Characterization of the Core Oligosaccharide and the O-Antigen Biological Repeating Unit from Halomonas stevensii Lipopolysaccharide: The First Case of O-Antigen Linked to the Inner Core

2012 ◽  
Vol 18 (12) ◽  
pp. 3729-3735 ◽  
Author(s):  
Giuseppina Pieretti ◽  
Sara Carillo ◽  
Buko Lindner ◽  
Kwang Kyu Kim ◽  
Keun Chul Lee ◽  
...  
Keyword(s):  
Inner Core ◽  
Core Oligosaccharide ◽  
The Core ◽  
O Antigen ◽  
First Case

scholarly journals Functional Characterization of MigA and WapR: Putative Rhamnosyltransferases Involved in Outer Core Oligosaccharide Biosynthesis of Pseudomonas aeruginosa

2008 ◽  
Vol 190 (6) ◽  
pp. 1857-1865 ◽  
Author(s):  
Karen K. H. Poon ◽  
Erin L. Westman ◽  
Evgeny Vinogradov ◽  
Shouguang Jin ◽  
Joseph S. Lam
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Functional Characterization ◽  
Outer Core ◽  
Resonance Spectroscopy ◽  
Core Oligosaccharide ◽  
Strain Pao1 ◽  
The Core ◽  
O Antigen ◽  
Knockout Mutants

ABSTRACT Pseudomonas aeruginosa lipopolysaccharide (LPS) contains two glycoforms of core oligosaccharide (OS); one form is capped with O antigen through an α-1,3-linked l-rhamnose (l-Rha), while the other is uncapped and contains an α-1,6-linked l-Rha. Two genes in strain PAO1, wapR (PA5000) and migA (PA0705), encode putative glycosyltransferases associated with core biosynthesis. We propose that WapR and MigA are the rhamnosyltransferases responsible for the two linkages of l-Rha to the core. Knockout mutants with mutations in both genes were generated. The wapR mutant produced LPS lacking O antigen, and addition of wapR in trans complemented this defect. The migA mutant produced LPS with a truncated outer core and showed no reactivity to outer core-specific monoclonal antibody (MAb) 5C101. Complementation of this mutant with migA restored reactivity of the LPS to MAb 5C101. Interestingly, LPS from the complemented migA strain was not reactive to MAb 18-19 (specific for the core-plus-one O repeat). This was due to overexpression of MigA in the complemented strain that caused an increase in the proportion of the uncapped core OS, thereby decreasing the amount of the core-plus-one O repeat, indicating that MigA has a regulatory role. The structures of LPS from both mutants were elucidated using nuclear magnetic resonance spectroscopy and mass spectrometry. The capped core of the wapR mutant was found to be truncated and lacked α-1,3-l-Rha. In contrast, uncapped core OS from the migA mutant lacked α-1,6-l-Rha. These results provide evidence that WapR is the α-1,3-rhamnosyltransferase, while MigA is the α-1,6-rhamnosyltransferase.

scholarly journals Structural Analysis of the Core Region of O-Lipopolysaccharide of Porphyromonas gingivalis from Mutants Defective in O-Antigen Ligase and O-Antigen Polymerase

2009 ◽  
Vol 191 (16) ◽  
pp. 5272-5282 ◽  
Author(s):  
Nikolay A. Paramonov ◽  
Joseph Aduse-Opoku ◽  
Ahmed Hashim ◽  
Minnie Rangarajan ◽  
Michael A. Curtis
Keyword(s):  
Porphyromonas Gingivalis ◽  
Inner Core ◽  
Outer Region ◽  
Outer Core ◽  
Core Region ◽  
Resonance Spectroscopy ◽  
Core Oligosaccharide ◽  
Unusual Structure ◽  
The Core ◽  
O Antigen

