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The retentates were then deuterium-exchanged for NMR analysis. The retentates after xylanase digestion total amount were deuterium exchanged 2 times by lyophilization in D 2 O.

For glycosyl linkage analysis, the samples were permethylated, depolymerized, reduced, and acetylated. Approximately 1 mg quantities of the samples were used for linkage analysis. Permethylation was effected by two rounds of treatment with sodium hydroxide 15 min and methyl iodide 45 min. Separation was performed on a 30 m Supelco SP bonded phase fused silica capillary column. To prepare the partially methylated alditol acetate PMAA derivatives of Kdo, the permethylated polysaccharide samples were subjected to sequential steps: reduction of the Kdo carboxymethyl groups with lithium triethylborodeuteride Superdeuteride, Aldrich, St.

After incubation, the mixtures were dried under a stream of dry nitrogen and then redried with absolute methanol. The same procedure was applied to authentic monosaccharide standards. Peaks were integrated using Chemstation software. A full FTMS spectrum was collected at 30, resolution with 20 microscans. PCR amplicons were sequenced, and the resulting sequence was the basis for design of interior primers specific for each of the four capsule synthesis loci.

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To screen for the presence of each locus, we used both universal flanking primers that amplified all capsule loci and locus-specific primers that annealed to the interior portion of each locus. The presence of either csaA , csbABC , cscABC , or csdABC the csa , csb , csc , or csd capsule synthesis locus was determined by PCR amplification using interior primers see Table 7 and confirmed by determining the size and restriction map of the flanking primer amplicon.

The digests were resolved on a 1. The statistical significance of the differences in the distribution of the different capsule types among carrier vs. The terminal residues t- arise from the non-reducing end of the polysaccharide; Kdo has two diastereomeric PMAAs due to the non-stereospecific reduction of C-2, which produces a new chiral center.

A Mass spectrum of the peak at This number is also equal to the charge state in the mass spectrum. Sequencing of the capsule synthesis locus flanking PCR amplicon from PYKK56 Panel B, Lane 5 revealed a csaA gene with a bp deletion in the open reading frame G , which also introduces a frameshift mutation leading to a predicted truncated CsaA protein of amino acids versus the wild type amino acid protein.

Funding acquisition: JWS. Project administration: JWS. Abstract Kingella kingae is an encapsulated gram-negative organism that is a common cause of osteoarticular infections in young children. Author Summary Kingella kingae is a gram-negative pathogen that is being recognized increasingly as a cause of joint, bone, and other bloodborne infections in young children, reflecting advances in cultivation techniques and molecular methods of detection.

Introduction Kingella kingae is being recognized increasingly as an important cause of bone and joint infections in young children, reflecting more sensitive cultivation techniques and increased availability of molecular-based diagnostic tools [ 1 , 2 ]. Results Four capsule synthesis loci are present in a diverse collection of K. Download: PPT. Fig 1. PCR screening of capsule synthesis genes reveals four loci. The four capsule synthesis loci are associated with four different polysaccharide capsule types To confirm that each of the four capsule synthesis loci is associated with a specific capsule type, we examined the glycosyl composition of purified polysaccharide capsule from representative strains that contain either the csa , csb , csc , or csd locus.

Table 1. Association between capsule locus screening and capsule composition. Fig 3. Two-dimensional NMR spectra of the polysaccharides isolated from K. Table 2. Chemical shift assignments of the type b capsular polysaccharide purified from PYKK Table 3. Chemical shift assignments of the type c capsular polysaccharide purified from PYKK Table 4. Chemical shift assignments of the type d capsular polysaccharide purified from BB The csa , csb , csc , and csd capsule synthesis loci are necessary and sufficient for polysaccharide capsule synthesis To confirm that the csa , csb , csc , and csd loci are essential for production of capsule, we deleted each of these loci and then examined the resulting strains for surface material that stains with Alcian blue.

Fig 5. Comparison of capsule migration pattern between capsule types. Table 5. Comparative molar ratio of main glycosyl residues in polysaccharide purified from the surface of the capsule swap strains as detected by 1-D Proton NMR. The type a and type b capsules are enriched in invasive isolates of K. Fig 7.

Capsule type diversity in K. Discussion In this study we examined a large collection of K. Methods Bacterial strains and growth conditions The strains representative of each capsule type that were used for the fundamental studies in the work are listed in Table 6.

Clinical isolate strain collection K. Ethics Statement The Israeli isolates used in this study are part of a preexisting anonymized collection and as such did not require IRB approval for use. Molecular methods and strain manipulation Targeted gene disruptions and complementation constructs in K. Polysaccharide capsule extraction, purification, and analysis In preparation for extraction and purification of capsule, the pam locus involved in synthesis of the galactan exopolysaccharide was deleted from the relevant strains [ 11 ].

Xylanase pretreatment.

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NMR spectroscopy. Linkage analysis. Linkage analysis of the Kdo residues. D and L determination. Size exclusion chromatography SEC. Genetic screen of capsule synthesis loci K.

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Statistical analysis The statistical significance of the differences in the distribution of the different capsule types among carrier vs. Supporting Information. S1 Fig. S2 Fig. S3 Fig. S4 Fig. S5 Fig. S6 Fig. S7 Fig. Analysis of non-encapsulated K. S1 Table. Distribution of capsular types among invasive and asymptomatically carried K. S2 Table. Distribution of capsular types by invasive K. S3 Table. Distribution of capsule types among the 16 most common K. S4 Table. Israeli strain collection used in this study.

References 1. Polymerase chain reaction diagnosis of Kingella kingae arthritis in a young child. Clin Infect Dis. Respiratory carriage of Kingella kingae among healthy children. Pediatr Infect Dis J.

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Genetic and molecular basis of Kingella kingae encapsulation. Infect Immun.

Modulation of Kingella kingae adherence to human epithelial cells by type IV pili, capsule, and a novel trimeric autotransporter. Roberts IS. The biochemistry and genetics of capsular polysaccharide production in bacteria. Annu Rev Microbiol.