Structural Determination and Genetic Identification of the O-Antigen from an Escherichia coli Strain, LL004, Representing a Novel Serogroup

The O-antigen is the outermost component of the lipopolysaccharide layer in Gram-negative bacteria, and the variation of O-antigen structure provides the basis for bacterial serological diversity. Here, we determined the O-antigen structure of an Escherichia coli strain, LL004, which is totally different from all of the E. coli serogroups. The tetrasaccharide repeating unit was determined as →4)-β-d-Galp-(1→3)-β-d-GlcpNAc6OAc(~70%)-(1→3)-β-d-GalpA-(1→3)-β-d-GalpNAc-(1→ with monosaccharide analysis and NMR spectra. We also characterized the O-antigen gene cluster of LL004, and sequence analysis showed that it correlated well with the O-antigen structure. Deletion and complementation testing further confirmed its role in O-antigen biosynthesis, and indicated that the O-antigen of LL004 is assembled via the Wzx/Wzy dependent pathway. Our findings, in combination, suggest that LL004 should represent a novel serogroup of E. coli.

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Human Neutrophil Chemotaxis Is Modulated by Capsule and O Antigen from an Extraintestinal Pathogenic Escherichia coli Strain

Infection and Immunity ◽

10.1128/iai.71.11.6435-6445.2003 ◽

2003 ◽

Vol 71 (11) ◽

pp. 6435-6445 ◽

Cited By ~ 9

Author(s):

Thomas A. Russo ◽

Bruce A. Davidson ◽

Diana M. Topolnycky ◽

Ruth Olson ◽

Stacy A. Morrill ◽

...

Keyword(s):

Escherichia Coli ◽

Ex Vivo ◽

Specific Antigen ◽

Lavage Fluid ◽

Coli Strain ◽

Neutrophil Chemotaxis ◽

Gram Negative ◽

E Coli ◽

O Antigen

ABSTRACT Gram-negative enteric bacilli, such as Escherichia coli, are common causes of nosocomial pneumonia. The interaction between pulmonary neutrophils and the infecting pathogen is a critical step in determining the outcome. Previous studies from our laboratory, for which a rat model of pneumonia was used, established that pulmonary neutrophil recruitment was modulated by the E. coli virulence factors capsule and O-specific antigen. To begin to understand the mechanism by which this recruitment occurs, we conducted in vitro and ex vivo chemotaxis assays, for which we used a clinically relevant E. coli isolate (CP9) and isogenic derivatives that were deficient in only the O antigen (CP921) or capsule (CP9.137) as chemoattractants with or without the high-affinity N-formylmethionyl-leucyl-phenylalanine receptor antagonist N-tert-butoxycarbonyl-methionine-leucine-phenylalanine (N-t-BOC). Given that only live E. coli was used for the initial in vitro chemotaxis assays, it was predicted that only N-t-BOC-sensitive chemotaxis would occur. However, both N-t-BOC-sensitive and -insensitive chemotaxis was observed. N-t-BOC-insensitive chemotaxis was mediated in part by interleukin 8, which was produced by neutrophils that had migrated toward E. coli. N-t-BOC-insensitive chemotaxis was only observed when live E. coli bacteria, not cell-free E. coli culture supernatants, were used as chemoattractants, suggesting that a direct E. coli-neutrophil interaction was necessary. The presence of both capsule and O antigen diminished total, N-t-BOC-sensitive, and N-t-BOC-insensitive neutrophil chemotaxis in vitro. The presence of capsule significantly decreased total, N-t-BOC-sensitive, and N-t-BOC-insensitive neutrophil chemotaxis ex vivo when cell-free bronchoalveolar lavage fluid from infected rats was used as the source of chemotactic factors. These effects of E. coli capsule and O antigen on neutrophil chemotaxis are novel, and they expand our understanding of the mechanisms by which these virulence traits contribute to the pathogenesis of gram-negative pneumonia and other extraintestinal infections.

