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

Abstract

Kingella kingae 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 csaA that is necessary for synthesis of the [3)-β-GalpNAc-(1→5)-β-Kdop-(2→] polysaccharide capsule (type a) in K. kingae strain 269-492. In the current study, we analyzed a large collection of invasive and carrier isolates from Israel and found that csaA was present in only 47% of the isolates. Further examination of this collection using primers based on the sequence that flanks csaA revealed three additional gene clusters (designated the csb, csc, and csd loci), all encoding predicted glycosyltransferases. The csb locus contains the csbA, csbB, and csbC genes and is associated with a capsule that is a polymer of [6)-α-GlcpNAc-(1→5)-β-(8-OAc)Kdop-(2→] (type b). The csc locus contains the cscA, cscB, and cscC genes and is associated with a capsule that is a polymer of [3)-β-Ribf-(1→2)-β-Ribf-(1→2)-β-Ribf-(1→4)-β-Kdop-(2→] (type c). The csd locus contains the csdA, csdB, and csdC genes and is associated with a capsule that is a polymer of [P-(O→3)[β-Galp-(1→4)]-β-GlcpNAc-(1→3)-α-GlcpNAc-1-] (type d). Introduction of the csa, csb, csc, and csd loci into strain KK01Δcsa, a strain 269-492 derivative that lacks the native csaA 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 K. kingae. 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 K. kingae capsule types and suggest that capsule type plays an important role in promoting K. kingae invasive disease.

Fig 1. PCR screening of capsule synthesis genes reveals four loci.

(A) PCR amplification across the variable synthesis region using flanking primers produces four amplicon sizes. (B) NruI digest of arg/hemB amplicon. (C) Illustration of the four capsule synthesis loci and the engineered empty locus. Each locus shows the capsule synthesis genes unique to each capsule type (white) and the highly homologous flanking regions shared among all strains (gray). Solid arrows (size not to scale) denote the approximate location of the screening primers that anneal to the homologous flanking regions used to amplify across the flanking genes irrespective of internal sequence (as shown in Panel A). The dashed arrows above each locus denote the approximate location of the locus-specific screening primers that generate amplicons for (D) the csa locus (~2400 bp), (E) the csb locus (~2200 bp), (F) the csc locus (~2750 bp), and (G) the csd locus (~4100 bp). Lane 1, ladder; lane 2, KK01; lane 3, PYKK98; lane 4, PYKK93; lane 5, PYKK89; lane 6, PYKK121; lane 7, PYKK58; lane 8, PYKK59; lane 9, PYKK60; lane 10, D7674; lane 11, E3339; lane 12, D7453; lane 13, BB270.

Fig 2. One-dimensional 1H-NMR spectra.

The one-dimensional 1H-NMR spectra of the type b (A), type c (B), de-O-acetylated type c (C), and type d (D) polysaccharides are shown.

Fig 3. Two-dimensional NMR spectra of the polysaccharides isolated from K. kingae clinical isolates.

(A) Overlay of 2-D ¹H-¹³C-HMQC (black) and HMBC (gray) NMR spectra of the type b capsule polysaccharide purified from the surface of PYKK58. The circled area is shown at a lower contour level because the peaks in this region were low in intensity. (B) Overlay of 2-D ¹H-¹³C-HSQC (black) and HMBC (gray) NMR spectra of the type C capsule polysaccharide purified from the surface of PYKK60. Since the Kdo residue does not have an anomeric proton, the HMBC cross peak from H3 to C2 is used to reference the Kdo anomeric carbons in (A) and (B). (C1) ¹H-³¹P-HMQC spectrum of the type d capsule polysaccharide purified from the surface of BB270. This spectrum shows that the polysacccharide consisting of Residues A, B, and C contains a phosphodiester linking together O-1 of A and O-3 of B and that the sequence consisting of Residues A’, B’, and C’ contains a phosphomonoester attached to O-3 of B’. (C2) Overlay of 2-D ¹H-¹³C-HSQC (black) and HMBC (gray) NMR spectra of the type d capsule polysaccharide purified from the surface of BB270. Dotted lines and gray labels indicate the inter-residue HMBC correlations showing the connections between residues and thus specifying the sequence of the polysaccharide.

Fig 4. Capsule polysaccharide repeating unit structures.

The capsule polysaccharide repeating unit structures for capsule type a (GalNAc-Kdo, panel A), capsule type b (GlcNAc-Kdo, panel B), capsule type c (ribose-Kdo, panel C), and capsule type d (galactose-GlcNAc, panel D) are shown.

Fig 5. Comparison of capsule migration pattern between capsule types.

Alcian blue stained gel depicting the migration pattern of capsule material purified from the surface of the source strains (lanes 2–5), capsule locus deletion mutants (lane 6–9), and the capsule complements (lanes 10–13). Lane 1, ladder; lane 2, KK01; lane 3, PYKK58; lane 4, PYKK60; lane 5, BB270; lane 6, KK01Δcsa; lane 7, PYKK58Δcsb; lane 8, PYKK60Δcsc; lane 9 BB270Δcsd; lane 10, KK01Δcsa(csa); lane 11 PYKK58Δcsb(csb); lane 12, PYKK60Δcsc(csc); lane 13 BB270Δcsd(csd).

Fig 6

(A) Illustration of the capsule swap vector in pUC19 harboring the csa, csb, csc, or csd locus with a Kan ^R marker for selection, and (B) the migration patterns of capsule material from isogenic capsule swaps. Alcian blue stained gel depicting the migration pattern of capsule material purified from the surface of the source strains (lanes 2–5) and the capsule swaps expressed in the isogenic KK01 background (lanes 7–10). Lane 1, ladder; lane 2, KK01; lane 3, PYKK58; lane 4, PYKK60; lane 5, PYKKBB270; lane 6, KK01Δcsa; lane 7, KK01Swapcsa; lane 8, KK01Swapcsb; lane 9, KK01Swapcsc; lane 10, KK01Swapcsd.

Fig 7. Capsule type diversity in K. kingae clinical isolates.

Type a is shown in dark gray, type b in light gray, type c in white, and type d in black. The number above each bar represents the number of isolates in each group. (A) The capsule type representation among carrier isolates (type a, 49.0%; type b, 19.2%; type c, 12.1%; type d, 19.7%) and invasive isolates (type a, 44.9%; type b, 51.5%; type c, 2.2%; type d, 1.7%) is shown. (B) The capsule type representation among common K. kingae clinical presentations is shown: bacteremia (type a, 37.7%; type b, 63.3%; type c, 0%; type d, 0%), endocarditis (type a, 54.5%; type b, 27.3%; type c, 18.2%; type d, 0%), and skeletal infections (type a, 50.0%; type b, 44.8%; type c, 2.1%; type d, 1.1%). (C) The capsule types among PFGE clonal groups containing ≥7 isolates are shown. Capsule type was determined by PCR screening for each of the four capsule synthesis loci.