Rapid detection of Mycobacterium tuberculosis in sputum with a solvatochromic trehalose probe

Abstract

Tuberculosis (TB) is the leading cause of death from an infectious bacterial disease. Poor diagnostic tools to detect active disease plague TB control programs and affect patient care. Accurate detection of live (Mtb), the causative agent of TB, could improve TB diagnosis and patient treatment. We report that mycobacteria and other corynebacteria can be specifically detected with a fluorogenic trehalose analog. We designed a 4--dimethylamino-1,8-naphthalimide-conjugated trehalose (DMN-Tre) probe that undergoes >700-fold increase in fluorescence intensity when transitioned from aqueous to hydrophobic environments. This enhancement occurs upon metabolic conversion of DMN-Tre to trehalose monomycolate and incorporation into the mycomembrane of Actinobacteria. DMN-Tre labeling enabled the rapid, no-wash visualization of mycobacterial and corynebacterial species without nonspecific labeling of Gram-positive or Gram-negative bacteria. DMN-Tre labeling was detected within minutes and was inhibited by heat killing of mycobacteria. Furthermore, DMN-Tre labeling was reduced by treatment with TB drugs, unlike the clinically used auramine stain. Lastly, DMN-Tre labeled Mtb in TB-positive human sputum samples comparably to auramine staining, suggesting that this operationally simple method may be deployable for TB diagnosis.

Engineering the Mycomembrane of Live Mycobacteria with an Expanded Set of Trehalose Monomycolate Analogues

“…Not only does this make the knockout of all mycoloyltransferases impossible due to lethality, but experiments involving partial blockage of mycoloyltransferase activity (e.g., by chemical inhibition or partial knockout) are complicated due to isoform redundancy and the resulting perturbation of cell envelope structure and permeability, which presumably can impact probe uptake and thus labeling efficiency. We attempted to use the Ag85C inhibitor ebselen and previously reported Msmeg mutants partially lacking mycoloyltransferase activity (e.g., Msmeg ag85a ::Tn and the triple knockout Msmeg ΔMSMEG _ 6396‐99 ), but the results were inconclusive. For example, we observed increases in cellular labeling for all of the TMM reporters in both Msmeg mutants, perhaps suggesting that partial mycoloyltransferase disruption leads to increased reporter uptake, followed by envelope incorporation via the remaining mycoloyltransferases.…”

“…QTF does not label mycomembrane components, rather it allows direct visualization of mycoloyltransferase activity, a novel and attractive feature that complements the TMM reporters developed by our group so far. Such fluorogenic probes that specifically detect mycobacteria in a single step are of interest as tuberculosis diagnostic tools, and trehalose ester‐based probes warrant further investigation in this regard.…”

“…Fluorine‐modified trehalose analogues can also be metabolized by mycobacteria, a finding which may pave the way for imaging mycobacterial infections in vivo using positron emission tomography (PET) . Recently, a trehalose analogue bearing a solvatochromic moiety was used as a fluorogenic probe for the rapid detection of Mtb in patient sputum samples . The latter two examples highlight the potential of trehalose‐based metabolic reporters for tuberculosis diagnostic applications.…”

Chemical Reporters for Exploring Microbiology and Microbiota Mechanisms

“…To visualize mycobacteria and monitor biosynthesis of its cell envelope, several reporters for trehalose and its derivatives have been developed by the Davis, Bertozzi, Swarts and Kiessling laboratories. For example, trehalose analogues have been developed for visualization of mycobacteria cell division (Figure E), visualization of antibiotic activity on mycobacterial cell wall (Figure C), and rapid detection of live Mycobacterium tuberculosis (Mtb) in sputum using solvatochromic dyes for clinical TB diagnosis (Figure D) . Azide and alkyne reporters of trehalose have also been developed for two‐step labeling of mycobacteria (Figure F) .…”

Glycopolymer Code: Programming Synthetic Macromolecules for Biological Targeting

“…[ 8 ] Last year, a team led by Bertozzi has achieved to develop a quick and simple way to detect the bacteria that causes tuberculosis using chemically tweaking a sugar molecule known as trehalose. [ 9 ] The in vivo and imaging results are very promising for the possible clinical applications to fight against tuberculosis.…”