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The Escherichia coli O111 and Salmonella enterica O35 Gene Clusters: Gene Clusters Encoding the Same Colitose-Containing O Antigen Are Highly Conserved

Journal of Bacteriology ◽

10.1128/jb.182.18.5256-5261.2000 ◽

2000 ◽

Vol 182 (18) ◽

pp. 5256-5261 ◽

Cited By ~ 30

Author(s):

Lei Wang ◽

Peter R. Reeves

Keyword(s):

Escherichia Coli ◽

Gene Cluster ◽

Salmonella Enterica ◽

Gc Content ◽

Gene Clusters ◽

Gram Negative Bacteria ◽

Normal Range ◽

E Coli ◽

O Antigen ◽

Antigen Structure

ABSTRACT O antigen is part of the lipopolysaccharide present in the outer membrane of gram-negative bacteria. Escherichia coli andSalmonella enterica each have many forms of O antigen, but only three are common to the two species. It has been found that, in general, O-antigen genes are of low GC content. This deviation in GC content from that of typical S. enterica or E. coli genes (51%) is thought to indicate that the O-antigen DNA originated in species other than S. enterica or E. coli and was captured by lateral transfer. The O-antigen structure of Salmonella enterica O35 is identical to that of E. coli O111, commonly found in enteropathogenicE. coli strains. This O antigen, which has been shown to be a virulence factor in E. coli, contains colitose, a 3,6-dideoxyhexose found only rarely in theEnterobacteriaceae. Sequencing of the O35-antigen gene cluster of S. enterica serovar Adelaide revealed the same gene order and flanking genes as in E. coli O111. The divergence between corresponding genes of these two gene clusters at the nucleotide level ranges from 21.8 to 11.7%, within the normal range of divergence between S. enterica and E. coli. We conclude that the ancestor of E. coli andS. enterica had an O antigen identical to the O111 and O35 antigens, respectively, of these species and that the gene cluster encoding it has survived in both species.

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Proteomic response of Escherichia coli to a membrane lytic and iron chelating truncated Amaranthus tricolor defensin

BMC Microbiology ◽

10.1186/s12866-021-02176-4 ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Tessa B. Moyer ◽

Ashleigh L. Purvis ◽

Andrew J. Wommack ◽

Leslie M. Hicks

Keyword(s):

Escherichia Coli ◽

Antibacterial Activity ◽

Antimicrobial Peptides ◽

Mechanism Of Action ◽

Gram Negative Bacteria ◽

Amaranthus Tricolor ◽

Core Motif ◽

Gram Negative ◽

E Coli ◽

Membrane Lysis

Abstract Background Plant defensins are a broadly distributed family of antimicrobial peptides which have been primarily studied for agriculturally relevant antifungal activity. Recent studies have probed defensins against Gram-negative bacteria revealing evidence for multiple mechanisms of action including membrane lysis and ribosomal inhibition. Herein, a truncated synthetic analog containing the γ-core motif of Amaranthus tricolor DEF2 (Atr-DEF2) reveals Gram-negative antibacterial activity and its mechanism of action is probed via proteomics, outer membrane permeability studies, and iron reduction/chelation assays. Results Atr-DEF2(G39-C54) demonstrated activity against two Gram-negative human bacterial pathogens, Escherichia coli and Klebsiella pneumoniae. Quantitative proteomics revealed changes in the E. coli proteome in response to treatment of sub-lethal concentrations of the truncated defensin, including bacterial outer membrane (OM) and iron acquisition/processing related proteins. Modification of OM charge is a common response of Gram-negative bacteria to membrane lytic antimicrobial peptides (AMPs) to reduce electrostatic interactions, and this mechanism of action was confirmed for Atr-DEF2(G39-C54) via an N-phenylnaphthalen-1-amine uptake assay. Additionally, in vitro assays confirmed the capacity of Atr-DEF2(G39-C54) to reduce Fe3+ and chelate Fe2+ at cell culture relevant concentrations, thus limiting the availability of essential enzymatic cofactors. Conclusions This study highlights the utility of plant defensin γ-core motif synthetic analogs for characterization of novel defensin activity. Proteomic changes in E. coli after treatment with Atr-DEF2(G39-C54) supported the hypothesis that membrane lysis is an important component of γ-core motif mediated antibacterial activity but also emphasized that other properties, such as metal sequestration, may contribute to a multifaceted mechanism of action.

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A High-Throughput Approach To Identify Compounds That Impair Envelope Integrity in Escherichia coli

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00537-16 ◽

2016 ◽

Vol 60 (10) ◽

pp. 5995-6002 ◽

Cited By ~ 7

Author(s):

Kristin R. Baker ◽

Bimal Jana ◽

Henrik Franzyk ◽

Luca Guardabassi

Keyword(s):

