Fluorinated trehalose analogues for cell surface engineering and imaging of Mycobacterium tuberculosis

Fluorinated trehalose analogues for cell surface engineering and imaging of Mycobacterium tuberculosis† Collette S. Guy, a James A. Gott, b Jonathan Ram´ ırez-C´ ardenas, c Christopher de Wolf, a Christopher M. Furze, a GeoffWest, b Juan C. Muñoz-Garc´ ıa, c Jesus Angulo c and Elizabeth Fullam * a The sensitive, rapid and accurate diagnosis of Mycobacterium tuberculosis (Mtb) infection is a central challenge in controlling the global tuberculosis (TB) pandemic. Yet the detection of mycobacteria is often made difficult by the low sensitivity of current diagnostic tools, with over 3.6 million TB cases missed each year. To overcome these limitations there is an urgent need for next-generation TB diagnostic technologies. Here we report the use of a discrete panel of native 19 F-trehalose (F-Tre) analogues to label and directly visualise Mtb by exploiting the uptake of fluorine-modified trehalose analogues via the mycobacterial trehalose LpqY-SugABC ATP-binding cassette (ABC) importer. We discovered the extent of modified F-Tre uptake correlates with LpqY substrate recognition and characterisation of the interacting sites by saturation transfer difference NMR coupled with molecular dynamics provides a unique glimpse into the molecular basis of fluorine-modified trehalose import in Mtb. Lipid profiling demonstrated that F-Tre analogues modified at positions 2, 3 and 6 are incorporated into mycobacterial cell-surface trehalose-containing glycolipids. This rapid one-step labelling approach facilitates the direct visualisation of F-Tre-labelled Mtb by Focused Ion Beam (FIB) Secondary Ion Mass Spectrometry (SIMS), enabling detection of the Mtb pathogen. Collectively, our findings highlight that F- Tre analogues have potential as tools to probe and unravel Mtb biology and can be exploited to detect and image TB. Introduction Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), continues to threaten humanity and remains a major health challenge, having claimed over 1.3 million lives in 2022. 1 Efforts to control the global TB pandemic have been jeopardised by the emergence of drug-resistant Mtb strains that are more complicated to treat and, in some instances, incurable. 1,2 Early diagnosis is crucial for the global management of TB since the timely intervention with antitubercular treatment regimens has the potential to prevent transmission, limit the emergence and spread of antibiotic resistant strains and save lives, yet each year over 3.6 million individuals do not receive treatment compounding the TB burden. 1,3,4 The current World Health Organisation approved TB diagnostics are limited to three main methods: microscopy, culture and nucleic acid amplication; 3,5 however, the low-cost microscopy-based tests used most widely in low-resource settings have limitations, which include low sensitivity. 6 Clearly, new molecular probes for improved, rapid, sensitive and accurate TB diagnostic technologies are needed. As the architecture of the Mtb cell envelope is distinct from other species, comprising an interconnected peptidoglycanarabinogalactan-mycolic acid (mAGP) core interspersed with a diverse array of mycobacterial specic‘free’lipids and glycolipids, 7–9 it provides a unique opportunity to develop Mtb pathogen specic reporter probes through utilisation of the specialised mycobacterial cell-wall assembly machinery for the direct incorporation of a detection moiety into the mycobacterial cell-envelope. Indeed, an array of uorescent-based probes have been developed to monitor Mtb metabolism and image cell-envelope components, which have provided new insights into fundamental aspects of Mtb biology, including enzyme function, metabolism, cell wall biosynthesis and cell division. 