Gene Acquisition by a Distinct Phyletic Group within Streptococcus pneumoniae Promotes Adhesion to the Ocular Epithelium

Abstract

Streptococcus pneumoniae (pneumococcus) displays broad tissue tropism and infects multiple body sites in the human host. However, infections of the conjunctiva are limited to strains within a distinct phyletic group with multilocus sequence types ST448, ST344, ST1186, ST1270, and ST2315. In this study, we sequenced the genomes of six pneumococcal strains isolated from eye infections. The conjunctivitis isolates are grouped in a distinct phyletic group together with a subset of nasopharyngeal isolates. The keratitis (infection of the cornea) and endophthalmitis (infection of the vitreous body) isolates are grouped with the remainder of pneumococcal strains. Phenotypic characterization is consistent with morphological differences associated with the distinct phyletic group. Specifically, isolates from the distinct phyletic group form aggregates in planktonic cultures and chain-like structures in biofilms grown on abiotic surfaces. To begin to investigate the association between genotype and epidemiology, we focused on a predicted surface-exposed adhesin (SspB) encoded exclusively by this distinct phyletic group. Phylogenetic analysis of the gene encoding SspB in the context of a streptococcal species tree suggests that sspB was acquired by lateral gene transfer from Streptococcus suis. Furthermore, an sspB deletion mutant displays decreased adherence to cultured cells from the ocular epithelium compared to the isogenic wild-type and complemented strains. Together these findings suggest that acquisition of genes from outside the species has contributed to pneumococcal tissue tropism by enhancing the ability of a subset of strains to infect the ocular epithelium causing conjunctivitis. IMPORTANCE Changes in the gene content of pathogens can modify their ability to colonize and/or survive in different body sites in the human host. In this study, we investigate a gene acquisition event and its role in the pathogenesis of Streptococccus pneumoniae (pneumococcus). Our findings suggest that the gene encoding the predicted surface protein SspB has been transferred from Streptococcus suis (a distantly related streptococcal species) into a distinct set of pneumococcal strains. This group of strains distinguishes itself from the remainder of pneumococcal strains by extensive differences in genomic composition and by the ability to cause conjunctivitis. We find that the presence of sspB increases adherence of pneumococcus to the ocular epithelium. Thus, our data support the hypothesis that a subset of pneumococcal strains has gained genes from neighboring species that enhance their ability to colonize the epithelium of the eye, thus expanding into a new niche.

Keywords: Streptococcus pneumoniae; gene transfer; genomics; host-pathogen interactions; phylogenetic analysis.

FIG 1

Phylogenetic analysis of pneumococcal strains. Maximum likelihood phylogeny of 40 pneumococcal strains generated from the core genome. Purple, distinct phyletic group; arrow, nasopharyngeal isolate; star, conjunctivitis isolates; yellow, endophthalmitis- and keratitis-associated strains. The scale bar indicates the number of substitutions per site. The numbers on the branch denote bootstrap values. The basal position of the distinct phyletic group presented in the context of other streptococcal species is illustrated in Fig. S3.

FIG 2

Conjunctivitis-associated strains B1599 and B1567 exhibit aggregates in planktonic culture and form abundant chain-like structures in biofilms. (A) Planktonic cultures of strains B1599, B1567, and D39. Conjunctivitis isolates precipitate at the bottom of the test tube, but no precipitate is observed for model strain D39. (B) Confocal images of 72-h biofilms fixed and stained with Syto59. Boxes on the bottom right display a magnified view. Conjunctivitis isolates B1599 and B1567 form chain-like structures (white arrows point to examples of chain-like structures); however, equivalent chains are not observed in the other eye-associated strains. The scale bar is the same for all micrographs.

FIG 3

Functional analyses of the sspB gene encoding the predicted SspB adhesin. (A) Schematic of the predicted SspB protein (GenBank accession no. KGI30072 and OYL08640.1) illustrating domains implicated in adhesion and biofilm formation. (B) Gene expression of sspB in S. pneumoniae B1599 (black bars); for comparison, we show the expression of a second related protein with SspC-C2 domains (WP_050568636) (gray bars). The y axis displays N0, the number of fluorescence units representing the RNA amount in the respective samples. (C) Role of sspB in attachment to HCLE cells. HCLE cells were exposed to S. pneumoniae strain B1599, B1599 ΔsspB, or B1599 ΔsspB::sspB for 30 min, and HCLE cells with bacteria attached were enumerated. Experiments were performed in triplicate, and a total of 460 HCLE cells were analyzed for presence of bacteria and the number of bacteria attached. The results are plotted using a violin plot, generated in the R statistical package. The violin plot displays the distribution of the data: the pink areas display the density plot, the thick black bars represent the midspread of the data (interquartile range), the thin black lines display the 95% confidence interval, and the white circles correspond to the median.

FIG 4

Phylogenetic reconciliation reveals a history of transfers in the origin of sspB. (A) Bayesian phylogeny of sspB gene sequences constructed with MrBayes v.3.2.6 from codon-aware multiple alignment of nucleic acid sequences. The tree is midpoint rooted. Branches are labeled with posterior probabilities representing statistical support. The scale bar represents the number of nucleotide substitutions per site. Colors are by species according to the labeling in panel B. (B) Interspecies sspB gene transfers in the context of the Streptococcus phylogeny. Transfers were inferred by reconciling the gene tree (A) with a species tree (see Fig. S4 in the supplemental material) based on the streptococcal species tree from Richards et al. (50), generated from a core set of 136 genes sampled from 44 streptococcal species. The evolutionary history contains all species in panel A (in color), plus additional species (in black) to provide a representative sample of well-studied taxa with at least one species for each major taxonomic group. The distinct phyletic lineage in pneumococcus is shown in dark blue. Four evolutionary scenarios were inferred with Notung 2.9 (see Fig. S5 in the supplemental material), each with 9 transfers, represented as arrows from the donor to the recipient species. (Solid and dashed lines represent transfers inferred in the upper and lower subtrees in panel A, respectively.) All evolutionary scenarios support a horizontal transfer from S. suis to the base of the distinct lineage (dark blue).