Fluorophages for TB drug screening using a whole-cell approach

ESTEFANÍA URDÁNIZ; MARIANA PIURI; FLORENCIA PAYASLIAN; LILIANA RONDON

Congreso; XXIV Congreso Latinoamericano de Microbiología; 2018

Asociacion Latinoamericana de Microbiologia

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. Our aim is to develop a novel, rapid and sensitive assay tobe used for in vitro activity testing of compounds. Fluorophages, mycobacteriophages carrying fluorescent genes, havebecome a useful tool to reveal the metabolic state of mycobacterial cells, and therefore their response to antibiotics. Weoptimized the conditions for automated fluorimetric detection with our fluorophage mCherrybombΦin a 96-well formatas a good alternative for in vitro extracellular activity testing of compounds. A decrease in the fluorescent signal wasobserved over time for increasing concentrations of compounds with different targets in M.tb allowing MIC calculation.To validate the methodology for drug screening, we miniaturized the assay to 384-well plates and evaluated GSK?s TBset of compounds (n=232) comparing results between M.tb H37Rv and M. bovis BCG. We set up the best conditions forinfection taking into account several parameters such as volume and UFC of inoculum, MOI, dispensation (manual vs.automated) and incubation time to achieve optimal fluorescence readout. Standard deviation and signal to backgroundratio (S:B) were not as good as expected, associated to a high background in the phage stock. Analysis were performedusing software Spotfire (TIBCO) including a correlation with the results previously obtained with this set of compoundsusing REMA methodology and we could create an order for activity of compounds. As expected, after preincubationfor 24 h of bacteria with drugs, compounds that showed best activity were those identified as inhibitors of essentialmembrane proteins, for example DprE, MmpL3, QcrB, and KasA. Using this methodology we were able to identifyseveral interesting compounds with high potency and unknown mechanism of action that could be further explored.We are developing a new generation of luciferase reporter phages that would increase the difference between controlsand increase the S:B ratio in order to get a rapid and robust assay for drug screening.Our research has been funded by several international and local agencies including: NIH, CONICET, ANPCyT, and TresCantos Open Lab Foundation.