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

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.

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

Journal of Bacteriology ◽

10.1128/jb.187.9.3002-3012.2005 ◽

2005 ◽

Vol 187 (9) ◽

pp. 3002-3012 ◽

Cited By ~ 84

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.

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

Journal of Bacteriology ◽

10.1128/jb.00019-09 ◽

2009 ◽

Vol 191 (16) ◽

pp. 5272-5282 ◽

Cited By ~ 35

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.

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

Journal of Bacteriology ◽

10.1128/jb.02562-14 ◽

2015 ◽

Vol 197 (10) ◽

pp. 1735-1746 ◽

Cited By ~ 13

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.

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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

Chemistry - A European Journal ◽

10.1002/chem.201102550 ◽

2012 ◽

Vol 18 (12) ◽

pp. 3729-3735 ◽

Cited By ~ 9

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

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

Canadian Journal of Microbiology ◽

10.1139/w08-082 ◽

2008 ◽

Vol 54 (11) ◽

pp. 906-917 ◽

Cited By ~ 8

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.

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