CRISPR influences the family relationships of virus DNA in Group A Streptococcus

Group A Streptococcus (GAS) is a major component of human pharyngeal microbiota. However, GAS is associated with a wide spectrum of invasive diseases, such as pharyngitis, suppurative skin inflammation, scarlet fever, rheumatic fever and, in rare cases, streptococcal toxic shock syndrome (STSS). A mutation in the Control of Virulence Sensor gene (covS), which is part of a 2-component control system in GAS, is thought to be involved in the onset of STSS [1]. Yet, this mechanism alone is insufficient to explain all cases of STSS. Moreover, the detailed pathogenic mechanism of STSS remains unclear. The serotypes of GAS strains are mainly characterized by two protein antigens expressed on the bacterial surface: M-type antigens, which are determined by the sequence of the emm gene encoding M protein; and T-type antigens, which are classified based on serological testing of enzyme-treated bacterial cells. Currently, most GAS strains associated with STSS worldwide have been classified as M1 T1 [2]. The M3-type strains are also frequently isolated from patients with STSS. The number of cases of STSS caused by M89- or M4-type strains is increasingly common in the United Kingdom and Australia [3, 4]. However, M-type antigen expression and pathogenicity are not necessarily correlated and therefore other factors should be considered to define the pathogenicity of GAS.

Several genome analyses of GAS have revealed that approximately 20% of its genome is composed of prophage genomes. These prophages encode various virulence factors, such as superantigens and DNases. Therefore, lateral transfer of virulence genes is associated with their dissemination and virulence [5]. Moreover, some GAS genomes also contain GAS clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems, which confer adaptive immunity against exogenic elements, including bacteriophages (phages). Therefore, exploration of the relationship between phage acquisition and CRISPR/Cas systems in GAS genomes may enable a better understanding of the evolutionary aspects of GAS pathogenicity.

CRISPRs are composed of several short repeat sequences of approximately 30–50 bp, separated by unique variable sequences. These sequences are widely distributed and present in 90% of archaea and 50% of eubacteria [6–8]. The CRISPR-based adaptive immune system functions in 3 stages; acquisition, expression, and interference [8–11]. In the acquisition stage, specific fragments of double-stranded DNA from a virus or plasmid are acquired at the leader end of a CRISPR array in the bacteria host DNA [8–10]. Therefore, spacer sequences may provide a historical perspective on foreign DNA exposure, and thereby are useful as an indicator of their evolution [12].

Phages, viruses that infect eubacteria, play crucial roles in regulating bacterial ecology, diversity, and virulence. For example, phage-derived toxin genes, such as sea, seg2, sek2, and sak, are expressed in Staphylococcus aureus following prophage induction [13]. Enterohemorrhagic Escherichia coli also contain more than 20 prophage or prophage-like regions in its genome, and a Shiga toxin-encoded gene (stx) is found in Stx prophage [14]. Thus, phages can act as carriers of virulence-related genes, and the integrated phage genome can influence genome diversification in bacteria, leading to the emergence of pathogenic strains. A previous study conducted by us, using 13 sequenced GAS genomes, demonstrated that GAS CRISPR spacers target phages and that a small number of spacers may be limiting certain phage infections. In other words, being subjected to phage infections may lead to spacer acquisition in survivors [15]. However, 32% of the target sequences in GAS spacers were not included in the database, suggesting the existence of unknown phage, plasmid, ICE or other sequences, although some phylogenetic relationships of known prophages were predicted. Therefore, additional prophage analysis should help to further elucidate CRISPR interactions in GAS, as more complete genome information on GAS is released. In this study, we investigated the characteristics of two genomic elements, prophages and CRISPR, in 118 GAS genomes. Three hundred and seventy-three prophages found in these 118 strains were classified into 14 groups, based on integration site. Correlation between the number, or diversification, of prophages and the phylogenetic relationships of GAS was analyzed.

The CRISPR/Cas system is widely conserved in archaea and bacteria. Moreover, type I-C CRISPR is widely conserved and considered to be an original model of CRISPR, whereas, type II CRISPR is only conserved in bacteria. Among them, type II-A CRISPR is mainly conserved in Firmicutes, including GAS [19]. Nozawa et al., reported that GAS contained both type I-C and type II-A CRISPR [15]. In the present study, we did not detect any novel CRISPR loci in the GAS genome. Therefore, the CRISPR locus in GAS may mainly consist of these two CRISPR systems.

In the present study, we inferred that the CRISPR/Cas system was functioning in the GAS genome, which was consistent with the findings of a previous study [15]. Moreover, we proposed that the main function of type II-A CRISPR is the elimination of phages, which protects the GAS genome more effectively than type I-C. Correlation between the number of type I-C spacer sequences and the number of prophages was relatively weak compared with that in type II-A (Fig. 3). This is inconsistent with the findings of our previous study [15]. Only 13 strains were used in our previous study, and most strains were isolated in the United States. Therefore, we speculated that the difference may be due to increased variation in M type and the isolated region used in this study.

We observed a significant difference in the number of prophages between type II-A CRISPR and no CRISPR strains. Therefore, our hypothesis that type II-A CRISPR may plays an important role in eliminating foreign DNA from GAS, was consistent with the findings of a study on S. mutans [20]. Therefore, we reached the conclusion that type II-A CRIPSR may be the main source of phage rejection and elimination in the GAS genome.

