Toxin-antitoxin-stabilized reporter plasmids for biophotonic imaging of Group A streptococcus.
Bioluminescence is a rapid and cost-efficient optical imaging technology that allows the detection of bacteria in real-time during disease development. Here, we report a novel strategy to generate a wide range of bioluminescent group A streptococcus (GAS) strains by using a toxin-antitoxin-stabilized plasmid. The bacterial luciferin-luciferase operon (lux) or the firefly luciferase gene (ffluc) was introduced into GAS via a stabilized plasmid. The FFluc reporter gave significantly stronger bioluminescent signals than the Lux reporter, and was generally more stable. Plasmid-based luciferase reporters could easily be introduced into a variety of GAS strains and the signals correlated linearly with viable cell counts. Co-expression of the streptococcal ω-ε-ζ toxin-antitoxin operon provided segregational stability in the absence of antibiotics for at least 17 passages in vitro and up to 7 days in a mouse infection model. In addition, genome-integrated reporter constructs were also generated by site-specific recombination, but were found to be technically more challenging. The quick and efficient generation of various M-type GAS strains expressing plasmid-based luciferase reporters with comparable and quantifiable bioluminescence signals allows for comparative analysis of different GAS strains in vitro and in vivo.
Department of Molecular Medicine and Pathology, School of Medical Sciences, University of Auckland, Private Bag 92019, Auckland, New Zealand.
Jacelyn M S
JM
Loh
Thomas
T
Proft
D000818:Animals; D001427:Bacterial Toxins / Q000235:genetics / Q000378:metabolism*; D001431:Bacteriological Techniques / Q000379:methods; D017930:Genes, Reporter; D042822:Genomic Instability; D007091:Image Processing, Computer-Assisted; D008156:Luciferases / Q000032:analysis* / Q000235:genetics; D008163:Luminescent Measurements; D051379:Mice; D010957:Plasmids; D013194:Staining and Labeling / Q000379:methods*; D013297:Streptococcus pyogenes / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism
D001427:Bacterial Toxins; D008156:Luciferases