Identification of Virulence-Associated Properties by Comparative Genome Analysis of Streptococcus pneumoniae, S. pseudopneumoniae, S. mitis, Three S. oralis Subspecies, and S. infantis

Abstract

From a common ancestor, Streptococcus pneumoniae and Streptococcus mitis evolved in parallel into one of the most important pathogens and a mutualistic colonizer of humans, respectively. This evolutionary scenario provides a unique basis for studies of both infection-associated properties and properties important for harmonious coexistence with the host. We performed detailed comparisons of 60 genomes of S. pneumoniae, S. mitis, Streptococcus pseudopneumoniae, the three Streptococcus oralis subspecies oralis, tigurinus, and dentisani, and Streptococcus infantis Nonfunctional remnants of ancestral genes in both S. pneumoniae and in S. mitis support the evolutionary model and the concept that evolutionary changes on both sides were required to reach their present relationship to the host. Confirmed by screening of >7,500 genomes, we identified 224 genes associated with virulence. The striking difference to commensal streptococci was the diversity of regulatory mechanisms, including regulation of capsule production, a significantly larger arsenal of enzymes involved in carbohydrate hydrolysis, and proteins known to interfere with innate immune factors. The exclusive presence of the virulence factors in S. pneumoniae enhances their potential as vaccine components, as a direct impact on beneficial members of the commensal microbiota can be excluded. In addition to loss of these virulence-associated genes, adaptation of S. mitis to a mutualistic relationship with the host apparently required preservation or acquisition of 25 genes lost or absent from S. pneumoniae Successful adaptation of S. mitis and other commensal streptococci to a harmonious relationship with the host relied on genetic stability and properties facilitating life in biofilms.IMPORTANCEStreptococcus pneumoniae is one of the most important human pathogens but is closely related to Streptococcus mitis, with which humans live in harmony. The fact that the two species evolved from a common ancestor provides a unique basis for studies of both infection-associated properties and properties important for harmonious coexistence with the host. By detailed comparisons of genomes of the two species and other related streptococci, we identified 224 genes associated with virulence and 25 genes unique to the mutualistic species. The exclusive presence of the virulence factors in S. pneumoniae enhances their potential as vaccine components, as a direct impact on beneficial members of the commensal microbiota can be excluded. Successful adaptation of S. mitis and other commensal streptococci to a harmonious relationship with the host relied on genetic stability and properties facilitating life in biofilms.

Keywords: Streptococcus mitis; Streptococcus oralis; Streptococcus pneumoniae; bacteremia; commensal; evolution; infective endocarditis; mutualism; virulence.

FIG 1

(A) Minimum evolution tree based on concatenated sequences of MLST loci showing the representability of the 13 S. pneumoniae indicated by colored dots. (B) Evolutionary relationships of genomes inferred from SplitsTree analysis. Phylogenetic trees of 60 genomes based on core sequence of 844,932 bp shared by all strains in the collection. The tree shows three major clusters supported by bootstrap values of 100, one consisting of S. mitis, S. pneumoniae, and S. pseudopneumoniae, a second composed of S. oralis (subsp. oralis, subsp. dentisani, subsp. tigurinus, and a genomosubspecies), and a third consisting of strains of S. infantis. The 20 S. mitis strains are segregated into two distinct clusters, one of them consisting of several minor subclusters.

FIG 2

Synteny gradient display visualizing changes in synteny of seven representative genomes relative to the reference, S. mitis NCTC12261. The reference genome’s genes are colored from yellow to blue on a gradient from left to right. The figure reveals conservation of the color gradient in syntenic regions, while shared genes located in rearrangements show up as color mismatches between the reference and query genomes. Gaps in query genomes (white spaces) represent genes that are present in the reference but not the query. Query genes with paralogs are displayed in black. The figure reveals conservation of gene synteny between the three gap-free S. mitis genomes. Synteny is still mostly conserved with S. oralis Uo5, although blocks of genes breaking the color gradient appear. Given that the leftmost blocks are dark blue and the rightmost ones are light green, it is likely that these nonsyntenic blocks are the results of inversions that are symmetrical with the chromosome’s start (or terminus) of replication. Two successive nested inversions will leave the tips of the first inversion in place (breaks of color gradient), while the second inversion will restore the inner part of the gradient. Many such nested symmetric inversions then leave many nonsyntenic blocks of different colors. Similar inversions are observed between the S. mitis reference and S. pneumoniae and S. pseudopneumoniae genomes, but syntenic blocks remain larger, indicative of fewer inversion events and the close evolutionary relationship.

FIG 3

Comparisons of the IgA1 protease/zmpA loci in representative strains of S. mitis demonstrate that the locus is a hot spot for recombination. (A) Cluster II strains of S. mitis (represented by SK667 and SK569) show the same structure as pneumococci (represented by TIGR4). In some strains, the locus includes the paralogous zmpD gene. Strains of S. mitis cluster I lack the zmpA gene at this site, but some strains instead have a gene encoding an unrelated LPxTG-anchored protein (SK637 and B6) or a complete capsular polysaccharide biosynthesis locus (SK321). (B) Some strains of S. mitis cluster I carry the zmpA gene in the same spot as S. oralis but without the paralogous zmpC gene.