title: A novel firefly luciferase biosensor enhances the detection of apoptosis induced by ESAT-6 family proteins of Mycobacterium tuberculosis.
abstract: The activation of caspase-3 is a key surrogate marker for detecting apoptosis. To quantitate caspase-3 activity, we constructed a biosensor comprising a recombinant firefly luciferase containing a caspase-3 cleavage site. When apoptosis was induced, caspase-3 cleavage of the biosensor activated firefly luciferase by a factor greater than 25. The assay conveniently detected apoptosis in real time, indicating that it will facilitate drug discovery. We screened ESAT-6 family proteins of Mycobacterium tuberculosis and found that esxA, esxT and esxL induced apoptosis. Further, activation of nuclear factor-κB (NF-κB) and the NF-κB-regulated genes encoding tumor necrosis factor-α (TNF-α) and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) participated in esxT-induced apoptosis. We conclude that this assay is useful for high-throughput screening to identify and characterize proteins and drugs that regulate apoptosis.
authors.affiliation: State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China.
authors.forename: Junwei
authors.initials: J
authors.lastname: Shi
authors.affiliation: State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China.
authors.forename: Huan
authors.initials: H
authors.lastname: Zhang
authors.affiliation: State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China.
authors.forename: Liurong
authors.initials: L
authors.lastname: Fang
authors.affiliation: State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China.
authors.forename: Yongqiang
authors.initials: Y
authors.lastname: Xi
authors.affiliation: State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China.
authors.forename: Yanrong
authors.initials: Y
authors.lastname: Zhou
authors.affiliation: State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China.
authors.forename: Rui
authors.initials: R
authors.lastname: Luo
authors.affiliation: State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China. Electronic address: wangdang511@126.com.
authors.forename: Dang
authors.initials: D
authors.lastname: Wang
authors.affiliation: State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China.
authors.forename: Shaobo
authors.initials: S
authors.lastname: Xiao
authors.affiliation: State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China.
authors.forename: Huanchun
authors.initials: H
authors.lastname: Chen
mesh_terms: D000942:Antigens, Bacterial / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D001426:Bacterial Proteins / Q000494:pharmacology*; D001681:Biological Assay / Q000379:methods*; D015374:Biosensing Techniques / Q000379:methods*; D006367:HeLa Cells; D006801:Humans; D049409:Luciferases, Firefly; D049408:Luminescent Agents; D008163:Luminescent Measurements / Q000379:methods*; D008856:Microscopy, Fluorescence / Q000379:methods*
chemical_list: D000942:Antigens, Bacterial; D001426:Bacterial Proteins; C093276:ESAT-6 protein, Mycobacterium tuberculosis; D049408:Luminescent Agents; D049409:Luciferases, Firefly
keywords: Apoptosis; Biosensor; Caspase-3; Firefly luciferase; Mycobacterium tuberculosis; NF-κB