Stable genetic integration of a red fluorescent protein in a virulent Group A Streptococcus strain

There are several advantages, both in vitro and in vivo, in utilizing bacteria that express a fluorescent protein. Such a protein can be transiently incorporated into the bacteria or integrated within the bacterial genome. The most widely utilized fluorescent protein is green fluorescent protein (GFP), but limitations exist on its use. Additional fluorescent proteins have been designed that have many advantages over GFP and technologies for their incorporation into bacteria have been optimized. In the current study, we report the successful integration and expression of a stable fluorescent reporter, mCherry (red fluorescent protein, RFP), into the genome of a human pathogen, Group A Streptococcus pyogenes (GAS) isolate AP53(S-). RFP was targeted at the atg codon of the fcR pseudogene that is present in the mga regulon of AP53(S-). Transcription of critical bacterial genes was not functionally altered by the genomic integration of mCherry. Host virulence both in vitro (keratinocyte infection and cytotoxicity) and in vivo (skin infection) was maintained in AP53(S-)-RFP. Additionally, survival of mice infected with either AP53(S-) or AP53(S-)-RFP was similar, demonstrating that overall pathogenicity of the AP53(S-) strain was not altered by the expression of mCherry. These studies demonstrate the feasibility of integrating a fluorescent reporter into the bacterial genome of a naturally virulent isolate of Group A S. pyogenes for comparative experimental studies.