GAS prophages were classified into 14 groups based on the presence or absence of ortholog clusters (Fig. 2). Prophage clustering raises issues due to the diversity of accessory genes and mosaic structures of prophages. The mosaic structure of prophages may induce genomic rearrangement via a ~ 5 kb sequence located near the 3′ terminus, as previously reported [21]. Therefore, only DNA alignment has been used to elucidate phylogenetic relationships in GAS prophages [16]. In this study, we used the GAS ortholog clusters for the clustering of prophages, and examined sequence similarities in each prophage group (Additional file 3: Figure S1 A-N). The sequence similarities of prophages in GAS can be roughly classified into two patterns: (1) the regions around 5′ and 3′ termini where genes encoding enzymes, such as integrase, and various kinds of virulence genes are conserved and (2) the central part of prophages encoding structural proteins is conserved. For example, 5′ and 3′ terminus regions tend to be conserved in Gps 4 and 10, whereas, regions with genes encoding structural proteins tend to be conserved in Gps 5, 6 and 14. These findings suggest the possibility of elucidating rearrangement regions systematically by examining prophage sequences and groups. A similar clustering method was used in a study on E. coli prophages, where E. coli Gps correlated with the shiga-toxin gene type [22]. Although, we were unable to find a relationship between GAS Gps and clinical symptoms, GAS Gps correlated with the integration site. Moreover, GAS strains harboring prophages that targeted the same genes that the integration site did, were not necessarily phylogenetically related. Therefore, we suggest that these genes tend to be the targets of phage integration.

The spacer sequences did not show homology with the Gps 1, 2, 3, and a majority of 4, characterized by mutL integration sites (Fig. 4). In previous reports, prophages such as SF370_4, Manfredo_5 and MGAS15252_1 were classified into streptococcal phage-like chromosomal islands (SpyCI) [16, 23]. Gps 3 and 4 lack structural proteins but harbored genes encoding DNA mismatch repair, multidrug efflux and Holliday-junction resolvase. Therefore, we speculated that Gps 3 and 4 consisted of SpyCI, since these characteristics are specifically found in SpyCI. As reported previously [24], SpyCI are believed to have originated from defective prophages. Also, the presence of a SpyCI in GAS correlates with a higher mutation rate and UV sensitivity compared to strains lacking SpyCI [25]. However, there have been no reports indicating that SpyCI are not targeted by CRISPR. One possible reason for these Gps not being recognized by CRISPR is that the spacer’s target region is absent or has mutated during evolution. Reportedly, deletion of spacer sequences may occur frequently [26]. Therefore, it may be speculated that GAS lacks spacers targeting these prophages. Furthermore, other non-CRISPR mechanisms, such as restriction-modification (R-M), toxin-antitoxin, and abortive infection systems may play a stronger role at phage rejection and elimination in GAS as well as CRISPR/Cas systems. For example, SF370 and MGAS10394 have type I and II R-M systems, respectively [27, 28]. Nonetheless, these results suggest that acceptance of foreign DNA by GAS may be related to the integration site.

Additionally, the study indicated that 3 different data types, M-type, spacer sequences, and prophage groups, strongly correlated with GAS phylogenetic relationships (Fig. 5 and Additional file 1: Table S6). These findings suggest that phage integration into the GAS genome is dependent on genomic characteristics of GAS such as M type antigens. For example, a previous study indicated that almost all M12 strains harbored Gp 9 prophages, and that this phage integration may have occurred before the 1950s [29]. Similarly, it has been reported that MGAS315_6, in prophage Gp 9, was integrated into the GAS genome before 1920 [30]. Considered together, Gp 9 prophages may have been integrated into the GAS genome before STSS and other streptococcal diseases emerged. In contrast, Davis et al., suggested that HKU360_2 (Gp 11) and HKU360_4 (Gp 5) triggered the expansion of M12 strains causing scarlet fever in Hong Kong in 2011 [31]. Although we did not find a clear correlation between Gps and STSS in this study, it may be possible to estimate pathogenic and non-pathogenic prophage groups via further analyses of the clustering/classification patterns of GAS prophages.

The strong correlation between the 3 data types, M-type, spacer sequences and Gps may be useful for estimating genetic characteristics and/or phylogenetic relationships of GAS. This approach was previously known as “CRISPR typing” and used in studies on Salmonella enterica [32], Mycobacterium tuberculosis [33], Yersinia pestis [34] and Corynebacterium diphtheriae [35]. For example, S. enterica strains were classified via conventional virulence gene analysis together with CRISPR typing [32]. Additionally, epidemiological trials inferring region-specific strains and estimating their dissemination routes have been conducted with Y. pestis [34].

The current study classified prophages found in 118 GAS strains and investigated correlation between spacer sequences and prophages. Results of phylogenetic and M-type analyses suggested that type II-A CRISPR may play an important role in eliminating phages, and that the integration site may be the deciding factor in acceptance of foreign DNA by GAS. It was also observed that clustered Gps correlated with M-type and their integration site. The present study provides novel information that will help analyze GAS phylogeny in detail based on M-type, spacer sequences and prophage distribution.