10–15 Since the Mtb cell envelope is highly abundant in trehalose-containing glycolipids that include the mycolic acid esters: trehalose monomycolates (TMM) and trehalose a School of Life Sciences, University of Warwick, Coventry, CV4 7AL, UK. E-mail: e. [email protected]; Tel: +44 (0)2476 574239 b WMG, University of Warwick, Coventry, CV4 7AL, UK c Instituto de Investigaciones Qu´ ımicas (IIQ), Consejo Superior de Investigaciones Cient´ ıcas, Universidad de Sevilla, Avenida Am´ erico Vespucio, 49, Sevilla, 41092, Spain †Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d4sc00721b Cite this: Chem. Sci.,2024,15, 13966 All publication charges for this article have been paid for by the Royal Society of Chemistry Received 30th January 2024 Accepted 13th June 2024 DOI: 10.1039/d4sc00721b rsc.li/chemical-science 13966 |Chem. Sci.,2024,15, 13966–13975 © 2024 The Author(s). Published by the Royal Society of Chemistry Chemical Science EDGE ARTICLE Open Access Article. Published on 12 August 2024. Downloaded on 4/29/2025 10:23:41 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. View Article Online View Journal | View Issue dimycolates (TDM), the acyltrehaloses comprising di- and polyacyltrehaloses and sulfoglycolipids, not typically found in other bacterial species, 7–9 recent attention has focused on the use of labelled trehalose derivatives for the visualisation of Mtb. 10–12,15–17 Incorporation of trehalose into mycobacterial cellenvelope glycolipids can proceed via two interlinked pathways: either through extracellular synthesis catalysed by the antigen 85 complex (Ag85) 18 and/or trehalose import 19 followed by biosynthesis of the TMM glycolipid within the cytoplasm (Fig. 1). The Ag85 complex, which include the secreted proteins Ag85A, Ag85B and Ag85C, is responsible for both the conjugation of extracellular mycolic acids to the arabinogalactan (AG) cell-wall core and the incorporation of a mycolic acid from one TMM into another TMM molecule to generate TDM. 18 Alternatively the ‘free’trehalose moiety, released concomitantly during Ag85 mediated cell-wall biosynthesis, can be recycled via the LpqY-SugABC ATP-binding cassette transporter 19 and used as a precursor to yield cytoplasmic TMM, which is subsequently shuttled across the membrane by the MmpL3 exporter for insertion into the mycobacterial cell-envelope. 20,21 Recent studies identied that both pathways can be hijacked to incorporate modied trehalose analogues into mycobacterial cell-envelope trehalose mycolates. The Ag85 complex is known to be particularly promiscuous for a wide panel of extensively modied trehalose substrates 12 whereas the LpqY-SugABC importer only tolerates minor modications. 11,16,22 Recent structural and functional analysis of the LpqY substrate binding lipoprotein has afforded molecular level insights into the rules governing the precise substrate selectivity for this transporter enabling the design of reporter mimics to hijack this pathway. 22 To date, trehalose probes possessing ‘clickable’azide and alkyne reactive handles have been used extensively for imaging of mycobacteria, 11,12,16,23,24 however this approach requires extensive wash steps and a secondary, bulky uorophore label for the direct visualisation of the cells, limiting their use. An 18 F trehalose-modied radioprobe can be used to label the nonpathogenic model Mycobacterium smegmatis organism. 17,25 However, this direct approach is challenging as uorine-18 has a short half-life (∼109 minutes) and it is not possible to infer labelling of pathogenic Mtb because of the signicant differences in nutrient uptake and processing between these diverse mycobacterial species. 26–28 To address these potential limitations we rationalised we could leverage the Mtb LpqY-SugABC transporter to develop and deliver native 19 F-modied trehalose reporter probes as a direct route to specically visualise mycobacteria by focused ion beam (FIB) secondary ion mass spectrometry (SIMS), a technique which is increasingly being applied as a powerful tool to map elemental ions and molecules in biological samples. 29–31 SIMs is an analytical technique that uses a focused ion beam to scan the sample surface, generating secondary ions detected by a mass analyser. 32 Analysis of the mass spectrum generated on a pixel-by-pixel basis produces an Fig. 1 Schematic illustrating the pathways involved in trehalose processing in mycobacteria. TMM: trehalose monomycolate; TDM: trehalose dimycolate. © 2024 The Author(s). Published by the Royal Society of Chemistry Chem. Sci.,2024,15,13966–13975 | 13967 Edge Article Chemical Science Open Access Article. Published on 12 August 2024. Downloaded on 4/29/2025 10:23:41 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. View Article Online ‘ion image’of the composition, distribution and intensity of elements/molecules with spatial resolution within a sample. We show here that a discrete panel of unnatural uorine modied trehalose analogues are indeed tolerated by the Mtb LpqY-SugABC transporter and