Streptococcus infantis, Streptococcus mitis, and Streptococcus oralis Strains With Highly Similar cps5 Loci and Antigenic Relatedness to Serotype 5 Pneumococci

Abstract

Streptococcus pneumoniae is a highly impactful bacterial pathogen on a global scale. The principal pneumococcal virulence factor and target of effective vaccines is its polysaccharide capsule, of which there are many structurally distinct forms. Here, we describe four distinct strains of three Mitis group commensal species (Streptococcus infantis, Streptococcus mitis, and Streptococcus oralis) recovered from upper respiratory tract specimens from adults in Kenya and the United States that were PCR-positive for the pneumococcal serotype 5 specific gene, wzy5. For each of the four strains, the 15 genes comprising the capsular polysaccharide biosynthetic gene cluster (cps5) shared the same order found in serotype 5 pneumococci, and each of the serotype 5-specific genes from the serotype 5 pneumococcal reference strain shared 76-99% sequence identity with the non-pneumococcal counterparts. Double-diffusion experiments demonstrated specific reactivity of the non-pneumococcal strains with pneumococcal serotype 5 typing sera. Antiserum raised against S. mitis strain KE67013 specifically reacted with serotype 5 pneumococci for a positive Quellung reaction and stimulated serotype 5 specific opsonophagocytic killing of pneumococci. Four additional commensal strains, identified using PCR serotyping assays on pharyngeal specimens, revealed loci highly homologous to those of pneumococci of serotypes 12F, 15A, 18C, and 33F. These data, in particular the species and strain diversity shown for serotype 5, highlight the existence of a broad non-pneumococcal species reservoir in the upper respiratory tract for the expression of capsular polysaccharides that are structurally related or identical to those corresponding to epidemiologically significant serotypes. Very little is known about the genetic and antigenic capsular diversity among the vast array of commensal streptococcal strains that represent multiple diverse species. The discovery of serotype 5 strains within three different commensal species suggests that extensive capsular serologic overlap exists between pneumococci and other members of the diverse Mitis group. These findings may have implications for our current understanding of naturally acquired immunity to S. pneumoniae and pneumococcal serotype distributions in different global regions. Further characterization of commensal strains carrying homologs of serotype-specific genes previously thought to be specific for pneumococci of known serotypes may shed light on the evolution of these important loci.

Keywords: capsular biosynthetic; capsular serotype; immunodiffusion analysis; opsonophagocytic killing (OPK); serotype-specific gene.

FIGURE 1

(A) Maximum parsimony tree based upon kSNP3.0 analysis (Gardner et al., 2015) of eight study genomes (indicated in red including wzy or wzx genes initially detected in PCR-serotyping assay) from this study, combined with 66 genomes from GenBank within the species S. mitis, S. oralis, and S. infantis described previously (Jensen et al., 2016). The k-mer size employed was 19. The scale is based upon 322 core SNPs. The indicated node support values were calculated by FastTreeMP as described (Gardner et al., 2015). The genome from strain F0392 obtained from NCBI (indicated in green) is also included. (B) Alignments of cps5 operons from five strains of non-pneumococcal species S. mitis (SM) 67013, S. oralis (SO, strains US0049 and F0392), and S. infantis (SI; strains US969j1 and US0024). The percent sequence identity of the entire biosynthetic cluster (wzg through fnlC) with the pneumococcal serotype 5 reference sequence (GenBank accession CR931637) is underlined at left. The percent identity of the allele over its entire overlap with the pneumococcal reference is indicated within each rectangle representing the indicated gene. For the S. oralis and S. infantis alignments that each depict a pair of distinct strains, each gene has the indicated conserved translational start and/or end. S. mitis 67013 is the only strain showing close linkage of the cps5 locus with upstream pbp2x and downstream pbp1a, while the two S. oralis cps5 loci lie 2–17 kb upstream of pbp1a. The position of conventional (c) and real time (rt) serotype 5 detection assays are indicated (c assay positive for all four strains tested, rt assay positive for all but S. mitis US67013). The percent identities are indicated between the two pairs of strains (US0049/F0392 and US969j1/US0024) below the indicated genes. The five cps5 genes that appear entirely serotype 5-specific (<54% identical to all other known pneumococcal cps genes) are indicated in orange. Phylogenetic analysis shows the relative relatedness of the five serotype 5 specific genes between the four species. Gene functions listed at bottom left are taken from accession CR931637. Black rectangles indicate transposase gene remnants. The orientations of pbp2x and pbp1a relative to cps5 are indicated at the right and left end, respectively, of each cps5 operon. The coordinates above the genes indicate base pairs (bp), also indicated as distances between the cps loci and flanking pbp genes.

FIGURE 2

(A) Reactivity of typing antisera raised against serotype 5 pneumococcal strain Ambrose (middle well) against wzy5-positive strains of S. infantis, S. oralis, S. mitis, and serotype 5 S. pneumoniae Ambrose. A serotype 4 pneumococcal strain is included as a negative control. In other experiments pneumococcal serotype 1 and S. mitis cps1-positive strain L006 (Lessa et al., 2018) were included as negative controls and showed no reactivity. (B) Top: Reactivity of pneumococcal serotype 5 typing antisera against S. mitis KE67013 (top grid, center well contains type 5 antisera; wells 1 and 2 contain type 5 pneumococcal extract. Wells 3 and 4 contain strain KE67013 extract. Middle and lower grids show reactivity of indicated dilutions of pneumococcal typing antisera (peripheral wells) against S. mitis KE67013 extract (middle grid, center well) and serotype 5 S. pneumoniae strain Ambrose extract (lower grid, center well). (C) Positive Quellung reactions of serotype 5 pneumococcal strain Ambrose when reacted with antisera raised against wzy5-positive S. mitis strain KE67013 (top panel) and with standard serotype 5 typing antiserum (middle panel). The same pneumococcal type 5 strain showed no reactivity when exposed to antisera prepared against an S. mitis strain L006 containing a cps1-like operon (Lessa et al., 2018) unrelated to cps5 (bottom panel). (D) Opsonophagocytic killing (OPK) activity of rabbit antisera raised against serotype 5 S. mitis KE67013 in three separate rabbits (1–3) and of pooled, clarified antisera from the three rabbits (pooled). Geometric Mean Titer (GMT) values of titers across 5–6 assay runs are shown with >50% killing compared with the growth in the complement control wells. Initial dilution was 1:400 with subsequent two-fold dilutions down to 1:51200. Test of the rabbit antiserum generated against serotype 5 S. pneumoniae gave optimal titer at 1:960 (not shown). KE67013 was included as the positive control in the OPK assay; KE67013 antiserum had very high (titer > 3000) opsonic activity against strain KE67013 (not shown). Pre-inoculation bleeding samples for all rabbits induced no killing (data not shown). Antisera prepared against control strain of S. mitis L006 carrying a full-length cps1-like operon (strain and antiserum described in Lessa et al., 2018) unrelated to cps5 showed no OPK activity against serotype 5 pneumococci. Coefficient of variation (CV) = standard deviation/GMT;SE (σ) = GMT ^∗ (exp (standard deviation) – 1)/sqrt (N) $\sigma =\sqrt{\frac{1}{N}{\displaystyle \sum _{i=1}^N}{\left(X_i-\mu \right)}^2.}$ Where, x_i is an individual natural-log transformed value μ is the mean/expected value and N is the total number of values. $\text{CV}=\sqrt{e^{{\sigma }^2}-1}.$