Structural characterization of Haemophilus parainfluenzae lipooligosaccharide and elucidation of its role in adherence using an outer core mutant

2008 ◽  
Vol 54 (11) ◽  
pp. 906-917 ◽  
Author(s):  
Angela Pollard ◽  
Frank St. Michael ◽  
Lynn Connor ◽  
Wade Nichols ◽  
Andrew Cox
Keyword(s):  
Inner Core ◽  
Structural Information ◽  
Outer Core ◽  
Epithelial Cell Line ◽  
Resonance Spectroscopy ◽  
Nontypeable Haemophilus Influenzae ◽  
Core Oligosaccharide ◽  
Oligosaccharide Structure ◽  
Haemophilus Parainfluenzae

The opportunistic pathogen Haemophilus parainfluenzae is a gram-negative bacterium found in the oropharynx of humans. Haemophilus parainfluenzae is a member of the Pasteurellaceae family in which it is most closely related to Haemophilus sengis and Actinobacillus . Characterization of surface displayed lipooligosaccharide has identified components that are crucial in adherence. We examined the oligosaccharide structure of lipooligosaccharide from 2 clinical isolates of H. parainfluenzae. Core oligosaccharide was isolated by standard methods from purified lipooligosaccharide. Structural information was established by a combination of monosaccharide and methylation analyses, nuclear magnetic resonance spectroscopy, and mass spectrometry revealing the following structures: R-(1-6)-β-Glc-(1-4)-d,d-α-Hep-(1-6)-β-Glc-(1-4)- substituting a tri-heptose-Kdo inner core of L,d-α-Hep-(1-2)-l,d-α-Hep-(1-3)-l,d-α-Hep-(1-5)-α-Kdo at the 4-position of the proximal l,d-α-Hep residue to Kdo, and with a PEtn residue at the 6-position of the central l,d-α-Hep residue. In strain 4282, the R substituent is β-galactose and in strain 4201 there is no substituent at the distal glucose. These analyses have revealed that multiple structural aspects of H. parainfluenzae lipooligosaccharide are comparable with nontypeable Haemophilus influenzae lipooligosaccharide. This study also identified a galactan in strain 4201 and a glucan in strain 4282. Haemophilus parainfluenzae was shown to adhere to a bronchial epithelial cell line to the same degree as nontypeable H. influenzae. However, an H. parainfluenzae mutant lacking the outer core of the lipooligosaccharide showed diminished adherence to the epithelial cells, suggesting that H. parainfluenzae lipooligosaccharide plays a role in tissue colonization.

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 Relative Contributions of Lipooligosaccharide Inner and Outer Core Modifications to Nontypeable Haemophilus influenzae Pathogenesis

2013 ◽  
Vol 81 (11) ◽  
pp. 4100-4111 ◽  
Author(s):  
Pau Morey ◽  
Cristina Viadas ◽  
Begoña Euba ◽  
Derek W. Hood ◽  
Montserrat Barberán ◽  
...  
Keyword(s):  
Haemophilus Influenzae ◽  
Bacterial Resistance ◽  
Inner Core ◽  
Outer Core ◽  
Nontypeable Haemophilus Influenzae ◽  
Core Oligosaccharide ◽  
Biofilm Growth ◽  
Content Type ◽  
Relative Contribution ◽  
Self Aggregation

ABSTRACTNontypeableHaemophilus influenzae(NTHi) is a frequent commensal of the human nasopharynx that causes opportunistic infection in immunocompromised individuals. Existing evidence associates lipooligosaccharide (LOS) with disease, but the specific and relative contributions of NTHi LOS modifications to virulence properties of the bacterium have not been comprehensively addressed. Using NTHi strain 375, an isolate for which the detailed LOS structure has been determined, we compared systematically a set of isogenic mutant strains expressing sequentially truncated LOS. The relative contributions of 2-keto-3-deoxyoctulosonic acid, the triheptose inner core, oligosaccharide extensions on heptoses I and III, phosphorylcholine, digalactose, and sialic acid to NTHi resistance to antimicrobial peptides (AMP), self-aggregation, biofilm formation, cultured human respiratory epithelial infection, and murine pulmonary infection were assessed. We show thatopsX,lgtF,lpsA,lic1, andlic2Acontribute to bacterial resistance to AMP;lic1is related to NTHi self-aggregation;lgtF,lic1, andsiaBare involved in biofilm growth;opsXandlgtFparticipate in epithelial infection; andopsX,lgtF, andlpsAcontribute to lung infection. Depending on the phenotype, the involvement of these LOS modifications occurs at different extents, independently or having an additive effect in combination. We discuss the relative contribution of LOS epitopes to NTHi virulence and frame a range of pathogenic traits in the context of infection.

