Establishment Of A Novel Firefly Luciferase Based Apoptosis Detection Method And Its Application In Screening Of Mycobacterium Tuberculosis Protein Regulating Apoptosis

Mycobacterium tuberculosis or Tubercle bacillus is the pathogenic bacteria of tuberculosis. Now about one-third of the world’s population is latently infected and 5%-10% of them will progress to active tuberculosis. Thus, there are about 880 million new infections and 3 million annual deaths caused by tuberculosis. Apoptosis is a type of programmed cell death and plays a key role in the physiological functions including tissue differentiation, organ development and general homeostasis. Although apoptosis is a normal process, dysregulated apoptosis contributes to a great many of diseases. Regulation of apoptosis is a part of the pathogenicity of Mycobacterium tuberculosis, which affects the survival, reproduction and release of Mycobacterium tuberculosis.Rapid and accurate apoptosis detection methods are the foundation for apoptosis mechanism related research and the evaluation of the impact of apoptosis on the diseases. The activation of caspase-3 is a key marker for apoptosis and methods for determining its activity are widely used to detect apoptosis. To quantitate caspase-3 activity, we constructed a biosensor comprising a recombinant firefly luciferase containing the caspase-3 cleavage site and screened some proteins encoded by Mycobacterium tuberculosis. The main studies were as following: 1. Generation and validation of caspase-3 reportersWe constructed four caspase-3 reporters and transfected the reporters in He La cells. The luciferase activities were measured with or without the positive stimulus inducing apoptosis. Higher value of luciferase and the cleavage of luciferase were detected with the positive stimulus, which indicated that the caspase-3 reporters were successfully constructed. When apoptosis was induced, the value of 233-Dna E-DEVDG increased over 25 folds and at the same time its control remained a low level. Our results indicated that with higher sensitivity and lower background value, 233-Dna E-DEVDG was more suitable for apoptosis detection. 2. Analysis of the ability of ESAT-6 family members encoded by Mycobacterium tuberculosis to induce apoptosis.In order to determine the level of apoptosis induced by ESAT-6 family proteins encoded by Mycobacterium tuberculosis, we co-transfected ESAT-6 family protein eukaryotic expression plasmids, 233-Dna E-DEVDG and p RL-TK. The results indicated that esx A, esx T and esx L could activate 233-Dna E-DEVDG with a dose dependent manner; esx C could not activate 233-Dna E-DEVDG. 3. Validation of the screening resultsIn this study, we used classical apoptosis detection method to validate the screening results. Caspase-3 plays a key role in apoptosis pathways and is the core indicator of apoptosis. We used Western Blot to detect the activation of caspase-3 and found that esx A and esx T could activate caspase-3 and esx C, a negative control, could not activate caspase-3. After strained by apoptosis assays kit, cells were detected by flow cytometry. The results indicated that esx A and esx T could increase the ratio of apoptotic cells. 4. Activation of nuclear factor-kappa B(NF-κB)mediates esx T-induced apoptosisNF-kB pathway is closely related to apoptosis and in some condition the activation of NF-kB is required for apoptosis. In order to investigate whether NF-kB pathway was involved in esx T-induced apoptosis, we used dual luciferase reporter assay and found that esx T induced a dose-dependent increase in NF-kB promoter activity. Increasing concentrations of NF-kB inhibitors reduced the level of esx T-induced apoptosis, indicating that the activation of NF-κB was involved.NF-kB pathway promotes apoptosis mainly by up-regulating the expression of apoptotic gene. To investigate which genes were involved, we used Real-Time PCR and Western Blot to detect the expression of NF-κB-regulated genes and found that esx T up-regulated tumor necrosis factor-α(TNF-α) and tumor necrosis factor-related apoptosis-inducing ligand(TRAIL). The studies showed that esx T might induce apoptosis by up-regulating the expression of NF-κB-regulated genes encoding TNF-α and TRAIL.