Tuberculosis is a common, and often lethal, infectious disease which has circulated since the dawn of mankind. It is caused by the bacteria, Mycobacterium tuberculosis; a small, aerobic, nonmotile bacillus, which tends to lodge in the pulmonary system and from where it then spreads through coughing or sneezing respiratory fluids through the air. It has been found in the bones of Egyptian mummies and was once the cause of nearly 25% of all deaths in Europe. TB remains the world’s second most lethal, infectious disease (behind HIV) and has a significantly high rate of occurrence in African, South American and Asian countries, where the rate of death is anywhere from 250-3000+ per 100,000. The World Health Organization (WHO) estimates that roughly 1/3 of the world’s population have been infected with TB (although 90-95% remain asymptomatic) and about 1.5 million people die from it every year. The resurgence of the disease, and its drug-resistant varieties, led the WHO to declare it a global health emergency in 1993. Tuberculosis is also one of the major research areas to which the Bill and Melinda Gates Foundation fund every year, which awards nearly $800M (total) to researchers through its Global Health Program.
In the lab, Tuberculosis can also be difficult to screen, with a 16-20 hour replication (compared to under an hour for most other bacteria), and can take 3-4 weeks to form on solid media before any in vivo testing can be done. This can be a significant impediment for fast, de novo antibiotic research and Nuria Andreu, from the Department of Medicine at Imperial College London, and associates wanted to use luciferase to change the score!
That’s not to say that bioluminescence hasn’t been used on TB in the past. Researchers have been using BLI on TB strains for almost 20 years, but not necessarily with the live strain of M. tuberculosis, or with in vivo imaging in live mice. Andreu wanted to create a reporter gene with FFluc in a virulent M. tuberculosis strain. First, the FFluc would need to be modified to red-shift the signal so that the signal would be more thermostable. This was was accomplished by mutating 6 amino acids in the sequence to develop a signal that shifted the emission signal from 560nm to 620nm. Second, they had to develop an integrase-free reporter into M. tuberculosis to stabilize FFluc reporter signal over successive generations. The integrase-free reporter responded with greater than 99% retention of the reporter gene after 3 months of in vitro growth, compared with just 60% retention rate for the parent strain!
Ultimately, this study shows great progress in creating a virulent reporter strain of M. tuberculosis which can be useful in drug research. Andreu was able to detect the presence of the bacteria in the lungs of live mice after only 2 weeks post infection (105 cfu), and as few as 103 cfu were detectable ex vivo in both lungs and spleens of the mice after dissection. Most importantly, they were able to make a rapid assessment of antibiotic efficacy, treating the diseased mice with Isoniazid (an organic compound often used as the first line medication in treatment of tuberculosis). They were able to see a nearly 9-fold decrease in the BLI signal for treated mice versus the control group after only 7 days of treatment.
Ultimately, this looks to be a great system for early lab development of new antibiotics. As resistance in TB continues to spread, we need new forms of treatment in order to stem the tide of resurgence and begin to eliminate the disease in the poorer sections of the world where TB remains rampant and the death toll remains high. Hopefully, this new reporter will help in developing those new drugs and we can begin to put an old disease to rest.
Andreu, N., Zelmer, A., Sampson, S. L., Ikeh, M., Bancroft, G. J., Schaible, U. E., Wiles, S., & Robertson, B. D. (2013). Rapid in vivo assessment of drug efficacy against Mycobacterium tuberculosis using an improved firefly luciferase. Journal of Antimicrobial Chemotherapy.
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