reveal the molecular architecture dictating their specic recognition. Subsequent characterisation demonstrated that this transport promiscuity can be harnessed to facilitate the uptake of uorinated trehalose probes into live Mtb cells, which are subsequently incorporated into extracellular mycobacterial trehalose containing mycolic acids, enabling detection of the Mtb pathogen. Taken together, these results highlight the possibility of exploiting native uorinated-trehalose derivatives to be used not only as tools for unravelling Mtb biology but also as new molecular probes for the direct visualisation and detection of Mtb. Results and discussion Panel of uorinated trehalose analogues To explore the potential of uorinated trehalose for Mtb detection and evaluate how selected modications inuence assimilation and cell-labelling, we synthesised a panel of uorinated trehalose analogues with systematic modications at either the 2-, 3-, 4-, or 6-position on one glucose subunit with retention of the natural stereochemistry (2–5). An established chemoenzymatic route 33 was employed to access 2-deoxy-2-uorotrehalose (2F-Tre) (2), 3-deoxy-3-uoro-trehalose (3F-Tre) (3) and 6-deoxy-6-uoro-trehalose (6F-Tre) (5)in42–49% yield, and a modied ve-step synthetic route 17,34 (Scheme S1†)afforded 4- deoxy-4-uoro-trehalose (4F-Tre) (4), giving the focused panel shown in Fig. 2 (details provided in ESI, Schemes S1, Fig. S12– S30 and characterisation data†). LpqY substrate specicity for F-Tre analogues As the mycobacterial specic LpqY-SugABC transporter is solely responsible for the import of trehalose, 11,19,22 our rst goal was to establish if the introduction, and position, of a uorine moiety impacts on substrate recognition. We assessed the binding affinity of each uorinated analogue (2–5) to LpqY, the substrate-binding domain that dictates substrate specicity and hence, subsequent transport, by microscale thermophoresis (MST). Highest binding affinities (K d ) were observed for 3F-Tre (110.8 ±7.1 mM) and 6F-Tre (90.3 ±0.9 mM) with comparable affinities to unmodied trehalose (109.5 ±3.2 mM) whereas 2F- Tre and 4F-Tre bind ∼8- and ∼16-fold more weakly, with 4F-Tre having affinity in the millimolar range (Table 1, Fig. S1†). Altogether, these ndings indicate that the position of the uorine modication is key to LpqY recognition with replacement at the 3- or 6-positions preferred. This is in direct contrast to previous LpqY-azide-trehalose binding analyses (Table 1) 22 that identied preferential binding of azide-trehalose modied at the 4- > 6- $3- > 2-positions, albeit with ∼2–4 weaker affinity than the corresponding uorinated versions, highlighting the importance of both the substituent type and position in transporter-substrate tolerance and recognition. Molecular basis of F-Tre analogue recognition Having established that LpqY has the capacity to recognise uorinated trehalose analogues we sought to determine the molecular rules of recognition in solution. Saturation transfer difference (STD) NMR conrmed binding for each uorinated trehalose analogue, verifying that switching a hydroxyl group to uorine at all possible positions is not detrimental for recognition by the mycobacterial trehalose transporter (Fig. 3). The STD NMR intensities differed depending on the temperature of the experiment (Fig. S2–S3 and Tables S1–S4†). More intense signal intensities were observed at low temperature (5 °C) for Fig. 2 Panel of fluorinated trehalose analogues. Table 1 Binding affinity analysis of LpqY to F-Tre analogues a Substrate K d (mM) Reference Trehalose 109.5 3.2 This study 2-Deoxy-2-uoro-trehalose (2F-Tre) 770.4 23.2 This study 2-Deoxy-2-azido-trehalose (2Az-Tre) 1915.6 7.7 22 3-Deoxy-3-uoro-trehalose (3F-Tre) 110.8 7.1 This study 3-Deoxy-3-azido-trehalose (3Az-Tre) 474.9 17.2 22 4-Deoxy-4-uoro-trehalose (4F-Tre) 1483.7 75.6 This study 4-Deoxy-4-azido-trehalose (4Az-Tre) 246.6 7.1 22 6-Deoxy-6-uoro-trehalose (6F-Tre) 90.3 0.9 This study 6-Deoxy-6-azido-trehalose (6Az-Tre) 442.7 5.3 22 a Mean ±standard error mean is from at least three independent experiments. The substrate structures are shown in Fig. 2. The binding affinity data is shown in Fig. S1. 