ABSTRACT Porphyromonas gingivalis synthesizes two lipopolysaccharides (LPSs), O-LPS and A-LPS. Here, we elucidate the structure of the core oligosaccharide (OS) of O-LPS from two mutants of P. gingivalis W50, ΔPG1051 (WaaL, O-antigen ligase) and ΔPG1142 (O-antigen polymerase), which synthesize R-type LPS (core devoid of O antigen) and SR-type LPS (core plus one repeating unit of O antigen), respectively. Structural analyses were performed using one-dimensional and two-dimensional nuclear magnetic resonance spectroscopy in combination with composition and methylation analysis. The outer core OS of O-LPS occurs in two glycoforms: an “uncapped core,” which is devoid of O polysaccharide (O-PS), and a “capped core,” which contains the site of O-PS attachment. The inner core region lacks l(d)-glycero-d(l)-manno-heptosyl residues and is linked to the outer core via 3-deoxy-d-manno-octulosonic acid, which is attached to a glycerol residue in the outer core via a monophosphodiester bridge. The outer region of the “uncapped core” is attached to the glycerol and is composed of a linear α-(1→3)-linked d-Man OS containing four or five mannopyranosyl residues, one-half of which are modified by phosphoethanolamine at position 6. An amino sugar, α-d-allosamine, is attached to the glycerol at position 3. In the “capped core,” there is a three- to five-residue extension of α-(1→3)-linked Man residues glycosylating the outer core at the nonreducing terminal residue. β-d-GalNAc from the O-PS repeating unit is attached to the nonreducing terminal Man at position 3. The core OS of P. gingivalis O-LPS is therefore a highly unusual structure, and it is the basis for further investigation of the mechanism of assembly of the outer membrane of this important periodontal bacterium.

scholarly journals Interactions of Pulmonary Collectins with Bordetella bronchiseptica and Bordetella pertussis Lipopolysaccharide Elucidate the Structural Basis of Their Antimicrobial Activities

2004 ◽  
Vol 72 (12) ◽  
pp. 7124-7130 ◽  
Author(s):  
Lyndsay M. Schaeffer ◽  
Francis X. McCormack ◽  
Huixing Wu ◽  
Alison A. Weiss
Keyword(s):  
Bordetella Pertussis ◽  
Lipid A ◽  
Antimicrobial Activities ◽  
Innate Immune ◽  
Bordetella Bronchiseptica ◽  
Structural Basis ◽  
Wild Type ◽  
Core Oligosaccharide ◽  
The Core ◽  
O Antigen

ABSTRACT Surfactant proteins A (SP-A) and D (SP-D) play an important role in the innate immune defenses of the respiratory tract. SP-A binds to the lipid A region of lipopolysaccharide (LPS), and SP-D binds to the core oligosaccharide region. Both proteins induce aggregation, act as opsonins for neutrophils and macrophages, and have direct antimicrobial activity. Bordetella pertussis LPS has a branched core structure and a nonrepeating terminal trisaccharide. Bordetella bronchiseptica LPS has the same structure, but lipid A is palmitoylated and there is a repeating O-antigen polysaccharide. The ability of SP-A and SP-D to agglutinate and permeabilize wild-type and LPS mutants of B. pertussis and B. bronchiseptica was examined. Previously, wild-type B. pertussis was shown to resist the effects of SP-A; however, LPS mutants lacking the terminal trisaccharide were susceptible to SP-A. In this study, SP-A was found to aggregate and permeabilize a B. bronchiseptica mutant lacking the terminal trisaccharide, while wild-type B. bronchiseptica and mutants lacking only the palmitoyl transferase or O antigen were resistant to SP-A. Wild-type B. pertussis and B. bronchiseptica were both resistant to SP-D; however, LPS mutants of either strain lacking the terminal trisaccharide were aggregated and permeabilized by SP-D. We conclude that the terminal trisaccharide protects Bordetella species from the bactericidal functions of SP-A and SP-D. The O antigen and palmitoylated lipid A of B. bronchiseptica play no role in this resistance.

scholarly journals Functional Characterization of WaaL, a Ligase Associated with Linking O-Antigen Polysaccharide to the Core of Pseudomonas aeruginosa Lipopolysaccharide

2005 ◽  
Vol 187 (9) ◽  
pp. 3002-3012 ◽  
Author(s):  
Priyanka D. Abeyrathne ◽  
Craig Daniels ◽  
Karen K. H. Poon ◽  
Mauricia J. Matewish ◽  
Joseph S. Lam
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Lipid A ◽  
Functional Characterization ◽  
Wild Type ◽  
The Core ◽  
O Antigen ◽  
Repeat Units ◽  
Cytoplasmic Face ◽  
O Antigens

