Abstract
The slow growth of Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), hinders development of new diagnostics, therapeutics and vaccines. Using non-invasive real-time imaging technologies to monitor the disease process in live animals would facilitate TB research in all areas. We developed fluorescent protein (FP) expressing Mycobacterium bovis BCG strains for in vivo imaging, which can be used to track bacterial location, and to quantify bacterial load in live animals. We selected an optimal FP for in vivo imaging, by first cloning six FPs: tdTomato, mCherry, mPlum, mKate, Katushka and mKeima, into mycobacteria under either a mycobacterial Hsp60 or L5 promoter, and compared their fluorescent signals in vitro and in vivo. Fluorescence from each FP-expressing strain was measured with a multimode reader using the optimal excitation and emission wavelengths for the FP. After normalizing bacterial numbers with optical density, the strain expressing L5-tdTomato displayed the highest fluorescence. We used the tdTomato-labeled M. bovis BCG to obtain real-time images of pulmonary infections in living mice and rapidly determined the number of bacteria present. Further comparison between L5-tdTomato and Hsp60-tdTomato revealed that L5-tdTomato carried four-fold more tdTomato gene copies than Hsp60-tdTomato, which eventually led to higher protein expression of tdTomato. Evaluating anti-TB efficacy of rifampicin and isoniazid therapy in vitro and in vivo using the L5-tdTomato strain demonstrated that this strain can be used to identify anti-TB therapeutic efficacy as quickly as 24 h post-treatment. These M. bovis BCG reporter strains represent a valuable new tool for evaluation of therapeutics, vaccines and virulence.
Highlights
Tuberculosis (TB) is a major cause of morbidity and mortality worldwide [1]
Noninvasive in vivo imaging technologies are emerging for quantification of bacterial loads in animals and has the potential to greatly accelerate TB research
We examined M. bovis BCG strains expressing various fluorescent protein (FP) to select an optimal FP and promoter to allow detection of mycobacteria in vitro and in vivo with fluorescence imaging
Summary
Tuberculosis (TB) is a major cause of morbidity and mortality worldwide [1]. The challenges faced in TB control are: lack of an effective vaccine, emergence of multidrug-resistant TB globally, and increasing cases of Mycobacterium tuberculosis and HIV co-infection in many regions. Optical imaging systems have been successfully used in real-time monitoring of tumorigenesis and a growing number of infectious diseases [4,5,6,7,8,9,10,11] Both bioluminescent imaging and fluorescent imaging probes have been developed for TB research [10,11,12,13,14,15,16,17,18,19], a number of which have been applied to in vivo imaging of M. tuberculosis [11, 12, 14, 18, 19]. The FP expressing strain having the highest sensitivity of detection was successfully applied to evaluation of the efficacy of anti-TB therapy in bacterial culture and in live animals
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