13968 |Chem. Sci.,2024,15,13966–13975 © 2024 The Author(s). Published by the Royal Society of Chemistry Chemical Science Edge Article Open Access Article. Published on 12 August 2024. Downloaded on 4/29/2025 10:23:41 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. View Article Online 2F-Tre and 4F-Tre, whereas a higher temperature of 30 °C was required for STD NMR observation of 3F-Tre and 6F-Tre, especially in the case of the 3-uoro derivative where the particularly low signal-to-noise ratio meant no STD NMR analysis was possible at 5 °C. These results indicate a much stronger affinity for 3F-Tre and 6F-Tre, in comparison with the 2- and 4-uoro derivatives, consistent with our binding affinity data (Table 1). STD binding epitope maps are unique for each F-Tre analogue (Fig. 3) and indicate that protons from both the unmodied and modied carbohydrate rings make distinct close contacts to LpqY relevant for recognition, with a signicant overall decrease in the relative STD intensities at the modied uorinated glucose ring (Fig. 3). Comparative DEEP-STD NMR analyses, 35 which assess the type of protein residue that makes contacts with the uorinated trehalose analogues (Fig. S4†), in combination with molecular dynamics simulations over 100 ns (Fig. S5†) revealed the relative orientations of the four ligands in the LpqY binding pocket Fig. 3 STD NMR binding epitope mapping of F-Tre analogues (2–5) to LpqY. Binding epitope map of (A) 2F-Tre, (B) 3F-Tre, (C) 4F-Tre and (D) 6F- Tre. Protein saturation was achieved by irradiation at 0.53 ppm for (A) and (C), whereas irradiation was at 0.84 ppm for (B) and (D). The coloured spheres represent normalized STD NMR intensities. STD responses are only indicated for protons that could accurately be measured. Asterisks indicate averaged STD values due to overlapping. Glc-1 (blue), Glc-2 (red), STD NMR, saturation transfer difference NMR. Fig. 4 F-Tre binding to LpqY. Model of the mode of binding of F-Tre analogues based on experimentally derived DEEP-STD NMR derived interactions and molecular dynamic simulations. (A) 2F-Tre (blue carbon atoms), (B) 3F-Tre (magenta carbon atoms), (C) 4F-Tre (dark grey carbon atoms), and (D) 6F-Tre (green carbon atoms). Cartoon representation of LpqY in grey (PDB 7APE), trehalose and binding site residues are represented as sticks (oxygen: red; fluorine: pale blue). © 2024 The Author(s). Published by the Royal Society of Chemistry Chem. Sci.,2024,15,13966–13975 | 13969 Edge Article Chemical Science Open Access Article. Published on 12 August 2024. Downloaded on 4/29/2025 10:23:41 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. View Article Online (Fig. 4). Each of the F-Tre analogues bind in similar relative orientations and sit in an analogous position to trehalose (PDB 7APE) 22 (Fig. S6†). The uorinated-modied glucose ring is located towards the entrance of the substrate binding cavity, similar to the orientation we observed previously for 6Az-Tre, 22 indicating that when the second glucose ring is modied it is preferentially accommodated within an expanded binding pocket at the channel entrance rather than buried at the base of the binding cle(Fig. 4). Because the sidechains of Asn25, Glu26, Gln59 and Asn134 make hydrogen bond contacts with the modied glucose ring it is clear this interaction network plays an important role in controlling uoro-modied trehalose specicity. Indeed, the signicantly reduced affinity of 4F-Tre could be attributed to weakened interactions through the loss of a hydrogen-bond partner with Glu26 when a uorine group is substituted at the 4-position. Mtb imports 2F-, 3F- and 6F-Tre analogues via the LpqY- SugABC transporter Having conrmed that the uorinated trehalose analogues bind to LpqY, and since not all substrates recognised by periplasmic binding domains are translocated by the cognate ABC transporter, we sought to evaluate whether these derivatives are taken up by live Mtb cellsbyionchromatography.TreatmentofMtb cells with each uorinated analogue led to a signicant dose-dependent increase in the accumulation of 2F-Tre, 3F-Tre and 6F-Tre in Mtb cells, with the assimilation of micromolar levels even aer exposure to the lowest concentration of the probe (25 mM) (Fig. 5). No uptake of 4F-Tre was detected, a nding consistent with its poor binding affinity to LpqY. The uptake pattern matches the entry of these analogues into non-pathogenic M. smegmatis cells, suggesting overlapping uorine-modied trehalose analogue recognition proles between these bacilli. 