Mosaicism in the alpha-like protein genes of group B streptococci

Abstract

Members of a family of repeat-containing surface proteins of group B streptococci (GBS) defined by the alpha C and Rib proteins exhibit size variability and cross-reactivity and have been studied as potential vaccine components. We report evidence of horizontal DNA transfer with subsequent recombination as a mechanism generating diversity within this antigen family. Alp2 and Alp3 are additional members of the alpha C protein family identified in strains of the emerging GBS serotypes V and VIII. Each contains an overall genetic organization highly similar to that of the alpha C and Rib proteins, including a tandem repeat region and conserved N- and C-terminal regions. Among different strains, protein size varies according to the number of tandem repeats within the corresponding gene. Unlike the alpha C and Rib proteins, however, the newly described alpha-like proteins contain other regions, including one similar to the IgA-binding region of the GBS beta C protein, a nontandem repeat region, and an isolated repeat highly homologous to the alpha C repeat. Sequence analysis of the regions flanking the alpha C protein gene on a 13.7-kb insert reveals several ORFs that are likely to be involved in basic metabolic pathways. Analysis of corresponding flanking regions in other GBS strains, including the parent strains of the newly described alpha-like proteins, shows striking conservation among all strains studied. These findings indicate that the alpha-like proteins are encoded by mosaic variants at a single genomic locus and suggest that recombination after horizontal DNA transfer is a means of generating diversity within this protein family.

Figure 1

Structure of alpha-like proteins from type V strain CJB-110 (Alp2) and type VIII strain JM9–130013 (Alp3). (A) Structure of Alp2 compared with the alpha C protein (Top) and protein Rib (Bottom). (B) Structure of Alp3 compared with Alp2 (Top) and protein Rib (Bottom). Percentages refer to amino acid identity between corresponding regions. Alpha C protein sequence is from ref. . Rib sequence is from ref. .

Figure 2

Correlation of the number of tandem repeats with size of alpha-like proteins from type V and VIII GBS strains. (A) Western blot of SDS extracts of clinical type V and VIII GBS strains with antiserum raised to purified alpha-like protein from strain JM9–130013 demonstrating protein size variation. (B) Structure of corresponding alpha-like protein genes. Except for the number of repeats in the tandem repeat region, all sequences were identical (>99%) to Alp3. The size of the tandem repeat region correlated with protein size for each strain. Generation of the laddering patterns on the Western blot is presumed to be a result of proteolysis at specific sites within each tandem repeat (10).

Figure 3

Homology of the flanking regions of the alpha C and alpha-like protein genes. The 13.7-kb insert of pJMS23, exclusive of the previously sequenced bca gene, was sequenced. pJMS23 originally was constructed from strain A909 (7). ORFs are designated by solid arrows. Numbers above the solid arrows correspond to the ORF numbers listed in Table 1. bca is the gene encoding the alpha C protein. PCR amplicons using primer pairs (N-1, N-2, C-1, C-2) constructed according to sequence from pJMS23 are designated by dashed lines with arrow ends at the top of the figure. Maps of the regions flanking the alpha-like protein genes in strains BM-110 (protein Rib), CJB-110 (Alp2), JM9–130013 (Alp3), and 18RS21 (protein nonexpresser) were constructed by using PCR and partial sequencing (see text) and are designated by solid lines below the pJMS23 sequence. Inverted hatches in the maps refer to insertions. Open rectangles indicate deletions. Dotted lines within the maps indicate regions that have not been analyzed. IS1381 and IS1191, insertion sequences (see text).

Figure 4

Multiple sequence alignments of the deduced amino acid sequences of the repeat regions of the alpha-like proteins. Alignments of the repeat regions illustrated conserved motifs near the beginnings and ends of the repeats. Black boxes indicate identity, and gray boxes indicate similarity. Al2alpha, alpha-like repeat element from Alp2; Alphac, alpha C protein repeat element; Al3b, B repeat element from Alp3; Rib, repeat element from protein Rib; Al2b, B repeat element from Al2; Espb, B repeat element from Esp; Espc, C repeat element from Esp; Al2au, A repeat element and beginning of U region from Alp2A. Because the sequence of R28 from GAS essentially is identical to that of Alp3 (98% overall, 96% within the region shown above), it was not included as a separate protein in this figure. Alpha C protein sequence is from ref. . Rib sequence is from ref. . Esp sequence is from ref. .