title: Application of Fluorescent Protein Expressing Strains to Evaluation of Anti-Tuberculosis Therapeutic Efficacy In Vitro and In Vivo.
abstract: The slow growth of Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), hinders development of new diagnostics, therapeutics and vaccines. Using non-invasive real-time imaging technologies to monitor the disease process in live animals would facilitate TB research in all areas. We developed fluorescent protein (FP) expressing Mycobacterium bovis BCG strains for in vivo imaging, which can be used to track bacterial location, and to quantify bacterial load in live animals. We selected an optimal FP for in vivo imaging, by first cloning six FPs: tdTomato, mCherry, mPlum, mKate, Katushka and mKeima, into mycobacteria under either a mycobacterial Hsp60 or L5 promoter, and compared their fluorescent signals in vitro and in vivo. Fluorescence from each FP-expressing strain was measured with a multimode reader using the optimal excitation and emission wavelengths for the FP. After normalizing bacterial numbers with optical density, the strain expressing L5-tdTomato displayed the highest fluorescence. We used the tdTomato-labeled M. bovis BCG to obtain real-time images of pulmonary infections in living mice and rapidly determined the number of bacteria present. Further comparison between L5-tdTomato and Hsp60-tdTomato revealed that L5-tdTomato carried four-fold more tdTomato gene copies than Hsp60-tdTomato, which eventually led to higher protein expression of tdTomato. Evaluating anti-TB efficacy of rifampicin and isoniazid therapy in vitro and in vivo using the L5-tdTomato strain demonstrated that this strain can be used to identify anti-TB therapeutic efficacy as quickly as 24 h post-treatment. These M. bovis BCG reporter strains represent a valuable new tool for evaluation of therapeutics, vaccines and virulence.
authors.affiliation: Department of Microbiology, Immunology, and Biochemistry, University of Tennessee Health Science Center, Memphis, Tennessee, United States of America.
authors.forename: Ying
authors.initials: Y
authors.lastname: Kong
authors.affiliation: Department of Microbiology, Immunology, and Biochemistry, University of Tennessee Health Science Center, Memphis, Tennessee, United States of America.
authors.forename: Dong
authors.initials: D
authors.lastname: Yang
authors.affiliation: Department of Microbial Pathogenesis and Immunology, Texas A & M Health Science Center, Bryan, Texas, United States of America.
authors.forename: Suat L G
authors.initials: SL
authors.lastname: Cirillo
authors.affiliation: Department of Microbiology, Immunology, and Biochemistry, University of Tennessee Health Science Center, Memphis, Tennessee, United States of America.
authors.forename: Shaoji
authors.initials: S
authors.lastname: Li
authors.affiliation: Caliper Life Sciences, PerkinElmer, Waltham, Massachusetts, United States of America.
authors.forename: Ali
authors.initials: A
authors.lastname: Akin
authors.affiliation: Caliper Life Sciences, PerkinElmer, Waltham, Massachusetts, United States of America.
authors.forename: Kevin P
authors.initials: KP
authors.lastname: Francis
authors.affiliation: Department of Microbial Pathogenesis and Immunology, Texas A & M Health Science Center, Bryan, Texas, United States of America.
authors.forename: Taylor
authors.initials: T
authors.lastname: Maloney
authors.affiliation: Department of Microbial Pathogenesis and Immunology, Texas A & M Health Science Center, Bryan, Texas, United States of America.
authors.forename: Jeffrey D
authors.initials: JD
authors.lastname: Cirillo
mesh_terms: D000818:Animals; D000995:Antitubercular Agents / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000235:genetics*; D002460:Cell Line; D018834:Chaperonin 60 / Q000235:genetics; D008164:Luminescent Proteins / Q000235:genetics*; D051379:Mice; D009163:Mycobacterium bovis / Q000187:drug effects / Q000235:genetics; D009169:Mycobacterium tuberculosis / Q000187:drug effects / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000187:drug effects / Q000235:genetics; D012293:Rifampin / Q000494:pharmacology; D014376:Tuberculosis / Q000188:drug therapy* / Q000235:genetics*
chemical_list: D000995:Antitubercular Agents; D001426:Bacterial Proteins; D018834:Chaperonin 60; D008164:Luminescent Proteins; D012293:Rifampin