17 These ndings, together with the determined F-Tre analogue affinities for LpqY, emphasise the importance of the LpqY substrate binding domain in controlling which substrates are translocated. As Mtb efficiently imports the 2-, 3- and 6-uorinated trehalose derivatives we then asked how rapidly each analogue is accumulated. We evaluated uptake in cytosolic extracts and calculated accumulation rates of 0.80 mMh −1 (2F-Tre) 0.94 mMh −1 (3F-Tre) and 0.82 mMh −1 (6F-Tre). Rapid uptake of each F-Tre derivative was observed at 30 min and at 8 hours ∼20% increased accumulation of 3F-Tre was detected compared to 2F- and 6F-Tre (Fig. 6, S8 and Table S6†). In agreement with previous studies that used radiolabelled 14 C-trehalose, 19 no saturation of uptake was observed for each F-Tre analogue 2,3and 5,indicatingthat an equilibrium between trehalose import and export has not yet been reached at the 8 hour timepoint, which may be consistent with the extremely slow growth rate of the Mtb bacillus or metabolic utilisation of the imported F-Tre analogues. Altogether, these observations establish that 2F-, 3F- and 6F-trehalose are rapidly transported, and accumulate, in live Mtb cells. We then asked whether the F-Tre analogues are imported via LpqY-SugABC transporter or via an alternative route and evaluated uptake in a Mycobacterium bovis BCG strain lacking the trehalose transporter (DlpqY-sugABC), 11 as a model for Mtb.In agreement with our Mtb data 2F-, 3F- and 6F-Tre accumulate in wild-type M. bovis BCG cytosolic extracts and not 4F-Tre (Fig. 7). Notably, uptake of each F-Tre derivative was completely abolished in the DlpqY-sugABC mutant and transport restored in the Fig. 5 Ion chromatography analysis of F-Tre labelled Mycobacterium tuberculosis.Mtb was cultured in the presence of F-Tre analogues 2–5 (0–200 mM) and the cytosolic extracts analysed by high performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD), and F-Tre concentration quantified. The ion-chroma- tography traces and retention times are shown in Fig. S7 and Table S5.† Fig. 6 Time dependent uptake of F-Tre analogues in Mycobacterium tuberculosis.Mtb was cultured in the presence of (A) 2F-Tre, (B) 3F-Tre and (C) 6F-Tre (100 mM) and the cytosolic extracts sampled at the time points indicated and analysed by high performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD). Error bars denote the standard deviation from duplicate experiments. Simple linear regression fit is shown with a black dotted line (A) y=80x; (B) y=0.94x; (C) y=0.82x. The concentration values are in Table S6.† Representative ion-chromatography traces are shown in Fig. S8†and retention times are in Table S7.† 13970 |Chem. Sci.,2024,15, 13966–13975 © 2024 The Author(s). Published by the Royal Society of Chemistry Chemical Science Edge Article Open Access Article. Published on 12 August 2024. Downloaded on 4/29/2025 10:23:41 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. View Article Online corresponding complemented strain. Taken together, as the mutant strain is unable to utilise F-Tre, these data clearly show that uptake of uorinated trehalose analogues occurs solely via the trehalose LpqY-SugABC import system. Mtb incorporates 2F-, 3F- and 6F-Tre analogues into trehalosecontaining glycolipids To determine the fate of F-Tre analogues following import across the mycobacterial cell-envelope Mtb and M. bovis BCG were exposed to each F-Tre analogue (2–5, 100 mM), alongside trehalose and untreated cells, and the extracted lipids evaluated by thin layer chromatography (TLC) and ion chromatography (Fig. 8, S9 and Table S9†). Treatment of Mtb and M. bovis BCG with 2F-, 3F- and 6F-Tre afforded new spots in the lipid extracts that were not present in the control samples (Fig. 8). No new spots were observed with 4F-Tre addition, consistent with its lack of uptake. Two additional lower polarity bands than TMM are present for the 2F- and 3F- samples and one for 6F-Tre (Fig. 8), indicating their incorporation into TMM (Fig. 8). This labelling pattern is consistent with two possible uorinated- TMM regiochemistries for 2F- and 3F-Tre, where the uorine is either on the same glucose moiety as the mycolic acid or the other subunit (Fig. 8). Since the TMM 6-position is blocked through mycolic attachment, only one 6 F trehalose isomer is possible, as observed (Fig. 8). The presence of one additional lower polarity TDM band for 2F- and 3F-Tre suggests these analogues are also incorporated into the TDMs. As expected, when mycobacteria are labelled with 6F-Tre no additional band in the TDM