ABSTRACT The O antigen of Pseudomonas aeruginosa B-band lipopolysaccharide is synthesized by assembling O-antigen-repeat units at the cytoplasmic face of the inner membrane by nonprocessive glycosyltransferases, followed by polymerization on the periplasmic face. The completed chains are covalently attached to lipid A core by the O-antigen ligase, WaaL. In P. aeruginosa the process of ligating these O-antigen molecules to lipid A core is not clearly defined, and an O-antigen ligase has not been identified until this study. Using the sequence of waaL from Salmonella enterica as a template in a BLAST search, a putative waaL gene was identified in the P. aeruginosa genome. The candidate gene was amplified and cloned, and a chromosomal knockout of PAO1 waaL was generated. Lipopolysaccharide (LPS) from this mutant is devoid of B-band O-polysaccharides and semirough (SR-LPS, or core-plus-one O-antigen). The mutant PAO1waaL is also deficient in the production of A-band polysaccharide, a homopolymer of d-rhamnose. Complementation of the mutant with pPAJL4 containing waaL restored the production of both A-band and B-band O antigens as well as SR-LPS, indicating that the knockout was nonpolar and waaL is required for the attachment of O-antigen repeat units to the core. Mutation of waaL in PAO1 and PA14, respectively, could be complemented with waaL from either strain to restore wild-type LPS production. The waaL mutation also drastically affected the swimming and twitching motilities of the bacteria. These results demonstrate that waaL in P. aeruginosa encodes a functional O-antigen ligase that is important for cell wall integrity and motility of the bacteria.

scholarly journals The lipopolysaccharide of the mastitis isolate Escherichia coli strain 1303 comprises a novel O-antigen and the rare K-12 core type

Microbiology ◽  
2011 ◽  
Vol 157 (6) ◽  
pp. 1750-1760 ◽  
Author(s):  
Katarzyna A. Duda ◽  
Buko Lindner ◽  
Helmut Brade ◽  
Andreas Leimbach ◽  
Elżbieta Brzuszkiewicz ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Inner Core ◽  
Bovine Mastitis ◽  
Outer Core ◽  
Coli Strain ◽  
Core Oligosaccharide ◽  
2D Nmr Spectroscopy ◽  
E Coli ◽  
O Antigen ◽  
K 12

Mastitis represents one of the most significant health problems of dairy herds. The two major causative agents of this disease are Escherichia coli and Staphylococcus aureus. Of the first, its lipopolysaccharide (LPS) is thought to play a prominent role during infection. Here, we report the O-antigen (OPS, O-specific polysaccharide) structure of the LPS from bovine mastitis isolate E. coli 1303. The structure was determined utilizing chemical analyses, mass spectrometry, and 1D and 2D NMR spectroscopy methods. The O-repeating unit was characterized as -[→4)-β-d-Quip3NAc-(1→3)-α-l-Fucp2OAc-(1→4)-β-d-Galp-(1→3)-α-d-GalpNAc-(1→]- in which the O-acetyl substitution was non-stoichiometric. The nucleotide sequence of the O-antigen gene cluster of E. coli 1303 was also determined. This cluster, located between the gnd and galF genes, contains 13 putative open reading frames, most of which represent unknown nucleotide sequences that have not been described before. The O-antigen of E. coli 1303 was shown to substitute O-7 of the terminal ld-heptose of the K-12 core oligosaccharide. Interestingly, the non-OPS-substituted core oligosaccharide represented a truncated version of the K-12 outer core – namely terminal ld-heptose and glucose were missing; however, it possessed a third Kdo residue in the inner core. On the basis of structural and genetic data we show that the mastitis isolate E. coli 1303 represents a new serotype and possesses the K-12 core type, which is rather uncommon among human and bovine isolates.

scholarly journals Identification of the Linkage between A-Polysaccharide and the Core in the A-Lipopolysaccharide of Porphyromonas gingivalis W50

