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.
Table 1.
Association between capsule locus screening and capsule composition.
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.
Table 2.
Chemical shift assignments of the type b capsular polysaccharide purified from PYKK58.
Table 3.
Chemical shift assignments of the type c capsular polysaccharide purified from PYKK60.
Table 4.
Chemical shift assignments of the type d capsular polysaccharide purified from BB270.
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 KanR 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.
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.
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.
Table 6.
Strains and plasmids used in this study.
Table 7.
Primers used in this study.