Cryptic prophages within a Streptococcus pyogenes genotype emm 4 lineage

Abstract

The major human pathogen Streptococcus pyogenes shares an intimate evolutionary history with mobile genetic elements, which in many cases carry genes encoding bacterial virulence factors. During recent whole-genome sequencing of a longitudinal sample of S. pyogenes isolates in England, we identified a lineage within emm4 that clustered with the reference genome MEW427. Like MEW427, this lineage was characterized by substantial gene loss within all three prophage regions, compared to MGAS10750 and isolates outside of the MEW427-like lineage. Gene loss primarily affected lysogeny, replicative and regulatory modules, and to a lesser and more variable extent, structural genes. Importantly, prophage-encoded superantigen and DNase genes were retained in all isolates. In isolates where the prophage elements were complete, like MGAS10750, they could be induced experimentally, but not in MEW427-like isolates with degraded prophages. We also found gene loss within the chromosomal island SpyCIM4 of MEW427-like isolates, although surprisingly, the SpyCIM4 element could not be experimentally induced in either MGAS10750-like or MEW427-like isolates. This did not, however, appear to abolish expression of the mismatch repair operon, within which this element resides. The inclusion of further emm4 genomes in our analyses ratified our observations and revealed an international emm4 lineage characterized by prophage degradation. Intriguingly, the USA population of emm4 S. pyogenes appeared to constitute predominantly MEW427-like isolates, whereas the UK population comprised both MEW427-like and MGAS10750-like isolates. The degraded and cryptic nature of these elements may have important phenotypic and fitness ramifications for emm4 S. pyogenes, and the geographical distribution of this lineage raises interesting questions on the population dynamics of the genotype.

Keywords: DNA mismatch repair; DNase; SpyCI; bacteriophage; group A Streptococcus; superantigen.

Fig. 1.

Recombination in prophage regions of isolates clustering with MEW427. Sequence data for all ten BSAC isolates were mapped to the reference isolate MGAS10750 along with a second reference isolate MEW427, and Gubbins analysis performed on the resulting alignment. Vertical red lines indicate recombination common to other isolates (internal nodes) and vertical blue lines indicate recombination on terminal branches. Six predicted regions of recombination were common to all five BSAC isolates and MEW427, which form a separate lineage to the other five BSAC isolates and MGAS10750. Four of these regions were within the three prophages (Φ10705.1, Φ10705.2, Φ10705.3) and the SpyCI element (Φ10705.4) (shaded grey). Two other regions of recombination were also identified within all MEW427-like isolates: *1, relating to the absence of MGAS10750 genes Spy485-488 in MEW427-like isolates; and *2, corresponding to the previously identified chimeric emm-enn gene found in MEW427-like isolates [36]. The phylogenetic tree was generated by Gubbins from 556 polymorphic sites following removal of regions of predicted recombination. The tree is mid-point rooted. The scale below boxed region represents genomic position relative to MGAS10750.

Fig. 2.

Gene loss within prophage regions. All genes (represented by arrows) present in the three prophages (a) Φ10705.1, (b) Φ10705.2 and (c) Φ10705.3 of MGAS10750 were also present in the five BSAC isolates BSAC_bs468, BSAC_bs916, BSAC_bs1052, BSAC_bs1379 and BSAC_bs1388 (M4_complete). However, fewer genes were present in the corresponding prophage regions (a) Φ427.1, (b) Φ427.2 and (c) Φ427.3 in MEW427 and the MEW427-like BSAC isolates BSAC_bs192, BSAC_bs400, BSAC_bs472 and BSAC_bs696 (M4_degraded). BSAC_bs1802 carried a different prophage associated with speC/spd1 and so is not shown in (a). Gene presence in each isolate is represented by corresponding horizontal lines. Some genes in MEW427 and M4_degraded isolates were truncated (shown in lighter colours), for example the integrase genes 03820 of Φ427.2 and 05920 of Φ427.3 (lighter shade of blue and shorter arrow/line). Within Φ427.1, the annotated gene 02770 actually comprised sections of spy609, spy0612 and spy614 of MGAS10750. In all isolate genomes and in MEW427, spy0619 of Φ10705.1 was divided into two genes. Locus numbers for some genes are provided above and below as a guide; MEW427 is annotated in increments of five. The asterisks indicate an additional gene that was present but not included in the reference annotation. Colours represent predicted gene function according to the key.

Fig. 3.

Phage induction in M4_complete isolates but not M4_degraded isolates. (a) Primers were designed to detect integrated (In) and excised (Ex) prophage. Primer pairs A+B and C+D spanned the attachment sites attL and attR, respectively, formed when the prophage (grey) is integrated into the chromosome (black). Primer pairs A+D and C+B spanned the attachment site attB on the bacterial chromosome and attP on the prophage, respectively, detecting prophage excision. (b) Both integrated prophage, as indicated by bands in the two ‘In’ lanes, and excised prophage, as indicated by bands in the two ‘Ex’ lanes (primer pairs A+D and C+B) were detected in M4_complete for all three prophages. Excision was enhanced by the addition of mitomycin C (+MC). No excision was detected for all three prophages in M4_degraded, as indicated by bands only in the In lanes but not the Ex lanes, even with mitomycin C. The exception was BSAC_bs1802, where excision of Φ10750.1/427.1 and Φ10750.3/427.3 was detected but excision of Φ10750.2/427.2 was not. Representative gels are shown for single isolates out of the five M4_complete BSAC isolates tested (BSAC_bs1388) and four M4_degraded tested (BSAC_bs472). Primer pairs were used in the following order: In lane 1, A+B (attL); In lane 2, C+D (attR); Ex lane 3, A+D (attB); Ex lane 4, C+B (attP).

Fig. 4.

Gene loss within the SpyCI. The SpyCIM4 element is integrated within the MMR operon, between mutL and mutS (red arrows), and comprises 25 genes at full-length (as in MGAS10750) in the genomes of M4_complete isolates. In the genomes of M4_degraded isolates, including MEW427, the SpyCI element consists of only 11 genes, representing ~56 % gene loss. The pattern of degradation was common to all M4_degraded. The SpyCI of BSAC_bs1802 is not shown, as this isolate contains a different SpyCI. Locus numbers for some genes are provided above and below; MEW427 is annotated in increments of five. The asterisks indicate an additional gene that was present but not included in the reference annotation. Colours represent predicted gene function according to the key.

Fig. 5.

Lineages within the emm4 population. Genome sequence data from 223 isolates were obtained and mapped against the reference genome MGAS10750 (black square) and 4994 concatenated core SNPs (excluding prophage and SpyCI regions) used to generate a maximum-likelihood phylogenetic tree. The second reference isolate MEW427 was also included (black triangle). Data comprised genomes from ABCs (USA) isolates (blue circles, n=48) [44], Canadian isolates (light blue circles, n=8) [45, 46], PHE (UK) isolates (red circles, n=153) [27, 47], as well as isolates from Cambridgeshire (CUH, UK) (pink circles, n=4) [48] and BSAC isolates (brown circles, n=10). Isolates in red and dark blue were obtained around a similar time period (2014–2015), but from either the UK (red) or the USA (blue). Two broad lineages exist, either associated with MEW427 (shaded grey) or MGAS10750 (unshaded). A small lineage was also identified (dotted line circle) and these isolates are all from the UK. Bar, number of substitutions per site.