Application of Fluorophages for full-scale in vitro activity testing of antitubercular drugs

MARIANA PIURI; MAR¨ªA JOS¨¦ REBOLLO-L¨®PEZ; PEDRO TORRES-GOMEZ; ESTEFANÍA URDÁNIZ; GRAHAM HATFULL; MAR¨ªA MARTINEZ-HOYOS

Conferencia; Gordon Research Conference: Tuberculosis Drug Discovery & Development.; 2017

Gordon Research Conferences

Tuberculosis (TB) is a major cause of human mortality. The emergence of resistant Mycobacterium tuberculosis (M.tb) strains has become a serious public health problem worldwide complicating treatment and control of the disease. Nowadays, there is a need for new and efficient anti-TB drugs. The bottleneck on the TB drug discovery pipeline still remains in the time-consuming activity testing of compounds. Our aim is to develop a novel, rapid and sensitive full-scale assay to be used for in vitro activity testing of compounds as part of the solution to the serious TB health problem. The development of fluorophages, mycobacteriophages carrying fluorescent genes, was described as a simple means of revealing the metabolic state of M.tb cells, and therefore their response to antibiotics. Recently, we have constructed an improved version of fluorophages with higher sensitivity and shorter time to detection of signal. Red bright M.tb cells were observed by fluorescent microscopy after only 5 hours of infection with the new mCherrybomb¦µ. We optimized the conditions for automated fluorimetric detection in a multiwell format as a good alternative for in vitro extracellular activity testing of compounds (Urd¨¢niz et al. 2016, AAC). Briefly, pure cultures of M.tb were infected at a MOI of 100 with mCherrybomb¦µ in the presence of the most common anti-TB drugs. A decrease in the fluorescent signal was observed over time for increasing concentrations of compounds with different targets in the bacteria and MICs were successfully determined. As a result, we are nowadays evaluating GSK¡¯s TB set of compounds to validate the methodology. The implementation of this methodology would allow to test activity of drugs in 48 hours. To test whether our methodology would also be useful for intracellular activity testing of compounds, we developed an infection assay in eukaryotic cells. We were able to detect intracellular M.tb by confocal microscopy after infection with mCherrybomb¦µ in THP-1 derived macrophages. Basically, differentiated cells were incubated with bacteria at a MOI 3 and 30 and after extensive washing, mCherrybomb¦µ was added overnight to reveal the presence of intracellular M.tb. After fixation, inmunofluorescence was performed using different cellular membrane antibodies for delimitation of intracellular space. Since this first cell infection protocol resulted in clumps of infected cells, we are currently evaluating a different protocol in which differentiation and infection are performed at the same time in a roller bottle using single cell suspension of bacteria. We intend to perform the infection with a GFP-expressing strain in order to quantify intracellular bacteria infected with mCherrybomb¦µ using a High Content Screening Opera platform (PerkinElmer).An automated infection assay in presence of different concentrations of drugs still must be evaluated. However, these promising results show that our phage-technology could be used for both extracellular and intracellular in vitro activity testing of compounds in a full-scale, fast and sensitive assay.