In vivo capsular switch in Streptococcus pneumoniae--analysis by whole genome sequencing

Abstract

Two multidrug resistant strains of Streptococcus pneumoniae - SV35-T23 (capsular type 23F) and SV36-T3 (capsular type 3) were recovered from the nasopharynx of two adult patients during an outbreak of pneumococcal disease in a New York hospital in 1996. Both strains belonged to the pandemic lineage PMEN1 but they differed strikingly in virulence when tested in the mouse model of IP infection: as few as 1000 CFU of SV36 killed all mice within 24 hours after inoculation while SV35-T23 was avirulent.Whole genome sequencing (WGS) of the two isolates was performed (i) to test if these two isolates belonging to the same clonal type and recovered from an identical epidemiological scenario only differed in their capsular genes? and (ii) to test if the vast difference in virulence between the strains was mostly - or exclusively - due to the type III capsule. WGS demonstrated extensive differences between the two isolates including over 2500 single nucleotide polymorphisms in core genes and also differences in 36 genetic determinants: 25 of which were unique to SV35-T23 and 11 unique to strain SV36-T3. Nineteen of these differences were capsular genes and 9 bacteriocin genes.Using genetic transformation in the laboratory, the capsular region of SV35-T23 was replaced by the type 3 capsular genes from SV36-T3 to generate the recombinant SV35-T3* which was as virulent as the parental strain SV36-T3* in the murine model and the type 3 capsule was the major virulence factor in the chinchilla model as well. On the other hand, a careful comparison of strains SV36-T3 and the laboratory constructed SV35-T3* in the chinchilla model suggested that some additional determinants present in SV36 but not in the laboratory recombinant may also contribute to the progression of middle ear disease. The nature of this determinants remains to be identified.

Figure 1. Annotation of the capsular biosynthetic gene regions of SV35-T23 and SV36-T3.

Whole genome sequencing using the 454 Life Sciences Titanium platform was followed by automated annotation and manual curation of all gene possession differences between SV35-T23 and SV36-T3 (gray: pseudogenes, blue: orthologous genes, yellow: unique genes, black: flanking genes; green: spectinomycin resistance cassette; white: genes present in both genomes and functional in at least one). These data were used to identify and characterize the type 23 and type 3 capsular biosynthetic genes, as well as to identify the contiguous regions of identity between the two strains. The panel shows a diagram of the capsular switch strategy highlighting the movement of the SV36-T3 capsular region containing the inserted spectinomycin-resistance cassette into the SV35-T23 where it replaces the type 23 capsule genes.

Figure 2. PCR confirmation of capsule-switch in SV35-T23 producing SV35-T3.

Six sets of primers (with one exception) were designed from the SV36-T3 genome to produce products that had small overlaps with the adjacent amplimers to ensure complete coverage of the entire capsular region. Diagnostic amplifications were done with each of the primer sets for each of the three pneumococcal strains: SV35Type3 spec (SV35-T3) (a), SV35-T23 (b), and SV36-T3 (c) from left to right. Primer pair #1 included a reverse primer in the spectinomycin resistance cassette explaining the missing SV36-T3 genomic DNA band. Primer pair #2 shows the presence of the spectinomycin resistance cassette in SV35-T3 which produces a larger band relative to SV36-T3. The SV35-T23 genomic DNA also produces a product, larger in size than the type 3 strains, because of homology between the capsular regions and the fact that the type 23 capsule region includes many more genes and is therefore larger. Primer pairs #3–6 show type 3 capsular DNA bands in the recombinant SV35-T3 strain that are identical in size to the SV36-T3 parental strain and which are absent in the SV35-T23 (type 23 capsule) genomic DNA.

Figure 3. Visualization of neighbor groups between strains SV35-T23 and SV36-T3 using the CIRCOS representation tool.

Each one of the 11 arches represents a neighboring group, the numbering around the circle indicates the position on the chromosome, and the width of the arch correlates to the size of the variant region. ‘*’mark the four regions that contain genic differences in addition to allelic differences. Regions are named relative to a coding sequence of interest, and abbreviations are as follows: restriction modification system (RM), cell wall associated protein (CWAP), phosphotransferase system (PTS), and bacteriocin locus (BLP).

Figure 4. Mortality Comparison of the virulence of SV strains in the mouse intraperitoneal infection model.

Bacterial strains grown in Todd-Hewitt broth were used to inoculate groups of mice (5 for each bacterial inoculum) – as described in the Methods. Strains expressing the type 3 capsular polysaccharide – SV36-T3 (○), SV36-T3* (carrying the spectinomycin resistance marker (•) and SV35-T3* (▪) – were each used at inoculum size of 10³ CFU per animal. Strain SV35-T23 (□) – expressing the capsular polysaccharide 23F – was used at inoculum size of 10⁷ CFU per animal. Survival rates were evaluated daily for 7 days – as described in the Methods.

Figure 5. Phenotypic variation caused by a capsular switch from type 23 to type 3.

Scatter plots illustrating differences in mortality and the outcome of otologic disease for animals infected with WT strainsSV35-T23 (blue symbols) and SV36-T3 (green symbols) and recombinant strains SV35-T3* (red symbols) and SV36-T3*(purple symbols) strains. Parameters are – as defined in Table 4– days to morbid state (A), days to onset of moderate or worst otologic disease (B), and maximal otologic score (C).