2015 ◽  
Vol 197 (10) ◽  
pp. 1735-1746 ◽  
Author(s):  
Nikolay Paramonov ◽  
Joseph Aduse-Opoku ◽  
Ahmed Hashim ◽  
Minnie Rangarajan ◽  
Michael A. Curtis
Keyword(s):  
Porphyromonas Gingivalis ◽  
Biosynthetic Pathway ◽  
Attachment Site ◽  
Outer Core ◽  
Etiologic Agent ◽  
Core Oligosaccharide ◽  
Content Type ◽  
The Core ◽  
O Antigen ◽  
Mutant Strains

ABSTRACTPorphyromonas gingivalissynthesizes two lipopolysaccharides (LPSs), O-LPS and A-LPS. The structure of the core oligosaccharide (OS) of O-LPS and the attachment site of the O-polysaccharide (O-PS) repeating unit [→3)-α-d-Galp-(1→6)-α-d-Glcp-(1→4)-α-l-Rhap-(1→3)-β-d-GalNAcp-(1→] to the core have been elucidated using the ΔPG1051 (WaaL, O-antigen ligase) and ΔPG1142 (Wzy, O-antigen polymerase) mutant strains, respectively. The core OS occurs as an “uncapped” glycoform devoid of O-PS and a “capped” glycoform that contains the attachment site of O-PS via β-d-GalNAc at position O-3 of the terminal α-(1→3)-linked mannose (Man) residue. In this study, the attachment site of A-PS to the core OS was determined based on structural analysis of SR-type LPS (O-LPS and A-LPS) isolated from aP. gingivalisΔPG1142 mutant strain by extraction with aqueous hot phenol to minimize the destruction of A-LPS. Application of one- and two-dimensional nuclear magnetic resonance (NMR) spectroscopy in combination with methylation analysis showed that the A-PS repeating unit is linked to a nonterminal α-(1→3)-linked Man of the “capped core” glycoform of outer core OS at position O-4 via a →6)-[α-d-Man-α-(1→2)-α-d-Man-1-phosphate→2]-α-d-Man-(1→ motif. In order to verify that O-PS and A-PS are attached to almost identical core glycoforms, we identified a putative α-mannosyltransferase (PG0129) inP. gingivalisW50 that may be involved in the formation of core OS. Inactivation of PG0129 led to the synthesis of deep-R-type LPS with a truncated core that lacks α-(1→3)-linked mannoses and is devoid of either O-PS or A-PS. This indicated that PG0129 is an α-1,3-mannosyltransferase required for synthesis of the outer core regions of both O-LPS and A-LPS inP. gingivalis.IMPORTANCEPorphyromonas gingivalis, a Gram-negative anaerobe, is considered to be an important etiologic agent in periodontal disease, and among the virulence factors produced by the organism are two lipopolysaccharides (LPSs), O-LPS and A-LPS. The structures of the O-PS and A-PS repeating units, the core oligosaccharide (OS), and the linkage of the O-PS repeating unit to the core OS in O-LPS have been elucidated by our group. It is important to establish whether the attachment site of the A-PS repeating unit to the core OS in A-LPS is similar to or differs from that of the O-PS repeating unit in O-LPS. As part of understanding the biosynthetic pathway of the two LPSs inP. gingivalis, PG0129 was identified as an α-mannosyltransferase that is involved in the synthesis of the outer core regions of both O-LPS and A-LPS.

scholarly journals Analysis of the Structure and Biosynthesis of the Lipopolysaccharide Core Oligosaccharide of Pseudomonas syringae pv. tomato DC3000

2021 ◽  
Vol 22 (6) ◽  
pp. 3250
Author(s):  
Alexander Kutschera ◽  
Ursula Schombel ◽  
Dominik Schwudke ◽  
Stefanie Ranf ◽  
Nicolas Gisch
Keyword(s):  
Pseudomonas Syringae ◽  
Species Complex ◽  
Inner Core ◽  
Divalent Cations ◽  
Membrane Integrity ◽  
Outer Core ◽  
Core Oligosaccharide ◽  
Structure Information ◽  
The Core ◽  
Genetic Components

