The serotype of type Ia and III group B streptococci is determined by the polymerase gene within the polycistronic capsule operon

Abstract

Streptococcus agalactiae is a primary cause of neonatal morbidity and mortality. Essential to the virulence of this pathogen is the production of a type-specific capsular polysaccharide (CPS) that enables the bacteria to evade host immune defenses. The identification, cloning, sequencing, and functional characterization of seven genes involved in type III capsule production have been previously reported. Here, we describe the cloning and sequencing of nine additional adjacent genes, cps(III)FGHIJKL, neu(III)B, and neu(III)C. Sequence comparisons suggested that these genes are involved in sialic acid synthesis, pentasaccharide repeating unit formation, and oligosaccharide transport and polymerization. The type III CPS (cpsIII) locus was comprised of 16 genes within 15.5 kb of contiguous chromosomal DNA. Primer extension analysis and investigation of mRNA from mutants with polar insertions in their cpsIII loci supported the hypothesis that the operon is transcribed as a single polycistronic message. The translated cpsIII sequences were compared to those of the S. agalactiae cpsIa locus, and the primary difference between the operons was found to reside in cps(III)H, the putative CPS polymerase gene. Expression of cps(III)H in a type Ia strain resulted in suppression of CPS Ia synthesis and in production of a CPS which reacted with type III-specific polyclonal antibody. Likewise, expression of the putative type Ia polymerase gene in a type III strain reduced synthesis of type III CPS with production of a type Ia immunoreactive capsule. Based on the similar structures of the oligosaccharide repeating units of the type Ia and III capsules, our observations demonstrated that cps(Ia)H and cps(III)H encoded the type Ia and III CPS polymerases, respectively. Additionally, these findings suggested that a single gene can confer serotype specificity in organisms that produce complex polysaccharides.

FIG. 1

GBS type Ia and III CPS repeating unit structures. Ia, S. agalactiae type Ia CPS subunit structure; III, S. agalactiae type III CPS subunit structure; Gal, galactose; Glc, glucose; GlcNAc, N-acetylglucosamine; NeuNAc, N-acetylneuraminic (sialic) acid. The critical linkages differentiating the type Ia and III CPS are shown in the shaded boxes.

FIG. 2

Organizational map of the type III GBS cps operon. ORFs within the operon, with the direction of transcription, are indicated by the open arrows. The corresponding gene designation is shown above each ORF. Restriction site designations: E, EcoRI; H, HindIII; S, SacI; X, XbaI. Plasmids used in sequencing the locus are depicted above the operon map. PCR products used to derive additional sequence are shown as solid bars above the operon map. The cpsIII transcriptional start site is indicated with an arrow in the cpsY-cpsA intergenic region. The ΩKm-2 insertion sites are indicated with triangles: filled triangle, cps_IIIA insertion; open triangle, cps_IIIB insertion; striped triangle, cps_IIIC insertion; dotted triangle, cps_IIID insertion. Sequences used in generating RNA probes are marked with open boxes. The location and orientation of the oligonucleotide used to initiate the primer extension reaction (primer 5a) is also shown.

FIG. 3

Schematic representation of the organization of the CPS synthesis loci of S. agalactiae type III (GBS type III), S. agalactiae type Ia (GBS type Ia) (60), and S. pneumoniae type 14 (Spn 14) (23). The unfilled arrows indicate ORFs within the operons and the direction of transcription. Filled arrows indicate ORFs not involved in CPS synthesis that flank the cps gene regions. Letters below the arrows indicate the gene designations. The organization of the cps loci into regions of associated gene function is indicated above the ORF diagrams.

FIG. 4

Capsule subunit assembly locus. Regions of divergent sequence between type Ia and III strains are shown either striped (type III) or checked (type Ia). The degree of homology between the conserved and nonconserved regions is shown at the bottom. Gene designations are shown as single letters above each ORF map, and the CPS polymerase (H) is also indicated. The plasmids used in the polymerase expression constructs are shown beneath each map. Sequences deleted in the construction of pDC123(H_III) are shown with dashes.

FIG. 5

(A) Primer extension reaction. Primer extension reactions were carried out on total cellular RNA derived from GBS strain COH1, using an antisense primer that hybridized to cps_IIIA mRNA. A sequencing ladder was generated from cps_IIIA double-stranded DNA using the same primer and was separated on a sequencing gel in lanes adjacent (lanes CTAG) to the primer extension products (p.ext.). The position of the apparent first ribonucleotide of the primer extension product (+1) is indicated to the right. The DNA sequence surrounding the start position of the mRNA is depicted vertically on the left, with the consensus −10 promoter sequence and the mRNA start site highlighted in bold. (B) RNA dot blots of total cellular RNA derived from the wt GBS strain COH1 and the ΩKm-2 cassette allelic exchange mutant strains COHY-107 (cps_IIIA), COHY-106 (cps_IIID), COHY-105 (cps_IIIC), and COHY-104 (cps_IIIB). The blots were hybridized to intragenic probes derived from the wt COH1 cps_IIIB, cps_IIIE, and neu_IIIA genes and exposed to X-ray film.

FIG. 6

Immunoblots directed against immobilized whole GBS utilizing rabbit polyclonal anti-CPS antisera, detected with HRP-conjugated, goat anti-rabbit secondary antibody and chemiluminescent HRP substrate. Strains used: COH1, GBS type III strain; A909, GBS type Ia strain; COH1(GH_Ia), strain COH1 containing pDC123(GH_Ia); COH1(H_Ia), strain COH1 containing pDC123(H_Ia); A909(GH_III), strain A909 containing pDC123(GH_III); A909(H_III), strain A909 containing pDC123(H_III). (A) Blot exposed to type Ia antisera; (B) blot exposed to type III antisera.

FIG. 7

Competitive ELISA inhibition curves produced from mutanolysin extracts of wt and heterologous CPS polymerase-expressing strains. CPS extracts were used to compete with immobilized purified type Ia CPS for anti-type Ia polyclonal antibody (A) or with immobilized purified type III CPS for anti-type III polyclonal antibody (B). Strains A909 and COH1 produce native type Ia and type III CPS, respectively. Strain A909(H_III) expresses the cps_IIIH allele from COH1, and strain COH1(H_Ia) expresses the cps_IaH allele from strain A909.