Streptococcus mitis Expressing Pneumococcal Serotype 1 Capsule

Abstract

Streptococcus pneumoniae's polysaccharide capsule is an important virulence factor; vaccine-induced immunity to specific capsular polysaccharide effectively prevents disease. Serotype 1 S. pneumoniae is rarely found in healthy persons, but is highly invasive and a common cause of meningitis outbreaks and invasive disease outside of the United States. Here we show that genes for polysaccharide capsule similar to those expressed by pneumococci were commonly detected by polymerase chain reaction among upper respiratory tract samples from older US adults not carrying pneumococci. Serotype 1-specific genes were predominantly detected. In five oropharyngeal samples tested, serotype 1 gene belonging to S. mitis expressed capsules immunologically indistinct from pneumococcal capsules. Whole genome sequencing revealed three distinct S. mitis clones, each representing a cps1 operon highly similar to the pneumococcal cps1 reference operon. These findings raise important questions about the contribution of commensal streptococci to natural immunity against pneumococci, a leading cause of mortality worldwide.

Figure 1

Genomic sequencing results showing alignment of cps1 regions between serotype 1 S. pneumoniae and S. mitis. Comparison of cps1-L polysaccharide synthetic cluster regions from S. mitis strains L006/L164 (16,613 bp), L115/L116 (16,617 bp) and L121 (16,626 bp) with the corresponding pneumococcal cps1 sequence. White rectangles represent open reading frames not shared between the species. Top diagram depicts ranges of sequence identity between pneumococcal cps1 genes and the S. mitis counterparts depicted on the bottom diagram. Ranges of sequence identity between the S. mitis alleles are given below S. mitis cps1 diagram. L006 and L164 shared identical 16613 sequence. L115 and L116 shared identical 16617 bp sequence. The connecting blue lines depict the alignment of the polysaccharide synthesis clusters. Black regions in the pneumococcal diagram depict remnants of transposase genes. Two inactive pneumococcal genes (aliB and rmlD, the latter renders the rhamnose biosynthesis cluster inactive) containing frameshift mutations are indicated, as well as the partial 5′ and 3′ ends of the dexB and aliA genes in both species (′). The region detected through use of PCR assays to first detect the presence of these strains in upper respiratory specimens is depicted by the red line above the wzy gene.

Figure 2

Species assignment of five Streptococcus mitis strains based upon phylogeny of concatenated housekeeping gene segments. Tree showing the positions of well-characterized strains described by Bishop et al. and the newly-identify S. mitis strains with pneumococcal cps1 homologues, shown in blue. Evolutionary distances were analyzed using MEGA 7.

Figure 3

(a) Quellung reaction using S. pneumoniae serotyping 1 anti-serum for (a.1) S. pneumoniae serotype 1 strain (positive control), (a.2) S. mitis serotype 1 (L121) strain, and (a.3) S. mitis non-serotype 1 (NCTC12261-negative control) strain – no capsule visualization; and Quellung reaction using S. mitis serotyping 1 rabbit anti-serum obtained from L121 strain for (a.4) S. pneumoniae serotype 1 strain. (b) Double immunodiffusion assay testing (b.1) cross-reactive of serotype 1 S. mitis and S. pneumoniae capsule extracts with serotype 1 S. pneumoniae typing anti-serum, and (b.2) control reaction using serotype 1 S. mitis and serotype 1 and non-serotype 1 S. pneumoniae extracts with serotype 5 S. pneumoniae typing anti-serum. Blank – wells with no bacteria; 1AS – serotype 1 S. pneumoniae anti-serum; Spn st1 – S. pneumoniae serotype 1; 5AS – serotype 5 S. pneumoniae anti-serum; Spn st5 – S. pneumoniae serotype 5 (S. mitis serotype 1 L006 strain used in reaction).

Figure 4

Opsonophagocytosis killing (OPK) testing of S. mitis and serotype 1 S. pneumoniae isolates using anti-serotype 1 pneumococcal and non-pneumococcal serum. Serotype 1 S. pneumoniae = positive control; S. mitis ATCC (NCTC 12261) strain confirmed to be wzy1-negative = negative control. Antibody source for OPK assay: 007 S. pneumoniae human reference serum. Complement: baby rabbit complement; OPK titer: reciprocal of serum dilution with >50% growth compared to serum free complement control. (S. mitis strain L006 and L121 representing two distinct S. mitis clones tested). Error bars represent the lower and upper limit of OPK titer based on the test runs.