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 Pasteurella multocida Expresses Two Lipopolysaccharide Glycoforms Simultaneously, but Only a Single Form Is Required for Virulence: Identification of Two Acceptor-Specific Heptosyl I Transferases

2007 ◽  
Vol 75 (8) ◽  
pp. 3885-3893 ◽  
Author(s):  
Marina Harper ◽  
John D. Boyce ◽  
Andrew D. Cox ◽  
Frank St. Michael ◽  
Ian W. Wilkie ◽  
...  
Keyword(s):  
Pasteurella Multocida ◽  
Inner Core ◽  
Phase Variation ◽  
Outer Core ◽  
Core Oligosaccharide ◽  
Bacterial Mutants ◽  
Major Antigen ◽  
First Time ◽  
Mucosal Pathogens

ABSTRACT Lipopolysaccharide (LPS) is a critical virulence determinant in Pasteurella multocida and a major antigen responsible for host protective immunity. In other mucosal pathogens, variation in LPS or lipooligosaccharide structure typically occurs in the outer core oligosaccharide regions due to phase variation. P. multocida elaborates a conserved oligosaccharide extension attached to two different, simultaneously expressed inner core structures, one containing a single phosphorylated 3-deoxy-d-manno-octulosonic acid (Kdo) residue and the other containing two Kdo residues. We demonstrate that two heptosyltransferases, HptA and HptB, add the first heptose molecule to the Kdo1 residue and that each exclusively recognizes different acceptor molecules. HptA is specific for the glycoform containing a single, phosphorylated Kdo residue (glycoform A), while HptB is specific for the glycoform containing two Kdo residues (glycoform B). In addition, KdkA was identified as a Kdo kinase, required for phosphorylation of the first Kdo molecule. Importantly, virulence data obtained from infected chickens showed that while wild-type P. multocida expresses both LPS glycoforms in vivo, bacterial mutants that produced only glycoform B were fully virulent, demonstrating for the first time that expression of a single LPS form is sufficient for P. multocida survival in vivo. We conclude that the ability of P. multocida to elaborate alternative inner core LPS structures is due to the simultaneous expression of two different heptosyltransferases that add the first heptose residue to the nascent LPS molecule and to the expression of both a bifunctional Kdo transferase and a Kdo kinase, which results in the initial assembly of two inner core structures.

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 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 Rapid Structural Analysis of a Synthetic Non-canonical Amino Acid by Microcrystal Electron Diffraction

2021 ◽  
Vol 7 ◽  
Author(s):  
Patrick R. Gleason ◽  
Brent L. Nannenga ◽  
Jeremy H. Mills
Keyword(s):  
Amino Acid ◽  
Electron Diffraction ◽  
Structure Determination ◽  
Structural Model ◽  
Structural Information ◽  
Enzymatic Reaction ◽  
Unnatural Amino Acid ◽  
Resonance Spectroscopy ◽  
Canonical Amino Acid

Structural characterization of small molecules is a crucial component of organic synthesis. In this work, we applied microcrystal electron diffraction (MicroED) to analyze the structure of the product of an enzymatic reaction that was intended to produce the unnatural amino acid 2,4-dihydroxyphenylalanine (24DHF). Characterization of our isolated product with nuclear magnetic resonance spectroscopy (NMR) and mass spectrometry (MS) suggested that an isomer of 24DHF had been formed. Microcrystals present in the isolated product were then used to determine its structure to 0.62 Å resolution, which confirmed its identity as 2-amino-2-(2,4-dihydroxyphenyl)propanoic acid (24DHPA). Moreover, the MicroED structural model indicated that both enantiomeric forms of 24DHPA were present in the asymmetric unit. Notably, the entire structure determination process including setup, data collection, and refinement was completed in ~1 h. The MicroED data not only bolstered previous results obtained using NMR and MS but also immediately provided information about the stereoisomers present in the product, which is difficult to achieve using NMR and MS alone. Our results therefore demonstrate that MicroED methods can provide useful structural information on timescales that are similar to many commonly used analytical methods and can be added to the existing suite of small molecule structure determination tools in future studies.

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

Conformational and Structural characterization of carbohydrates and their interactions studied by NMR

2021 ◽  
Vol 28 ◽  
Author(s):  
Francisco Javier Cañada ◽  
Ángeles Canales ◽  
Pablo Valverde ◽  
Beatriz Fernández de Toro ◽  
Mónica Martínez-Orts ◽  
...  
Keyword(s):  
Structural Information ◽  
Theoretical Calculations ◽  
Chemical Shifts ◽  
Structural Complexity ◽  
Coupling Constants ◽  
Resonance Spectroscopy ◽  
Additional Information ◽  
Nuclear Overhauser ◽  
Nuclear Overhauser Effects

: Carbohydrates, either free or as glycans conjugated with other biomolecules, participate in many essential biological processes. Their apparent simplicity in terms of chemical functionality hides an extraordinary diversity and structural complexity. Deeply deciphering at the atomic level their structures is essential to understand their biological function and activities, but it is still a challenging task in need of complementary approaches and no generalized procedures are available to address the study of such complex, natural glycans. The versatility of Nuclear Magnetic Resonance spectroscopy (NMR) often makes it the preferred choice to study glycans and carbohydrates in solution media. The most basic NMR parameters, namely chemical shifts, coupling constants and nuclear Overhauser effects, allow defining short or repetitive chain sequences and characterize their structures and local geometries either in the free state or when interacting with other biomolecules, rendering additional information on the molecular recognition processes. The increased accessibility to carbohydrate molecules extensively or selectively labeled with 13C boosts the resolution and detail that analyzed glycan structures can reach. In turn, structural information derived from NMR, complemented with molecular modeling and theoretical calculations can also provide dynamic information on the conformational flexibility of carbohydrate structures. Furthermore, using partially oriented media or paramagnetic perturbations, it has been possible to introduce additional long-range observables rendering structural information on longer and branched glycan chains. In this review, we provide examples of these studies and an overview of the recent and most relevant NMR applications in the glycobiology field.

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