Escherichia Coli ◽

High Throughput ◽

High Throughput Screening ◽

Gram Negative Bacteria ◽

Chromogenic Substrate ◽

Intrinsic Resistance ◽

Gram Negative ◽

Content Type ◽

E Coli ◽

Screening Platform

ABSTRACTThe envelope of Gram-negative bacteria constitutes an impenetrable barrier to numerous classes of antimicrobials. This intrinsic resistance, coupled with acquired multidrug resistance, has drastically limited the treatment options against Gram-negative pathogens. The aim of the present study was to develop and validate an assay for identifying compounds that increase envelope permeability, thereby conferring antimicrobial susceptibility by weakening of the cell envelope barrier in Gram-negative bacteria. A high-throughput whole-cell screening platform was developed to measureEscherichia colienvelope permeability to a β-galactosidase chromogenic substrate. The signal produced by cytoplasmic β-galactosidase-dependent cleavage of the chromogenic substrate was used to determine the degree of envelope permeabilization. The assay was optimized by using known envelope-permeabilizing compounds andE. coligene deletion mutants with impaired envelope integrity. As a proof of concept, a compound library comprising 36 peptides and 45 peptidomimetics was screened, leading to identification of two peptides that substantially increased envelope permeability. Compound 79 reduced significantly (from 8- to 125-fold) the MICs of erythromycin, fusidic acid, novobiocin and rifampin and displayed synergy (fractional inhibitory concentration index, <0.2) with these antibiotics by checkerboard assays in two genetically distinctE. colistrains, including the high-risk multidrug-resistant, CTX-M-15-producing sequence type 131 clone. Notably, in the presence of 0.25 μM of this peptide, both strains were susceptible to rifampin according to the resistance breakpoints (R> 0.5 μg/ml) for Gram-positive bacterial pathogens. The high-throughput screening platform developed in this study can be applied to accelerate the discovery of antimicrobial helper drug candidates and targets that enhance the delivery of existing antibiotics by impairing envelope integrity in Gram-negative bacteria.

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Characterization of the β-lactamase specified by the resistance factor R-1818 in Escherichia coli K12 and other Gram-negative bacteria

Biochemical Journal ◽

10.1042/bj1230501 ◽

1971 ◽

Vol 123 (4) ◽

pp. 501-505 ◽

Cited By ~ 13

Author(s):

J. W. Dale

Keyword(s):

Escherichia Coli ◽

Host Species ◽

Host Bacterium ◽

Gram Negative Bacteria ◽

Gram Negative ◽

E Coli ◽

R Factor ◽

Relationship Of ◽

The Relationship

1. The amino acid composition of the β-lactamase from E. coli (R-1818) was determined. 2. The R-1818 β-lactamase is inhibited by formaldehyde, hydroxylamine, sodium azide, iodoacetamide, iodine and sodium chloride. 3. The Km values for benzylpenicillin, ampicillin and oxacillin have been determined by using the R-factor enzyme from different host species. The same values were obtained, irrespective of the host bacterium. 4. The molecular weight of the enzyme was found to be 44600, and was the same for all host species. 5. The relationship of R-1818 and R-GN238 β-lactamases is discussed.

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Heterogeneous and Flexible Transmission ofmcr-1in Hospital-AssociatedEscherichia coli

mBio ◽

10.1128/mbio.00943-18 ◽

2018 ◽

Vol 9 (4) ◽

Cited By ~ 23

Author(s):

Yingbo Shen ◽

Zuowei Wu ◽

Yang Wang ◽

Rong Zhang ◽

Hong-Wei Zhou ◽

...

Keyword(s):

