Detection of large numbers of pneumococcal virulence genes in streptococci of the mitis group

Abstract

Seven streptococcal isolates from the mitis group were analyzed for the presence of pneumococcal gene homologues by comparative genomic hybridization studies with microarrays based on open reading frames from the genomes of Streptococcus pneumoniae TIGR4 and R6. The diversity of pneumolysin (ply) and neuraminidase A (nanA) gene sequences was explored in more detail in a collection of 14 S. pseudopneumoniae and 29 mitis group isolates, respectively. The mitis group isolates used in the microarray experiments included a type strain (NCTC 12261), two S. mitis isolates from the nasopharynxes of children, one S. mitis isolate from a case of infective endocarditis, one S. mitis isolate from a dental abscess, and one S. oralis isolate and one S. pseudopneumoniae isolate from the nasopharynxes of children. The results of the microarray study showed that the 5 S. mitis isolates had homologues to between 67 and 82% of pneumococcal virulence genes, S. oralis hybridized to 83% of pneumococcal virulence genes, and S. pseudopneumoniae hybridized to 92% of identified pneumococcal virulence genes. Comparison of the pneumolysin, mitilysin (mly), and newly identified pseudopneumolysin (pply) gene sequences revealed that mly and pply genes are more closely related to each other than either is to ply. In contrast, the nanA gene sequences in the pneumococcus and streptococci from the mitis group are closely clustered together, sharing 99.4 to 99.7% sequence identity with pneumococcal nanA alleles.

FIG. 1.

Comparison of percentages of genes hybridized to pneumococcal microarrays for different VGS species (reference genome, S. pneumoniae TIGR4). S. pneumoniae hybridization results are representative of hybridization values from previous studies of S. pneumoniae strains, using the same arrays, in our laboratory.

FIG. 2.

Presence of pneumococcal virulence gene homologues in related VGS species by comparative genomic hybridization. The presence of a homologue by hybridization is represented by a black box, and its absence is shown by a white box. The final five genes shown are essential virulence genes of unknown function present on identified regions of pneumococcal diversity (33).

FIG. 3.

Minimum evolution tree of ply DNA alleles and homologues in related streptococcal species. ply and mly alleles were previously described (20, 21). The tree was drawn using MEGA4 software and demonstrates the relatedness of these toxins at the DNA level.

FIG. 4.

Four VGS NanA alleles identified in isolates of S. mitis and S. pseudopneumoniae. Alleles 1 to 3 were aligned with a pneumococcal NanA sequence from a clinical isolate of serotype 19A, ST1201 (GenBank accession no. FJ440122.1), and allele 4 was aligned with the pneumococcal NanA allele of sequenced strain G54. Row 2 represents the amino acid position in the pneumococcal NanA protein, and row 3 represents the amino acid present in the pneumococcal NanA allele. Next to each strain is the mutant amino acid present at each position along the protein. The amino acid sequence identity between the NanA alleles is noted as a percentage.

FIG. 5.

Minimum evolution tree of nanA DNA alleles and homologues in related streptococcal species. Pneumococcal nanA alleles include those from sequenced pneumococcal strains. The tree was drawn using MEGA4 software and demonstrates the relatedness of these enzymes at the DNA level.