region is observed, since both 6-positions are inaccessible due to the attachment of mycolic acids on both glucose rings preventing the production of 6-uorinated TDM. Altogether, these experiments demonstrate that, unless the 4-position is modied, mycobacteria have the capacity to take up uorinated trehalose analogues and incorporate these modied sugars into cell-surface trehalose mycolates. It is notable that 4F-Tre is not found in any trehalose glycolipid in either Mtb or M. bovis BCG as this differs from the situation found for the analogous azido-trehalose derivatives which are Fig. 7 Comparison of F-Tre uptake in Mycobacterium bovis BCG and LpqY-SugABC mutant strains. Wild-type Mycobacterium bovis BCG and the DlpqY-sugABC Mycobacterium bovis BCG mutant and complemented strain Mycobacterium bovis BCG DlpqY-sugABC::lpqY-SugABC were cultured in the presence of F-Tre analogues 2,3,4and 5(100 mM) and the cytosolic extracts analysed by high performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD). Retention times and peak areas are in Table S8.† © 2024 The Author(s). Published by the Royal Society of Chemistry Chem. Sci.,2024,15,13966–13975 | 13971 Edge Article Chemical Science Open Access Article. Published on 12 August 2024. Downloaded on 4/29/2025 10:23:41 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. View Article Online all incorporated into mycobacterial glycolipids, albeit via different routes, depending on the mycobacterial species. 11,17 For example, in M. bovis BCG, 3- and 4-azido trehalose are not recycled via LpqY-SugABC but, instead, incorporated into TMM and TDM by the extracellular Ag85 complex. 11 Yet this does not occur for any F-Tre analogue. Thus, these ndings reveal distinct mechanistic pathways for trehalose analogue recognition and use, which depend on both the type and position of modication and the mycobacterial species, highlighting an evolutionary divergence of trehalose transport across mycobacterial species. Mtb does not readily metabolise 2F-, 3F- and 6F-Tre analogues into corresponding glucose analogues Next, to determine if the uorinated-trehalose derivatives are processed further we analysed the same cytosolic extracts of Mtb exposed to 2,3, and 5(100 mM) for the presence of uorinatedglucose break-down products that would result from trehalase activity: 2-deoxy-2-uoro-glucose (2F-Glc), 3-deoxy-3-uoroglucose (3F-Glc) and 6-deoxy-6-uoro-glucose (6F-Glc) respectively (Fig. S10 and Table S10†). No cytosolic 3F- or 6F-glucose were found, indicating that the cleavage of 3F- and 6F-Tre does not occur. This suggests that intact 3F- and 6F-Tre are predominantly processed by pathways that ultimately incorporate these derivatives into cell-surface trehalose glycolipids. In contrast, Mtb labelled with 2F-Tre revealed the presence of low levels of 2F-Glc, indicating some degradation occurs, likely catalysed by the Mtb trehalase. 36 It is possible that the 2F-Glc metabolite is then diverted into alternative glucose utilisation pathways and not solely incorporated into cell-surface trehalose opening up potential off-target labelling routes. As the F-Tre analogues effectively label Mtb glycolipids, we did not explore additional metabolic pathways. However the F-Tre analogues might also be metabolised by intracellular enzymes such as Fig. 8 Thin layer chromatography analysis of Mtb and M. bovis BCG lipids after labelling with F-Tre analogues. (A) Mtb extracted apolar lipids. (B) M. bovis BCG extracted apolar lipids. (C) Structures illustrating potential F-Tre TMM and TDM modifications. Red arrows indicate new spots with intermediate polarity compared to TMM and TDM standards in region 1; green arrows indicated new spots which are less polar than TDM in region 2; orange arrows indicate new spots with intermediate polarity compared to TMM and TDM standards in region 1 that are visible directly on the TLC plate but not as clearly visible on the image. The image has been contrast adjusted to improve the visualization of spots. TMM: trehalose monomycolate standard, TDM: trehalose dimycolate standard. 