title: A Fluorescent Probe for DetectingMycobacterium tuberculosisand Identifying Genes Critical for Cell Entry.
abstract: The conventional method for quantitatingMycobacterium tuberculosis(Mtb)in vitroandin vivorelies on bacterial colony forming unit (CFU) enumeration on agar plates. Due to the slow growth rate ofMtb, it takes 3-6 weeks to observe visible colonies on agar plates. Imaging technologies that are capable of quickly quantitating both active and dormant tubercle bacilliin vitroandin vivowould accelerate research toward the development of anti-TB chemotherapies and vaccines. We have developed a fluorescent probe that can directly label theMtbcell wall components. The fluorescent probe, designated as DLF-1, has a strong affinity to the D-Ala-D-Ala unit of the late peptidoglycan intermediates in the bacterial cell wall. We demonstrate that DLF-1 is capable of detectingMtbin both the actively replicating and dormant statesin vitroat 100 nM without inhibiting bacterial growth. The DLF-1 fluorescence signal correlated well with CFU of the labeled bacteria (R2= 1 and 0.99 for actively replicating and dormantMtb, respectively). DLF-1 can also quantitate labeledMtbinside of cells. The utility of DLF-1 probe to quantitateMtbwas successfully applied to identify genes critical for cell invasion. In conclusion, this novel near infrared imaging probe provides a powerful new tool for enumeratingMtbwith potential future use in bacterial virulence study.
authors.affiliation: Department of Microbiology, Immunology and Biochemistry, University of Tennessee Health Science Center Memphis, TN, USA.
authors.forename: Dong
authors.initials: D
authors.lastname: Yang
authors.affiliation: Department of Microbiology, Immunology and Biochemistry, University of Tennessee Health Science Center Memphis, TN, USA.
authors.forename: Feng
authors.initials: F
authors.lastname: Ding
authors.affiliation: Department of Pharmaceutical Sciences, University of Tennessee Health Science Center Memphis, TN, USA.
authors.forename: Katsuhiko
authors.initials: K
authors.lastname: Mitachi
authors.affiliation: Department of Pharmaceutical Sciences, University of Tennessee Health Science Center Memphis, TN, USA.
authors.forename: Michio
authors.initials: M
authors.lastname: Kurosu
authors.affiliation: Chemical Biology and Therapeutics Department, St. Jude Children's Research Hospital Memphis, TN, USA.
authors.forename: Richard E
authors.initials: RE
authors.lastname: Lee
authors.affiliation: Department of Microbiology, Immunology and Biochemistry, University of Tennessee Health Science Center Memphis, TN, USA.
authors.forename: Ying
authors.initials: Y
authors.lastname: Kong
keywords: Mycobacterium tuberculosis; dormant M. tuberculosis; fluorescence; imaging