Characterization of the Kingella kingae Polysaccharide Capsule and Exopolysaccharide

Abstract

Recent evidence indicates that Kingella kingae produces a polysaccharide capsule. In an effort to determine the composition and structure of this polysaccharide capsule, in the current study we purified capsular material from the surface of K. kingae strain 269–492 variant KK01 using acidic conditions to release the capsule and a series of steps to remove DNA, RNA, and protein. Analysis of the resulting material by gas chromatography and mass spectrometry revealed N-acetyl galactosamine (GalNAc), 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo), and galactose (Gal). Further analysis by NMR demonstrated two distinct polysaccharides, one consisting of GalNAc and Kdo with the structure →3)-β-GalpNAc-(1→5)-β-Kdop-(2→ and the other containing galactose alone with the structure →5)-β-Galf-(1→. Disruption of the ctrA gene required for surface localization of the K. kingae polysaccharide capsule resulted in elimination of GalNAc and Kdo but had no effect on the presence of Gal in bacterial surface extracts. In contrast, deletion of the pamABCDE locus involved in production of a reported galactan exopolysaccharide eliminated Gal but had no effect on the presence of GalNAc and Kdo in surface extracts. Disruption of ctrA and deletion of pamABCDE resulted in a loss of all carbohydrates in surface extracts. These results establish that K. kingae strain KK01 produces a polysaccharide capsule with the structure →3)-β-GalpNAc-(1→5)-β-Kdop-(2→ and a separate exopolysaccharide with the structure →5)-β-Galf-(1→. The polysaccharide capsule and the exopolysaccharide require distinct genetic loci for surface localization.

Oropharyngeal Kingella kingae carriage in children: characteristics and correlation with osteoarticular infections

“…Previous evidence suggested differences in virulence, colonization capacity and affinity for osteoarticular tissue among different K. kingae strains (23,34). This raises the question of whether age, gender, or season influences the susceptibility of an organism to be colonized with a more or less virulent clone.…”

“…The available data suggest that by 24 mo most children who will be infected by K. kingae already have been infected resulting in acquired immunity (15,22). Furthermore, recent investigation demonstrated the presence of a carbohydrate capsule in K kingae (23). Since immunity to polysaccharides in humans matures between the ages of 2-4 y (24), this may explain the increased risk of clinical apparent invasive infection in colonized children below the age of 24 mo.…”

Genetic and Molecular Basis of Kingella kingae Encapsulation

“…The gene disruptions and complementation constructs were confirmed by PCR. The ctrA and pamABCDE disruptions used in this study were generated previously (3,4). To delete the lipA, lipB, and csaA loci, fragments corresponding to the surrounding 5= and 3= regions were PCR amplified by using the primers shown in Table 2, and a resistance cassette was ligated between the flanking regions.…”

“…In previous work, our group described a series of surface factors expressed by K. kingae that influence adherence to epithelial cells in vitro, including type IV pili and a trimeric autotransporter protein called Knh. Adherence by our prototype K. kingae strain is impeded by a polysaccharide capsule containing N-acetylgalactosamine (GalNAc) and ␤-3-deoxy-Dmanno-octulosonic acid (␤Kdo) with the structure [¡3)-␤-GalpNAc-(1¡5)-␤-Kdop-(2¡] n , which masks the adhesive properties of Knh and is likely displaced when pili bind to the cell surface and retract (3,4).…”

Quality control of sweet medicines based on gas chromatography-mass spectrometry

“…Besides, anhydrosugars that are common in red/brown algal cell walls such as 3,6-anhydrogalactose need to be analyzed by reductive hydrolysis (64). Mild methanolysis is required for the detection of 3-deoxy-D-manno-oct-2-ulosonic acid (KDO), which is used by bacteria in the synthesis of lipopolysaccharides (65)(66)(67). GlcNAc also easily destroyed during hydrolysis therefore mild acid condition or methanolysis is needed (68)(69)(70)(71).…”