title: Single-Nucleotide Polymorphisms Found in the migA and wbpX Glycosyltransferase Genes Account for the Intrinsic Lipopolysaccharide Defects Exhibited by Pseudomonas aeruginosa PA14.
abstract: Pseudomonas aeruginosa PA14 is widely used by researchers in many laboratories because of its enhanced virulence over strain PAO1 in a wide range of hosts. Although lipopolysaccharide (LPS) is an important virulence factor of all P. aeruginosa strains, the LPS of PA14 has not been characterized fully. A recent study showed that the structure of its O-specific antigen (OSA) belongs to serotype O19. We found that the OSA gene cluster of PA14 shares ∼99% identity with those of the O10/O19 group. These two serotypes share the same O-unit structure, except for an O-acetyl substitution in one of the sugars in O10. Here we showed that both PA14 and O19 LPS cross-reacted with the O10-specific monoclonal antibody MF76-2 in Western blots. Analysis by SDS-PAGE and silver staining showed that PA14 LPS exhibited modal chain lengths that were different from those of O19 LPS, in that only "very long" and "short" chain lengths were observed, while "medium" and "long" chain lengths were not detected. Two other novel observations included the lack of the uncapped core oligosaccharide epitope and of common polysaccharide antigen (CPA) LPS. The lack of the uncapped core oligosaccharide was caused by point mutations in the glycosyltransferase gene migA, while the CPA-negative phenotype was correlated with a single amino acid substitution, G20R, in the glycosyltransferase WbpX. Additionally, we showed that restoring CPA biosynthesis in PA14 significantly stimulated mature biofilm formation after 72 h, while outer membrane vesicle production was not affected.
  
  
  OBJECTIVE
  P. aeruginosa PA14 is a clinical isolate that has become an important reference strain used by many researchers worldwide. LPS of PA14 has not been characterized fully, and hence, confusion about its phenotype exists in the literature. In the present study, we set out to characterize the O-specific antigen (OSA), the common polysaccharide antigen (CPA), and the core oligosaccharide produced by PA14. We present evidence that PA14 produces an LPS consisting of "very-long-chain" and some "short-chain" OSA belonging to the O19 serotype but is devoid of CPA and the uncapped core oligosaccharide epitope. These intrinsic defects in PA14 LPS were due to single-nucleotide polymorphisms (SNPs) in the genes that encode glycosyltransferases in the corresponding biosynthesis pathways. Since sugars in CPA and the uncapped core are receptors for different bacteriocins and pyocins, the lack of CPA and an intact core may contribute to the increased virulence of PA14. Restoring CPA production in PA14 was found to stimulate mature biofilm formation.
authors.affiliation: Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario, Canada.
authors.forename: Youai
authors.initials: Y
authors.lastname: Hao
authors.affiliation: Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario, Canada.
authors.forename: Kathleen
authors.initials: K
authors.lastname: Murphy
authors.affiliation: Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario, Canada.
authors.forename: Reggie Y
authors.initials: RY
authors.lastname: Lo
authors.affiliation: Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario, Canada ckhursig@uoguelph.ca jlam@uoguelph.ca.
authors.forename: Cezar M
authors.identifier: http://orcid.org/0000-0001-9263-7604
authors.initials: CM
authors.lastname: Khursigara
authors.affiliation: Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario, Canada ckhursig@uoguelph.ca jlam@uoguelph.ca.
authors.forename: Joseph S
authors.identifier: http://orcid.org/0000-0002-1822-1165
authors.initials: JS
authors.lastname: Lam
mesh_terms: D000942:Antigens, Bacterial / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D018441:Biofilms; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology; D016695:Glycosyltransferases / Q000235:genetics* / Q000378:metabolism; D008070:Lipopolysaccharides / Q000737:chemistry / Q000378:metabolism*; D017354:Point Mutation; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*; D011550:Pseudomonas aeruginosa / Q000145:classification / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D065288:Serogroup
chemical_list: D000942:Antigens, Bacterial; D001426:Bacterial Proteins; D008070:Lipopolysaccharides; D016695:Glycosyltransferases