Application of Fluorescent Protein Expressing Strains to Evaluation of Anti-Tuberculosis Therapeutic Efficacy <i>In Vitro</i> and <i>In Vivo</i>

<div><p>The slow growth of <i>Mycobacterium tuberculosis</i> (<i>Mtb</i>), 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 <i>Mycobacterium bovis</i> BCG strains for <i>in vivo</i> imaging, which can be used to track bacterial location, and to quantify bacterial load in live animals. We selected an optimal FP for <i>in vivo</i> 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 <i>in vitro</i> and <i>in vivo</i>. 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 <i>M</i>. <i>bovis</i> 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 <i>in vitro</i> and <i>in vivo</i> 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 <i>M</i>. <i>bovis</i> BCG reporter strains represent a valuable new tool for evaluation of therapeutics, vaccines and virulence.</p>