The recent re-emergence of scarlet fever is a rising global health challenge. Streptococcus pyogenes emm1 variant, M1UK, has been associated with seasonal scarlet fever and invasive disease outbreaks in the United Kingdom and is distinguished by the upregulated expression of the superantigen SpeA. M1UK has spread globally, with reports in Australia, where circulating sublineages harbor a unique toxin repertoire associated with scarlet fever outbreaks in Hong Kong and China. Analysis of the Australian emm1 population identified 26% of all Australian M1UK strains had acquired the scarlet fever-associated prophage ΦSP1380.vir, a homologue of ΦHKU.vir (95% similarity), previously linked to M1 and M12 scarlet fever outbreaks in Hong Kong and China. This prophage carries a distinct toxin gene repertoire, including secreted superantigens SSA and SpeC and the DNase Spd1.
To investigate the role of prophage-encoded exotoxins in M1UK pathogenesis, we generated isogenic mutants of the Australian M1UK isolate SP1380, containing the full exotoxin repertoire of superantigens SpeA, SpeC and SSA, and DNase Spd1. Mutants were generated using Gibson assembly cloning with a temperature-sensitive plasmid pLZts to delete (i) speA, (ii) the ΦHKU.vir-associated toxins (ssa, speC, and spd1), and (iii) all four toxins simultaneously. DNA sequence analysis verified the integrity of the isogenic mutant genomes and Western blot analysis confirmed the absence of exotoxin expression. These isogenic mutants provide a platform to dissect the contribution of prophage-encoded exotoxins to M1UK virulence in vivo and in vitro and help to advance our understanding of the factors driving the re-emergence of scarlet fever.