A FRET-Based Fluorogenic Trehalose Dimycolate Analogue for Probing Mycomembrane-Remodeling Enzymes of Mycobacteria

Abstract

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.

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

“…In related work, we have now reported a fluorescence‐quenched analogue of TDM, named FRET‐TDM, that reports on the mycomembrane‐remodeling activity of trehalose dimycolate hydrolase (Tdmh) . In addition, the Theriot group have reported the use of fluorophore‐modified TMM analogues (synthesized by CuAAC between O‐AlkTMM‐C7 and azido fluorophores) in an elegant investigation of cell envelope assembly and V snapping in Cglut , which further illustrated O‐linked TMM analogues’ applicability in cellular imaging experiments.…”

Trehalose recycling promotes energy-efficient mycomembrane reorganization in nutrient-limited mycobacteria

“…At least two different processes liberate trehalose: ligation of mycolic acids from TMM to arabinogalactan to form AGM and transfer of mycolic acids from TMM to another molecule of TMM to form TDM. Breakdown of TDM by the TDM hydrolase (TDMH) yields TMM and mycolic acids (Holmes et al, 2019; Ojha et al, 2010; Yang et al, 2013), so subsequent use of TMM in the forgoing reactions would also release trehalose. Our metabolic labeling results suggested that all of these processes are active as M. smegmatis adapts to carbon limitation ( Figure 1 ).…”

“…Perhaps the more interesting question, however, is the source of the energetically-expensive TMM building blocks. Breakdown of TDM by TDMH furnishes free mycolic acids and TMM, the latter of which could serve as a donor for sidewall AGM synthesis (Holmes et al, 2019; Ojha et al, 2010). While such a pathway would not require ATP, it would be limited by the amount of TDM loss that can be tolerated without lysis (Ojha et al, 2010; Yang et al, 2013) or reduced resilience to host stress (Yang et al, 2014).…”

“…Both processes release free trehalose. TDM can also be degraded by TDM hydrolase (TDMH) into TMM and free mycolic acids, the latter of which are an important component of biofilm extracellular matrix in mycobacteria (Holmes et al, 2019; Ojha et al, 2010). While a salvage mechanism for mycolic acids is still under debate (Cantrell et al, 2013; Dunphy et al, 2010; Forrellad et al, 2014; Wilburn et al, 2018), recapture of trehalose occurs via the LpqY-SugABC transporter (Kalscheuer et al, 2010b).…”

Metabolic labeling probes for interrogation of the host–pathogen interaction

“…Finally, the Swarts-group recently reported a similar approach, in which cleavage of a quencher group from a construct containing fluorescein and a DABCYL quencher by the mycomembrane remodeling hydrolase Tdmh, led to a the enhancement of green fluorescence. 113 One downside to all these probes is that they exclusively label metaboli-Organic & Biomolecular Chemistry Review cally active cells. As such, they can only be used to diagnose dividing Mtb and monitor treatment efficacy.…”