A fluorogenic trehalose probe for tracking phagocytosed mycobacterium tuberculosis Tingting Dai1, Jinghang Xie2, Qihua Zhu2,3,4, Mireille Kamariza5, Ke Jiang2, Carolyn R. Bertozzi1,6, Jianghong Rao1,2* 1Department of Chemistry, Stanford University, Stanford, CA 94305, USA.; 2Department of Radiology, Molecular Imaging Program at Stanford, Stanford University School of Medicine, Stanford, CA 94305, USA.; 3State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing, 211198, China.; 4Jiangsu Key Laboratory of Drug Design and Optimization, Department of Medicinal Chemistry, China Pharmaceutical University, Nanjing 211198, China.; 5Department of Biology, Stanford University, Stanford, CA 94305, USA.; 6Howard Hughes Medical Institute, Stanford University, Stanford, CA 94305, USA. Tuberculosis (TB) disease caused by Mycobacterium tuberculosis (Mtb) is responsible for approximately 1.6 million deaths every year. Metabolic labeling studies that provide on the intracellular replication status of viable Mtb are crucial for the elucidation of host-pathogen interactions. We have here developed a cephalosphorinase-digestable green trehalose (CDG-Tre) fluorogenic probe that enabled the visualization of single live Bacille Calmette-Guérin (BCG) cells within macrophages at concentrations as low as 2 µM. Upon activation by BlaC, the -lactamase uniquely expressed by Mtb, CDG-Tre releases a fluorescent trehalose analogue that will be subsequently incorporated within the bacterial cell wall via trehalose metabolic pathway. CDG-Tre showed better selectivity for mycobacteria over other clinically prevalent species in the Corynebacterineae suborder. The designed CDG-Tre probe offers a unique metabolism-based strategy to label BCG. It enables to track a single Mtb cell in both pre- and post-phagocytosis and potentially could be used to help elucidate fundamental physiological and pathological process related to the mycomembrane.