Construction of Staphylococcus aureus bioluminescence tracing system based on Antares2

Abstract

Objective To construct a new fusion luciferase-labeled Staphylococcus aureus (S. aureus) tracing system. Methods An S. aureus tracer strain was constructed by using DNA homologous recombination technique. The gene encoding fusion luciferase Antares2 was knocked in the genome of S. aureus strain USA300 by fusing with the enolase (Eno) encoding gene. The bioluminescence intensity was analyzed by mixing with the tracer strain and different substrates. The suitable tracer/substrate system was selected based on the detection of bioluminescence intensity. The sensitivity of the tracer/substrate system in vitro and in vivo was also evaluated. Results The knock-in plasmid pBT2-eno-antares2 was constructed. After transformed into S. aureus USA300, the Antares2 knock-in strain was screened out. The tracer strain USA300/Eno-Antares2 was identified by DNA sequencing as well as Western blot detection of Eno-Antares2 fusion proteins. Bacterial growth curve and hemolytic activity tests showed comparable results between USA300/Eno-Antares2 tracer strain and its wild-type USA300. USA300/Eno-Antares2 catalyzed substrates diphenylterazine (DTZ), furimazine (FUR), and hydrofurimazine (HFZ) to produce macroscopical bioluminescence. Analysis of different substrates at diverse concentrations revealed that USA300/Eno-Antares2/HFZ system exhibited the best performance. This system enabled reliable bioluminescence tracking of S. aureus with sensitivities of approximately 50 colony forming unit (CFU) in vitro and 100 CFU in vivo. Conclusion S. aureus USA300/Eno-Antares2/HFZ tracing system is a good tool, which can be used for the study of S. aureus infection, colonization, and dissemination.

Keywords

• staphylococcus aureus
• bioluminescence
• tracing system
• luciferases