Toxin–antitoxin-stabilized reporter plasmids for biophotonic imaging of Group A streptococcus

• Published in: Applied microbiology and biotechnology Vol. 97; no. 22; pp. 9737 - 9745
• Main Authors: Loh, Jacelyn M. S, Proft, Thomas
• Format: Journal Article
• Language: English
• Published: Berlin/Heidelberg Springer-Verlag 01-11-2013; Springer Berlin Heidelberg; Springer; Springer Nature B.V
• Subjects: Analysis; Animals; Antibiotics; Antitoxins; Applied Genetics and Molecular Biotechnology; Bacteria; Bacterial Toxins - genetics; Bacterial Toxins - metabolism; Bacteriological Techniques - methods; Binding sites; Bioluminescence; Biomedical and Life Sciences; Biotechnology; Cardiovascular disease; E coli; Gene expression; Genes; Genes, Reporter; Genomes; Genomic Instability; Gram-positive bacteria; image analysis; Image Processing, Computer-Assisted; Insects; Kinases; Life Sciences; luciferase; Luciferases - analysis; Luciferases - genetics; Luciferin; Luminescent Measurements; Mice; Microbial Genetics and Genomics; Microbiology; operon; Pathogens; Physiological aspects; Plasmids; Proteins; site-specific recombination; Staining and Labeling - methods; Streptococcus; Streptococcus infections; Streptococcus pyogenes - enzymology; Streptococcus pyogenes - genetics; Streptococcus pyogenes - metabolism; Studies; Toxins; Virulence; Group A streptococcus; Bioluminescence; Toxin–antitoxin system; Firefly luciferase; Biophotonic imaging
• ISSN: 0175-7598; 1432-0614
• DOI: 10.1007/s00253-013-5200-7

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.