Structure of capsule polysaccharide repeating unit (top) and galactan exopolysaccharide repeating unit (bottom).

<p>Structure of capsule polysaccharide repeating unit (top) and galactan exopolysaccharide repeating unit (bottom).</p

Strains used in this study ?.

<p>Strains used in this study ?.</p

Glycosyl composition of extracellular preparations of <i>K. kingae</i> strains used in this study.

<p>Mass in µg is listed first, and percentage is shown in parentheses. Mass and percentage are per 10 µg polysaccharide material analyzed.</p>*<p>3-Deoxy-D-Manno-oct-2-ulosonic acid.</p

Complete chemical shift assignment of the isolated polysaccharides.

<p>N-acetyl signal: 2.04/25.0 ppm.</p><p>Carbon chemical shifts are in italics. Carbon chemical shifts with characteristic downfield displacement due to glycosylation are in bold.</p

Colony morphology of <i>K. kingae</i> strains used in this study.

<p>Strain KK01 (A) and KK01<i>pamABCDE</i> (B) form mucoid colonies consistent with encapsulation. KK01<i>ctrA</i> (C) and KK01<i>ctrA pamABCDE</i> (D) form rough, non-mucoid colonies consistent with loss of encapsulation.</p

2-D HSQC NMR spectrum of the polysaccharide with assignments of all the signals of the three major residues.

<p>For numbering, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075409#pone-0075409-t003" target="_blank">Table 3</a>.</p

2-D NOESY NMR spectrum of the polysaccharide.

<p>The sequence-determining correlations are labeled. For numbering, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075409#pone-0075409-t003" target="_blank">Table 3</a>.</p

Staining profile and purity of polysaccharide material used for analysis.

<p>Alcian blue staining is shown on the top, and silver staining is on the bottom. Lane 1, extract from KK01 using heat; lane 2, extract from KK01 using Tris acetate; lane 3, purified material from KK01 using Tris acetate; lane 4, purified material from KK01<i>pamABCDE</i> using Tris acetate; lane 5, purified material from KK01<i>ctrA</i> using Tris acetate; lane 6, purified material from KK01<i>ctrA</i> using surface PBS extraction; lane 7, purified material from KK01<i>ctrA pamABCDE</i> using Tris acetate. MS = molecular size (in kDa).</p

1-D proton NMR spectrum of the polysaccharide.

<p>The integration values show the relative molar ratio of the two polysaccharides, A_m and (B–C)_n of about 1∶3. For numbering, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075409#pone-0075409-t003" target="_blank">Table 3</a> (C3e and C3a designate the equatorial and axial proton, respectively, of the C-3 methylene group).</p

<i>Kingella kingae</i> Expresses Four Structurally Distinct Polysaccharide Capsules That Differ in Their Correlation with Invasive Disease

<div><p><i>Kingella kingae</i> is an encapsulated gram-negative organism that is a common cause of osteoarticular infections in young children. In earlier work, we identified a glycosyltransferase gene called <i>csaA</i> that is necessary for synthesis of the [3)-β-Gal<i>p</i>NAc-(1→5)-β-Kdo<i>p</i>-(2→] polysaccharide capsule (type a) in <i>K</i>. <i>kingae</i> strain 269–492. In the current study, we analyzed a large collection of invasive and carrier isolates from Israel and found that <i>csaA</i> was present in only 47% of the isolates. Further examination of this collection using primers based on the sequence that flanks <i>csaA</i> revealed three additional gene clusters (designated the <i>csb</i>, <i>csc</i>, and <i>csd</i> loci), all encoding predicted glycosyltransferases. The <i>csb</i> locus contains the <i>csbA</i>, <i>csbB</i>, and <i>csbC</i> genes and is associated with a capsule that is a polymer of [6)-α-Glc<i>p</i>NAc-(1→5)-β-(8-OAc)Kdo<i>p</i>-(2→] (type b). The <i>csc</i> locus contains the <i>cscA</i>, <i>cscB</i>, and <i>cscC</i> genes and is associated with a capsule that is a polymer of [3)-β-Rib<i>f</i>-(1→2)-β-Rib<i>f</i>-(1→2)-β-Rib<i>f</i>-(1→4)-β-Kdo<i>p</i>-(2→] (type c). The <i>csd</i> locus contains the <i>csdA</i>, <i>csdB</i>, and <i>csdC</i> genes and is associated with a capsule that is a polymer of [P-(O→3)[β-Gal<i>p</i>-(1→4)]-β-Glc<i>p</i>NAc-(1→3)-α-Glc<i>p</i>NAc-1-] (type d). Introduction of the <i>csa</i>, <i>csb</i>, <i>csc</i>, and <i>csd</i> loci into strain KK01Δ<i>csa</i>, a strain 269–492 derivative that lacks the native <i>csaA</i> gene, was sufficient to produce the type a capsule, type b capsule, type c capsule, and type d capsule, respectively, indicating that these loci are solely responsible for determining capsule type in <i>K</i>. <i>kingae</i>. Further analysis demonstrated that 96% of the invasive isolates express either the type a or type b capsule and that a disproportionate percentage of carrier isolates express the type c or type d capsule. These results establish that there are at least four structurally distinct <i>K</i>. <i>kingae</i> capsule types and suggest that capsule type plays an important role in promoting <i>K</i>. <i>kingae</i> invasive disease.</p></div