid undecaprenyl pyrophosphate (C-55PP) to the lipid A moiety of lipopolysaccharide, generating a lipid A tris-phosphorylated species. The protein is encoded by the non-essential lpxT gene, which is conserved in distantly related Gram-negative bacteria. In this work, we investigated the phenotypic effect of lpxT ectopic expression from a plasmid in Escherichia coli. We found that lpxT induction inhibited cell division and led to the formation of elongated cells, mostly with absent or altered septa. Moreover, the cells became sensitive to detergents and to hypo-osmotic shock, indicating that they had cell envelope defects. These effects were not due to lipid A hyperphosphorylation or C-55PP sequestering, but most likely to defective lipopolysaccharide transport. Indeed, lpxT overexpression in mutants lacking the L,D-transpeptidase LdtD and LdtE, which protect cells with outer membrane defects from osmotic lysis, caused cell envelope defects. Moreover, we found that pyrophosphorylated lipid A was also produced in a lpxT deletion mutant, indicating that LpxT is not the only protein able to perform such lipid A modification in E. coli.

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Section: Resultssupporting

confidence: 78%

Overexpression of lpxT Gene in Escherichia coli Inhibits Cell Division and Causes Envelope Defects without Changing the Overall Phosphorylation Level of Lipid A

et al. 2020

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“…Figure A shows the chemical space of lipid A from all six bacterial species. In total, 218 potential lipid A species with 188 distinct structures were annotated in this single study (Supporting Information Tables S8 and S9),with several structures corroborated by earlier studies. ,,,,,− ,− …”

Section: Resultssupporting

confidence: 72%

LipidA-IDER to Explore the Global Lipid A Repertoire of Drug-Resistant Gram-Negative Bacteria

et al. 2023

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With the global emergence of drug-resistant bacteria causing difficult-to-treat infections, there is an urgent need for a tool to facilitate studies on key virulence and antimicrobial resistant factors. Mass spectrometry (MS) has contributed substantially to the elucidation of the structure-function relationships of lipid A, the endotoxic component of lipopolysaccharide which also serves as an important protective barrier against antimicrobials. Here, we present LipidA-IDER, an automated structure annotation tool for system-level scale identification of lipid A from high-resolution tandem mass spectrometry (MS 2 ) data. LipidA-IDER was validated against previously reported structures of lipid A in the reference bacteria, Escherichia coli and Pseudomonas aeruginosa . Using MS 2 data of variable quality, we demonstrated LipidA-IDER annotated lipid A with a performance of 71.2% specificity and 70.9% sensitivity, offering greater accuracy than existing lipidomics software. The organism-independent workflow was further applied to a panel of six bacterial species: E. coli and Gram-negative members of ESKAPE pathogens. A comprehensive atlas comprising 188 distinct lipid A species, including remodeling intermediates, was generated and can be integrated with software including MS-DIAL and Metabokit for identification and semiquantitation. Systematic comparison of a pair of polymyxin-sensitive and polymyxin-resistant Acinetobacter baumannii isolated from a human patient unraveled multiple key lipid A structural features of polymyxin resistance within a single analysis. Probing the lipid A landscape of bacteria using LipidA-IDER thus holds immense potential for advancing our understanding of the vast diversity and structural complexity of a key lipid virulence and antimicrobial-resistant factor. LipidA-IDER is freely available at .

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“…Lipid A from E. coli Nissle has a hexa-acylated and bisphosphorylated disaccharide backbone with fatty acids containing 12−14 carbon atoms. 48 LPS from E. coli Nissle have a core OS (R1-type) comprising of glucose, galactose, heptose, Kdo, phosphate, and phosphoethanolamine (PEtN) residues. 31 Additionally, LPS from E. coli Nissle contain one O-antigen repeating unit (O-6 type) with glucose, mannose, N-acetyl galactosamine, and N-acetyl glucosamine residues.…”

Section: ■ Results and Discussionmentioning

confidence: 99%

“…Based on our MS 1 and MS/MS characterization, here, we characterized LPS from E. coli Nissle and revealed positively charged pEtN modifications known to disrupt interactions with and confer resistance to cationic antimicrobial peptides (CAMPs). 48 Because PEtN modulates LPS recognition and toxicity by introducing positive charge, the presence of multiple PEtN modification sites may play a significant role in the mechanism of LPS antibiotic resistance and pathogenesis. It would be interesting to study the effect of PEtN modification site, core OS heptose site versus lipid A site, to better understand the mechanisms of PEtN-mediated antimicrobial resistance in gut microbiota.…”

Section: ■ Conclusionmentioning

confidence: 99%

Top-Down Characterization of Bacterial Lipopolysaccharides and Lipooligosaccharides Using Activated-Electron Photodetachment Mass Spectrometry

Keener,

Goh,

Yoo

et al. 2024

Anal. Chem.

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Lipopolysaccharides (LPS) and lipooligosaccharides (LOS) are located in the outer membrane of Gram-negative bacteria and are comprised of three distinctive parts: lipid A, core oligosaccharide (OS), and O-antigen. The structure of each region influences bacterial stability, toxicity, and pathogenesis. Here, we highlight the use of targeted activatedelectron photodetachment (a-EPD) tandem mass spectrometry to characterize LPS and LOS from two crucial players in the human gut microbiota, Escherichia coli Nissle and Bacteroides fragilis. a-EPD is a hybrid activation method that uses ultraviolet photoirradiation to generate charge-reduced radical ions followed by collisional activation to produce informative fragmentation patterns. We benchmark the a-EPD method for top-down characterization of triacyl LOS from E. coli R2, then focus on characterization of LPS from E. coli Nissle and B. fragilis. Notably, a-EPD affords extensive fragmentation throughout the backbone of the core OS and O-antigen regions of LPS from E. coli Nissle. This hybrid approach facilitated the elucidation of structural details for LPS from B. fragilis, revealing a putative hexuronic acid (HexA) conjugated to lipid A.

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Structural characterization of phosphoethanolamine-modified lipid A from probiotic Escherichia coli strain Nissle 1917

Abstract: The probiotic Escherichia coli strain Nissle 1917 is able to colonize the gut more efficiently through lipid A mediated cationic antimicrobial peptide tolerance.

Cited by 7 publications

References 50 publications

Overexpression of lpxT Gene in Escherichia coli Inhibits Cell Division and Causes Envelope Defects without Changing the Overall Phosphorylation Level of Lipid A

Overexpression of lpxT Gene in Escherichia coli Inhibits Cell Division and Causes Envelope Defects without Changing the Overall Phosphorylation Level of Lipid A

LipidA-IDER to Explore the Global Lipid A Repertoire of Drug-Resistant Gram-Negative Bacteria

Top-Down Characterization of Bacterial Lipopolysaccharides and Lipooligosaccharides Using Activated-Electron Photodetachment Mass Spectrometry

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