Lipopolysaccharide (LPS), the major component of the outer membrane of Gram-negative bacteria, is important for bacterial viability in general and host–pathogen interactions in particular. Negative charges at its core oligosaccharide (core-OS) contribute to membrane integrity through bridging interactions with divalent cations. The molecular structure and synthesis of the core-OS have been resolved in various bacteria including the mammalian pathogen Pseudomonas aeruginosa. A few core-OS structures of plant-associated Pseudomonas strains have been solved to date, but the genetic components of the underlying biosynthesis remained unclear. We conducted a comparative genome analysis of the core-OS gene cluster in Pseudomonas syringae pv. tomato (Pst) DC3000, a widely used model pathogen in plant–microbe interactions, within the P. syringae species complex and to other plant-associated Pseudomonas strains. Our results suggest a genetic and structural conservation of the inner core-OS but variation in outer core-OS composition within the P. syringae species complex. Structural analysis of the core-OS of Pst DC3000 shows an uncommonly high phosphorylation and presence of an O-acetylated sugar. Finally, we combined the results of our genomic survey with available structure information to estimate the core-OS composition of other Pseudomonas species.

scholarly journals Characterization of Vibrio cholerae O1 Antigen as the Bacteriophage K139 Receptor and Identification of IS1004Insertions Aborting O1 Antigen Biosynthesis

2000 ◽  
Vol 182 (18) ◽  
pp. 5097-5104 ◽  
Author(s):  
Jutta Nesper ◽  
Dagmar Kapfhammer ◽  
Karl E. Klose ◽  
Hilde Merkert ◽  
Joachim Reidl
Keyword(s):  
Vibrio Cholerae ◽  
Binding Studies ◽  
Core Oligosaccharide ◽  
O Antigen ◽  
Mouse Small Intestine ◽  
Normal Human ◽  
Vibrio Cholerae O1 ◽  
Phage Receptor ◽  
El Tor

ABSTRACT Bacteriophage K139 was recently characterized as a temperate phage of O1 Vibrio cholerae. In this study we have determined the phage adsorption site on the bacterial cell surface. Phage-binding studies with purified lipopolysaccharide (LPS) of different O1 serotypes and biotypes revealed that the O1 antigen serves as the phage receptor. In addition, phage-resistant O1 El Tor strains were screened by using a virulent isolate of phage K139. Analysis of the LPS of such spontaneous phage-resistant mutants revealed that most of them synthesize incomplete LPS molecules, composed of either defective O1 antigen or core oligosaccharide. By applying phage-binding studies, it was possible to distinguish between receptor mutants and mutations which probably caused abortion of later steps of phage infection. Furthermore, we investigated the genetic nature of O1-negative strains by Southern hybridization with probes specific for the O antigen biosynthesis cluster (rfb region). Two of the investigated O1 antigen-negative mutants revealed insertions of element IS1004 into the rfb gene cluster. Treating onewbeW::IS1004 serum-sensitive mutant with normal human serum, we found that several survivors showed precise excision of IS1004, restoring O antigen biosynthesis and serum resistance. Investigation of clinical isolates by screening for phage resistance and performing LPS analysis of nonlysogenic strains led to the identification of a strain with decreased O1 antigen presentation. This strain had a significant reduction in its ability to colonize the mouse small intestine.

Isolation and characterization of 3-acetamido-3,6-dideoxy-L-glucose from the core oligosaccharides obtained from the aquatic gram-negative bacteria Aeromonas hydrophila and Vibrio anguillarum

1981 ◽  
Vol 59 (11-12) ◽  
pp. 877-879 ◽  
Author(s):  
Joseph H. Banoub ◽  
Derek H. Shaw
Keyword(s):  
Aeromonas Hydrophila ◽  
Vibrio Anguillarum ◽  
Amino Sugar ◽  
Gram Negative Bacteria ◽  
Core Oligosaccharide ◽  
Gram Negative ◽  
The Core ◽  
Isolation And Characterization ◽  
Bacterial Lipopolysaccharides

The amino sugar 3-acetamido-3,6-dideoxy-L-glucose has been isolated and characterized from the core oligosaccharide obtained from the bacterial lipopolysaccharides of Aeromonas hydrophila and Vibrio anguillarum. This is the first occasion in which a dideoxyamino sugar has been confirmed as a constituent of the core oligosaccharide rather than the O-polysaccharide.

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