Escherichia Coli ◽

Genomic Analysis ◽

Multidrug Resistant ◽

Fluoroquinolone Resistance ◽

Gram Negative Bacteria ◽

Colistin Resistance ◽

Gram Negative ◽

Content Type ◽

E Coli ◽

Genes Encoding

ABSTRACTThe recent emergence of a transferable colistin resistance mechanism, MCR-1, has gained global attention because of its threat to clinical treatment of infections caused by multidrug-resistant Gram-negative bacteria. However, the possible transmission route ofmcr-1amongEnterobacteriaceaespecies in clinical settings is largely unknown. Here, we present a comprehensive genomic analysis ofEscherichia coliisolates collected in a hospital in Hangzhou, China. We found thatmcr-1-carrying isolates from clinical infections and feces of inpatients and healthy volunteers were genetically diverse and were not closely related phylogenetically, suggesting that clonal expansion is not involved in the spread ofmcr-1. Themcr-1gene was found on either chromosomes or plasmids, but in most of theE. coliisolates,mcr-1was carried on plasmids. The genetic context of the plasmids showed considerable diversity as evidenced by the different functional insertion sequence (IS) elements, toxin-antitoxin (TA) systems, heavy metal resistance determinants, and Rep proteins of broad-host-range plasmids. Additionally, the genomic analysis revealed nosocomial transmission ofmcr-1and the coexistence ofmcr-1with other genes encoding β-lactamases and fluoroquinolone resistance in theE. coliisolates. These findings indicate thatmcr-1is heterogeneously disseminated in both commensal and pathogenic strains ofE. coli, suggest the high flexibility of this gene in its association with diverse genetic backgrounds of the hosts, and provide new insights into the genome epidemiology ofmcr-1among hospital-associatedE. colistrains.IMPORTANCEColistin represents one of the very few available drugs for treating infections caused by extensively multidrug-resistant Gram-negative bacteria. The recently emergentmcr-1colistin resistance gene threatens the clinical utility of colistin and has gained global attention. Howmcr-1spreads in hospital settings remains unknown and was investigated by whole-genome sequencing ofmcr-1-carryingEscherichia coliin this study. The findings revealed extraordinary flexibility ofmcr-1in its spread among genetically diverseE. colihosts and plasmids, nosocomial transmission ofmcr-1-carryingE. coli, and the continuous emergence of novel Inc types of plasmids carryingmcr-1and newmcr-1variants. Additionally,mcr-1was found to be frequently associated with other genes encoding β-lactams and fluoroquinolone resistance. These findings provide important information on the transmission and epidemiology ofmcr-1and are of significant public health importance as the information is expected to facilitate the control of this significant antibiotic resistance threat.

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National Surveillance of Antimicrobial Susceptibility of Bacteremic Gram-Negative Bacteria with Emphasis on Community-Acquired Resistant Isolates: Report from the 2019 Surveillance of Multicenter Antimicrobial Resistance in Taiwan (SMART)

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01089-20 ◽

2020 ◽

Vol 64 (10) ◽

Author(s):

Po-Yu Liu ◽

Yu-Lin Lee ◽

Min-Chi Lu ◽

Pei-Lan Shao ◽

Po-Liang Lu ◽

...

Keyword(s):

Escherichia Coli ◽

Pseudomonas Aeruginosa ◽

Antimicrobial Resistance ◽

Klebsiella Pneumoniae ◽

Gram Negative Bacteria ◽

National Surveillance ◽

Gram Negative ◽

Content Type ◽

E Coli ◽

Carbapenem Resistant

ABSTRACT A multicenter collection of bacteremic isolates of Escherichia coli (n = 423), Klebsiella pneumoniae (n = 372), Pseudomonas aeruginosa (n = 300), and Acinetobacter baumannii complex (n = 199) was analyzed for susceptibility. Xpert Carba-R assay and sequencing for mcr genes were performed for carbapenem- or colistin-resistant isolates. Nineteen (67.8%) carbapenem-resistant K. pneumoniae (n = 28) and one (20%) carbapenem-resistant E. coli (n = 5) isolate harbored blaKPC (n = 17), blaOXA-48 (n = 2), and blaVIM (n = 1) genes.

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Metabolic phospholipid labeling of intact bacteria enables a fluorescence assay that detects compromised outer membranes

The Journal of Lipid Research ◽

10.1194/jlr.ra120000654 ◽

2020 ◽

Vol 61 (6) ◽

pp. 870-883 ◽

Cited By ~ 2

Author(s):

Inga Nilsson ◽

Sheng Y. Lee ◽

William S. Sawyer ◽

Christopher M. Baxter Rath ◽

Guillaume Lapointe ◽

...

Keyword(s):

Escherichia Coli ◽

Gram Negative Bacteria ◽

Metabolic Labeling ◽

Wild Type ◽

Transport Pathways ◽

Gram Negative ◽

E Coli ◽

Outer Membranes ◽

Outer Leaflet ◽

Promising Tool

Gram-negative bacteria possess an asymmetric outer membrane (OM) composed primarily of lipopolysaccharides (LPSs) on the outer leaflet and phospholipids (PLs) on the inner leaflet. The loss of this asymmetry due to mutations in the LPS biosynthesis or transport pathways causes the externalization of PLs to the outer leaflet of the OM and leads to OM permeability defects. Here, we used metabolic labeling to detect a compromised OM in intact bacteria. Phosphatidylcholine synthase expression in Escherichia coli allowed for the incorporation of exogenous propargylcholine into phosphatidyl(propargyl)choline and exogenous 1-azidoethyl-choline (AECho) into phosphatidyl(azidoethyl)choline (AEPC), as confirmed by LC/MS analyses. A fluorescent copper-free click reagent poorly labeled AEPC in intact wild-type cells but readily labeled AEPC from lysed cells. Fluorescence microscopy and flow cytometry analyses confirmed the absence of significant AEPC labeling from intact wild-type E. coli strains and revealed significant AEPC labeling in an E. coli LPS transport mutant (lptD4213) and an LPS biosynthesis mutant (E. coli lpxC101). Our results suggest that metabolic PL labeling with AECho is a promising tool for detecting a compromised bacterial OM, revealing aberrant PL externalization, and identifying or characterizing novel cell-active inhibitors of LPS biosynthesis or transport.

