Mycobacterium tuberculosis, the etiological agent of human tuberculosis, requires the non-mammalian disaccharide trehalose for growth and virulence. Recently, detectable trehalose analogues have gained attention as probes for studying trehalose metabolism and as potential diagnostic imaging agents for mycobacterial infections. Of particular interest are deoxy-[18F]fluoro-D-trehalose (18F-FDTre) analogues, which have been suggested as possible positron emission tomography (PET) probes for in vivo imaging of M. tuberculosis infection. Here, we report progress toward this objective, including the synthesis and conformational analysis of four non-radioactive deoxy-[19F]fluoro-D-trehalose (19F-FDTre) analogues, as well as evaluation of their uptake by M. smegmatis. The rapid synthesis and purification of several 19F-FDTre analogues was accomplished in high yield using a one-step chemoenzymatic method. Conformational analysis of the 19F-FDTre analogues using NMR and molecular modeling methods showed that fluorine substitution had a negligible effect on the conformation of the native disaccharide, suggesting that fluorinated analogues may be successfully recognized and processed by trehalose metabolic machinery in mycobacteria. To test this hypothesis and to evaluate a possible route for delivery of FDTre probes specifically to mycobacteria, we showed that 19F-FDTre analogues are actively imported into M. smegmatis via the trehalose-specific transporter SugABC-LpqY. Finally, to demonstrate the applicability of these results to the efficient preparation and use of short-lived 18F-FDTre PET radiotracers, we carried out 19F-FDTre synthesis, purification, and administration to M. smegmatis in 1 hour.
The mycobacterial outer membrane, or mycomembrane, is essential for the viability and virulence of Mycobacterium tuberculosis and related pathogens. The mycomembrane is a dynamic structure, whose chemical composition and biophysical properties can change during stress to give an advantage to the bacterium. However, the mechanisms that govern mycomembrane remodeling and their significance to mycobacterial pathogenesis are still not well characterized. Recent studies have shown that trehalose dimycolate (TDM), a major glycolipid of the mycomembrane, is broken down by the mycobacteria-specific enzyme TDM hydrolase (Tdmh) in response to nutrient deprivation, a process which appears to modulate the mycomembrane to increase nutrient acquisition, but at the expense of stress tolerance. Tdmh activity thus balances the growth of M. tuberculosis during infection in a manner that is contingent upon host immunity. Current methods to probe Tdmh activity are limited, impeding the development of inhibitors and the investigation of the role of Tdmh in bacterial growth and persistence. Here, we describe the synthesis and evaluation of FRET-TDM, which is a fluorescence-quenched analogue of TDM that is designed to fluoresce upon hydrolysis by Tdmh and potentially other trehalose ester-degrading hydrolases involved in mycomembrane remodeling. We found that FRET-TDM was efficiently activated in vitro by recombinant Tdmh, generating a 100-fold increase in fluorescence. FRET-TDM was also efficiently activated in the presence of whole cells of Mycobacterium smegmatis and M. tuberculosis , but the observed signal was predominantly Tdmh-independent, suggesting that physiological levels of Tdmh are low and that other mycobacterial enzymes also hydrolyze the probe. The latter notion was confirmed by employing a native protein gel-based fluorescence assay to profile FRET-TDM-activating enzymes from M. smegmatis lysates. On the other hand, FRET-TDM was capable of detecting the activity of Tdmh in cells when it was overexpressed. Together, our data demonstrate that FRET-TDM is a convenient and sensitive in vitro probe of Tdmh activity, which will be beneficial for Tdmh enzymatic characterization and inhibitor screening. In more complex samples, for example, live cells or cell lysates, FRET-TDM can serve as a tool to probe Tdmh activity at elevated enzyme levels, and it may facilitate the identification and characterization of related hydrolases that are involved in mycomembrane remodeling. Our study also provides insights as to how the structure of FRET-TDM or related fluorogenic probes can be optimized to achieve improved specificity and sensitivity for detecting mycobacteria.
Trehalosamine (2-amino-2-deoxy-α,α-d-trehalose) is an aminoglycoside with antimicrobial activity against Mycobacterium tuberculosis, and it is also a versatile synthetic intermediate used to access imaging probes for mycobacteria. To overcome inefficient chemical synthesis approaches, we report a two-step chemoenzymatic synthesis of trehalosamine that features trehalose synthase (TreT)-catalyzed glycosylation as the key transformation. Soluble and recyclable immobilized forms of TreT were successfully employed. We demonstrate that chemoenzymatically synthesized trehalosamine can be elaborated to two complementary imaging probes, which label mycobacteria via distinct pathways.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.