Single-cell phenotyping for better drug discovery

Abstract Mycobacterium tuberculosis (Mtb) infects nearly 10 million people yearly and kills almost 1.5 million. Treating even drug-susceptible tuberculosis (TB) is difficult because subpopulations of bacteria, while not genetically resistant, can evade killing by antibiotics. To discover small molecule inhibitors for these tolerant subpopulations, we need to understand where they are located and what is their physiology. Directly identifying tolerant bacteria is impossible with conventional methods that rely on measurements from millions or billions of cells. Here, to overcome these limitations we have assembled a team with expertise in single-cell measurements, live-cell microscopy, and TB pathogenesis and metabolism. Leveraging these skills, we have succeeded in quantifying the growth and metabolism of individual mycobacteria in different subcellular environments. We propose to build upon these results and establish which subcellular environment is most associated with drug tolerance, and what carbon source is used by drug-tolerant M. tuberculosis. Then, we will design in vitro culture conditions mimicking this environment. These culture conditions will be used in future drug screening efforts designed to find small molecule inhibitors of drug tolerant mycobacteria.