Sialylation of group B streptococcal capsular polysaccharide is mediated by cpsK and is required for optimal capsule polymerization and expression - PubMed (original) (raw)

Comparative Study

Sialylation of group B streptococcal capsular polysaccharide is mediated by cpsK and is required for optimal capsule polymerization and expression

D O Chaffin et al. J Bacteriol. 2005 Jul.

Abstract

Several bacterial pathogens have evolved the means to escape immune detection by mimicking host cell surface carbohydrates that are crucial for self/non-self recognition. Sialic acid, a terminal residue on these carbohydrates, inhibits activation of the alternate pathway of complement by recruiting the immune modulating molecule factors H, I, and iC3b. Sialylation of capsular polysaccharide (CPS) is important for virulence of group B streptococci (GBS), a significant human pathogen. We previously reported that cpsK, a gene within the cps locus of type III GBS, could complement a sialyltransferase deficient lst mutant of Haemophilus ducreyi, implicating its role in sialylation of the GBS capsule. To explore the function of cpsK in GBS capsule production, we created a mutant in cpsK. Immunoblot analysis and enzyme-linked immunosorbent assay using anti-type III CPS antisera demonstrated that the mutant CPS did not contain sialic acid. This was confirmed by high-performance liquid chromatography after mild acid hydrolysis of the CPS. Although increased CPS chain length was seen for this strain, CPS production was <20% of the parental isolate. An episomal cpsK copy restored synthesis of sialo-CPS to wild-type levels. These data support our hypothesis that cpsK encodes the GBS CPS sialyltransferase and provide further evidence that lack of CPS oligosaccharide sialylation reduces the amount of CPS expressed on the cell surface. These observations also imply that one or more of the components involved in synthesis or transport of oligosaccharide repeating units requires a sialo-oligosaccharide for complete activity.

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Figures

FIG. 1.

Organization of the Streptococcus agalactiae serotype III CPS synthesis operon. Proposed functions of the cps gene products are indicated at the top of the diagram. Locations of the Tn_916_ΔE transposon and _km_-Ω-2 cassette insertions in strains COH1-11 (black triangle), COH1-13 (open triangle), and COH1-355 (shaded triangle) are indicated. The site of the chloramphenicol acetyltransferase allelic exchange replacement of cpsK in COH1-350 and the cps locus expressed in pLM104 are also indicated. The cps gene designations are indicated below the arrows representing each open reading frame. The locations of the cpsK and cpsL probes for Northern dot blot analysis are shown as grey or open bars, respectively, above the schematic of the cps operon.

FIG. 2.

RNA dot blots of total cellular RNA from the COH1 or COH1-350 strains. Total RNA was hybridized to antisense RNA probes that were generated from in vitro transcription of cpsK, cpsL, or rpsL (a ribosomal subunit housekeeping gene). Either 4 or 2 μg of cellular RNA was transferred to a nitrocellulose membrane and hybridized with the probes. Strain designations: COH1, wt; COH1-350, Δ_cpsK_.

FIG. 3.

GBS serotype III and Spn_14 CPS RPU structures, adapted from reference . Gal_p, galactose; Glc_p_, glucose; Glc_p_NAc, _N_-acetylglucosamine; Neu_p_NAc, _N_-acetylneuraminic (sialic) acid. The brackets to the right delineate the portions of the structure, corresponding to the CPS RPUs produced by S. pneumoniae serotype 14 and serotype III GBS, respectively. Structural motifs recognized by anti-_Spn_14 monoclonal antibody HASP-15 (†), anti-type III GBS sialo-CPS monoclonal antibody S9 (§), and anti-type III GBS polyclonal antibody (*) are as indicated.

FIG. 4.

Whole cell immuno-dot blots using either anti-serotype III polyclonal (A), anti-serotype III sialo-CPS monoclonal (B), or anti-Spn_14 CPS monoclonal (C) antibodies. Dilutions of cells were made in PBS and spotted on nitrocellulose membranes (1:1 = 106 CFU), and the cells were fixed and then probed with the various anti-CPS antibodies. Strains shown are COH1 (wt), COH1-350 (Δ_cpsK), COH1-350(pLM104) (Δ_cpsK/cpsK_), COH1-11 (Δ_neuA_), and S. pneumoniae NCTC11902 (_Spn_14).

FIG. 5.

Thin-layer chromatography of purified CPS acid hydrolysates. (Left) Mild acid hydrolysis of CPS from strain COH1 (wt), COH1-350 (Δ_cpsK_), and COH1-350(pLM104) (Δ_cpsK_/cpsK). The migration of pure sialic acid (Neu5Ac) is seen in the first lane (5 μg Neu5Ac). The Neu5Ac lactone is an artifact of the hydrolysis. (Right) Strong acid hydrolysis of CPS from strain COH1 (wt), COH1-350 (Δ_cpsK_), and COH1-350(pLM104) (Δ_cpsK_/cpsK), serotype 14 S. pneumoniae NCTC11902 (_Spn_14), or _N-_acetylglucosamine hydrolyzed under identical conditions. The migration distances of authentic glucosamine (GlcNH), galactose (Gal), glucose (Glc), and _N-_acetylglucosamine (GlcNAc) are indicated.

FIG. 6.

Competitive inhibition ELISA curves of mutanolysin cell wall extracts. (A) Competition between extracts (or purified GBS CPS) and immobilized GBS serotype III CPS for anti-serotype III polyclonal antibody. (B) Competition between extracts (or purified Spn_14 CPS) and immobilized S. pneumoniae serotype 14 CPS for monoclonal anti-serotype 14 pneumococcal antibody. Grey diamonds, purified GBS serotype III CPS; open squares, strain COH1 CPS extract (wt); grey triangles, strain COH1-350 CPS extract (Δ_cpsK); open circles, strain COH1-11 CPS extract (Δ_neuA_); black triangles, strain COH1-350(pLM104) CPS extract (Δ_cpsK_/cpsK); open diamonds, purified _Spn_14 CPS.

FIG. 7.

Immuno-dot blots of culture supernatants and cell pellets. (A) Clarified culture supernatants, either unconcentrated (neet) [strains COH1 and COH1-350(pLM104)] or 100-fold concentrated (strains COH1-350, COH1-11, and COH1-13) versus (B) whole cells washed with PBS. (C) Purified type III GBS and _Spn_14 CPS. Samples were serially diluted twofold, spotted onto a nitrocellulose membrane, and tested for the presence of CPS with anti-serotype III polyclonal antiserum.

FIG. 8.

SDS-PAGE of cell wall and protoplast fractions from mutanolysin extracts of whole cells. After centrifugation, the cell wall fraction (cw) or protoplasts (p) were separated on 5 to 20% gradient gels and blotted, and CPS was detected with anti-type III CPS Ab. The migration distance of the CPS is shown relative to that of protein standards run on the same gel. The arrow indicates the location of the CPS associated with the cell wall fraction in strain COH1-350.

FIG. 9.

Sephacryl S-400 size exclusion chromatograph of strain COH1 (wt) and COH1-350 (Δ_cpsK_) CPS. COH1 and COH1-350 CPS (300 μg and 172 μg, respectively) were fractionated over a Sephacryl S-400 column (1.6 by 65 cm). Collected fractions were assayed for CPS by quantitative immunofluorescent dot blot analysis. The molecular mass of the CPS was determined relative to dextran standards, and the number of RPUs was calculated based on the predicted molecular weight of the sialo (COH1) or asialo (COH1-350) RPUs.

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Sialylation of group B streptococcal capsular polysaccharide is mediated by cpsK and is required for optimal capsule polymerization and expression - PubMed (original) (raw)