title: Real-time Imaging of Mycobacterium tuberculosis, Using a Novel Near-Infrared Fluorescent Substrate.
abstract: Slow growth of Mycobacterium tuberculosis, the causative agent of tuberculosis, hinders advancement in all areas of research toward prevention and treatment. Real-time imaging with reporter enzyme fluorescence (REF) that uses custom fluorogenic substrates for bacterial enzymes allows rapid and specific detection of M. tuberculosis in live animals. We have synthesized a novel REF substrate, CNIR800, that carries a near-infrared (NIR) fluorochrome IRDye 800CW, with a quencher connected through the lactam ring that is hydrolyzed by the enzyme BlaC (β-lactamase) that is naturally expressed by M. tuberculosis. CNIR800 produces long-wavelength emission at 795 nm upon excitation (745 nm) and exhibits significantly improved signal to noise ratios for detection of M. tuberculosis. The detection threshold with CNIR800 is approximately 100 colony-forming units (CFU) in vitro and <1000 CFU in the lungs of mice. Additionally, fluorescence signal from cleaved CNIR800 reaches maximal levels 4-6 hours after administration in live animals, allowing accurate evaluation of antituberculous drug efficacy. Thus, CNIR800 represents an excellent substrate for accurate detection of M. tuberculosis rapidly and specifically in animals, facilitating research toward understanding pathogenic mechanisms, evaluation of therapeutic outcomes, and screening new vaccines.
authors.affiliation: Department of Microbial Pathogenesis and Immunology, Texas A&M Health Science Center, Bryan.
authors.forename: Hee-Jeong
authors.initials: HJ
authors.lastname: Yang
authors.affiliation: Department of Microbiology, Immunology, and Biochemistry, University of Tennessee Health Science Center, Memphis.
authors.forename: Ying
authors.initials: Y
authors.lastname: Kong
authors.affiliation: Department of Radiology, Stanford University, California.
authors.forename: Yunfeng
authors.initials: Y
authors.lastname: Cheng
authors.affiliation: Department of Microbial Pathogenesis and Immunology, Texas A&M Health Science Center, Bryan.
authors.forename: Harish
authors.initials: H
authors.lastname: Janagama
authors.affiliation: Department of Microbial Pathogenesis and Immunology, Texas A&M Health Science Center, Bryan.
authors.forename: Hany
authors.initials: H
authors.lastname: Hassounah
authors.affiliation: Department of Radiology, Stanford University, California.
authors.forename: Hexin
authors.initials: H
authors.lastname: Xie
authors.affiliation: Department of Radiology, Stanford University, California.
authors.forename: Jianghong
authors.initials: J
authors.lastname: Rao
authors.affiliation: Department of Microbial Pathogenesis and Immunology, Texas A&M Health Science Center, Bryan.
authors.forename: Jeffrey D
authors.initials: JD
authors.lastname: Cirillo
mesh_terms: D000818:Animals; D001557:Benzenesulfonates / Q000737:chemistry / Q000378:metabolism; D002511:Cephalosporins / Q000737:chemistry; D005260:Female; D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism; D007211:Indoles / Q000737:chemistry / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D009169:Mycobacterium tuberculosis / Q000201:enzymology / Q000302:isolation & purification*; D001618:beta-Lactamases / Q000032:analysis
chemical_list: D001557:Benzenesulfonates; C000619935:CNIR800; D002511:Cephalosporins; D005456:Fluorescent Dyes; C562366:IRDye 800CW; D007211:Indoles; D001618:beta-Lactamases
keywords: Mycobacterium; antimicrobials; fluorescence; in vivo imaging; infection