A Fluorescent Probe for Detecting Mycobacterium tuberculosis and Identifying Genes Critical for Cell Entry

Abstract

The conventional method for quantitating Mycobacterium tuberculosis (Mtb) in vitro and in vivo relies on bacterial colony forming unit (CFU) enumeration on agar plates. Due to the slow growth rate of Mtb, it takes 3–6 weeks to observe visible colonies on agar plates. Imaging technologies that are capable of quickly quantitating both active and dormant tubercle bacilli in vitro and in vivo would accelerate research toward the development of anti-TB chemotherapies and vaccines. We have developed a fluorescent probe that can directly label the Mtb cell wall components. The fluorescent probe, designated as DLF-1, has a strong affinity to the D-Ala-D-Ala unit of the late peptidoglycan intermediates in the bacterial cell wall. We demonstrate that DLF-1 is capable of detecting Mtb in both the actively replicating and dormant states in vitro at 100 nM without inhibiting bacterial growth. The DLF-1 fluorescence signal correlated well with CFU of the labeled bacteria (R2 = 1 and 0.99 for actively replicating and dormant Mtb, respectively). DLF-1 can also quantitate labeled Mtb inside of cells. The utility of DLF-1 probe to quantitate Mtb was successfully applied to identify genes critical for cell invasion. In conclusion, this novel near infrared imaging probe provides a powerful new tool for enumerating Mtb with potential future use in bacterial virulence study.

Improved Conventional and New Approaches in the Diagnosis of Tuberculosis

“…DLF-1 is a high affinity stoichiometric probe for the D-Ala-D-Ala motif of bacterial peptidoglycan. The fluorescent probe directly labels the cell wall components of M. tuberculosis , representing an alternative approach for a rapid quantitative analysis of active and dormant M. tuberculosis in vitro and in vivo ( Yang et al, 2016 ). The D-Ala-D-Ala motif is also present in other bacterial strains besides M. tuberculosis ; thus, DLF-1 cannot be specifically used to label M. tuberculosis .…”

Chemical Tools for Illumination of Tuberculosis Biology, Virulence Mechanisms, and Diagnosis

“…Copyright 2019 American Chemical Society. Representative fluorescent images of A549 human cell lines treated with M. smegmatis expressing tdTomato protein (in green), probe 31 + Cy5.5 (in red), and DAPI (in blue) showing detection of replicating bacteria using probe 31 inside human cells (scale bar: 10 μm) are reproduced from ref . Copyright 2016 Yang et al…”

Bioorthogonal Correlative Light-Electron Microscopy of Mycobacterium tuberculosis in Macrophages Reveals the Effect of Antituberculosis Drugs on Subcellular Bacterial Distribution

“…Fluorescent protein-modified Mtb has allowed its imaging by confocal microscopy, but the reliability of fluorescent proteins varies and the fluorescence is lost upon degradation of the protein by the host . Metabolic labeling of the mycobacterial cell wall, using fluorescent or bioorthogonal analogues of d -alanine , or trehalose, − or using fluorescent antibiotic analogues has allowed the study of growing and dividing Mtb . − This has, for example, allowed the discrimination of live from dead Mtb in sputum samples . Finally, a dual-targeting activity-based probe was recently reported, combining the activity of two Mtb -specific enzymes to obtain extremely high specificity for Mtb over other mycobacteria .…”