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Phospholipid distribution in the cytoplasmic membrane of Gram-negative bacteria is highly asymmetric, dynamic, and cell shape-dependent

Science Advances ◽

10.1126/sciadv.aaz6333 ◽

2020 ◽

Vol 6 (23) ◽

pp. eaaz6333 ◽

Cited By ~ 6

Author(s):

Mikhail Bogdanov ◽

Kyrylo Pyrshev ◽

Semen Yesylevskyy ◽

Sergey Ryabichko ◽

Vitalii Boiko ◽

...

Keyword(s):

Escherichia Coli ◽

Physical Properties ◽

Cell Shape ◽

Yersinia Pseudotuberculosis ◽

Cytoplasmic Membrane ◽

The Other ◽

Gram Negative Bacteria ◽

Gram Negative ◽

E Coli ◽

Phospholipid Distribution

The distribution of phospholipids across the inner membrane (IM) of Gram-negative bacteria is unknown. We demonstrate that the IMs of Escherichia coli and Yersinia pseudotuberculosis are asymmetric, with a 75%/25% (cytoplasmic/periplasmic leaflet) distribution of phosphatidylethanolamine (PE) in rod-shaped cells and an opposite distribution in E. coli filamentous cells. In initially filamentous PE-lacking E. coli cells, nascent PE appears first in the periplasmic leaflet. As the total PE content increases from nearly zero to 75%, cells progressively adopt a rod shape and PE appears in the cytoplasmic leaflet of the IM. The redistribution of PE influences the distribution of the other lipids between the leaflets. This correlates with the tendency of PE and cardiolipin to regulate antagonistically lipid order of the bilayer. The results suggest that PE asymmetry is metabolically controlled to balance temporally the net rates of synthesis and translocation, satisfy envelope growth capacity, and adjust bilayer chemical and physical properties.

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Variations in O-Antigen Biosynthesis and O-Acetylation Associated with Altered Phage Sensitivity in Escherichia coli 4s

Journal of Bacteriology ◽

10.1128/jb.02398-14 ◽

2014 ◽

Vol 197 (5) ◽

pp. 905-912 ◽

Cited By ~ 33

Author(s):

Yuriy A. Knirel ◽

Nikolai S. Prokhorov ◽

Alexander S. Shashkov ◽

Olga G. Ovchinnikova ◽

Evelina L. Zdorovenko ◽

...

Keyword(s):

Escherichia Coli ◽

Host Cells ◽

Side Chain ◽

Gram Negative Bacteria ◽

Gene Encoding ◽

Content Type ◽

Surface Receptors ◽

E Coli ◽

O Antigen ◽

Phage Sensitivity

The O polysaccharide of the lipopolysaccharide (O antigen) of Gram-negative bacteria often serves as a receptor for bacteriophages that can make the phage dependent on a given O-antigen type, thus supporting the concept of the adaptive significance of the O-antigen variability in bacteria. The O-antigen layer also modulates interactions of many bacteriophages with their hosts, limiting the access of the viruses to other cell surface receptors. Here we report variations of O-antigen synthesis and structure in an environmentalEscherichia coliisolate, 4s, obtained from horse feces, and its mutants selected for resistance to bacteriophage G7C, isolated from the same fecal sample. The 4s O antigen was found to be serologically, structurally, and genetically related to the O antigen ofE. coliO22, differing only in side-chain α-d-glucosylation in the former, mediated by agtrlocus on the chromosome. Spontaneous mutations ofE. coli4s occurring with an unusually high frequency affected either O-antigen synthesis or O-acetylation due to the inactivation of the gene encoding the putative glycosyltransferase WclH or the putative acetyltransferase WclK, respectively, by the insertion of IS1-like elements. These mutations induced resistance to bacteriophage G7C and also modified interactions ofE. coli4s with several other bacteriophages conferring either resistance or sensitivity to the host. These findings suggest that O-antigen synthesis and O-acetylation can both ensure the specific recognition of the O-antigen receptor following infection by some phages and provide protection of the host cells against attack by other phages.

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