13972 |Chem. Sci.,2024,15, 13966–13975 © 2024 The Author(s). Published by the Royal Society of Chemistry Chemical Science Edge Article Open Access Article. Published on 12 August 2024. Downloaded on 4/29/2025 10:23:41 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. View Article Online TreS, which isomerises trehalose to maltose, or through phosphorylation. Imaging F-Tre labelled Mtb by secondary ion mass spectrometry As uorine-modied trehalose analogues are incorporated into cell-surface trehalose-containing Mtb glycolipids, we next examined the ability to visualise and image F-Tre labelled Mtb cells using focused ion beam (FIB) secondary ion mass spectrometry (SIMS), focused on 19 F − ions. Elemental scanning for negative ions in the mass range 1–100 detected 19 F − uorine ions in all Mtb samples exposed to F-Tre and only trace levels in non-treated cells (Fig. S11†and 9). We then mapped the distribution of uorine in the samples, rst imaging the Mtb cells by scanning electron microscopy (SEM) to show the morphology of the cells and then analysis of the same section by FIB-SIMS. The intensity of the SIMS 19 F − signal correlated with the scanning electron micrograph and showed a clear pattern of accumulation and localisation of F-Tre in treated Mtb cells (Fig. 9). In contrast only background 19 F − levels were observed in untreated cells. Crucially, our ndings establish that F-Tre labelling of live Mtb cells affords a prominent 19 F − SIMS signal and low background demonstrating the feasibility of this approach for the direct detection and imaging of mycobacteria. Whilst robust labelling is observed for each probe, it is conceivable that the incorporation of multiple uorine atoms on the same glucose ring that combines modications at either the 2- 3-, or 6-positions could enhance the signal intensity further. Conclusions One of the major hurdles in controlling the TB pandemic is the need for improved TB diagnosis. One route to achieve this is to transition from sputum microscopy to molecular testing. Yet molecular probes for the direct detection of TB are lacking. To address this deciency, we used native uorinated trehalose probes for imaging by FIB-SIMS, providing unique opportunities to specically visualise and detect the Mtb pathogen. Given the requirement for specialised mass-spectrometry imaging equipment, optimisation will be necessary for use in resource limited settings. However 19-uorine labelled probes may offer advantages over 18-uorine methods, which have short halflives, especially where point-of-care analysis is unfeasible and instead requires sample collection and central testing facilities. In conclusion, here we have clearly shown that we can hijack the promiscuity of the mycobacterial specic LpqY-SugABC trehalose transport machinery to incorporate uorinated reporters into mycobacterial specic cell-surface glycolipids, combined with FIB-SIMS imaging to resolve 19 F − localisation and directly image Mtb cells. Moreover, our ndings demonstrate that F-Tre probes allow for the detection of the Mtb pathogen with high sensitivity. Expanding on this, our studies reveal a discernible dependence on the position of uorine atom modication for efficient Mtb labelling. Specically, we have uncovered the molecular framework that dictates probe recognition and import allowing us to explain F-Tre selectivity. We have shown that a single uorine modication at the 2-, 3- and 6-positions of Fig. 9 F-Tre labelled Mtb scanning electron microscope (SEM) images and focused ion beam-secondary ion mass spectrometry (FIB-SIMS) maps of Mtb cells treated with (a) 2F-Tre, (c) 3F-Tre (e) 6F-Tre (100 mM) and untreated control cells (b), (d) and (f). Colour scale represents 19 F − SIMS counts, black =lowest intensity, yellow =highest intensity. White scale bar 10 mm (a, c, d and e), 20 mm (b and d). © 2024 The Author(s). Published by the Royal Society of Chemistry Chem. Sci.,2024,15, 13966–13975 | 13973 Edge Article Chemical Science Open Access Article. Published on 12 August 2024. Downloaded on 4/29/2025 10:23:41 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. View Article Online one glucose ring is optimal since substitution at the 4-position disrupts a critical hydrogen bond interaction that signicantly weakens interactions with LpqY, impeding 4F-Tre import. The structural insights into F-Tre recognition and uptake analysis provided by this study, combined with FIB-SIMS imaging of Mtb cells provides a rm foundation that open opportunities for the rational development of high-contrast molecular probes for the detection of mycobacteria and provides an important step towards expanding the existing toolbox of molecular reporters for TB diagnosis. Data availability The data supporting this article have been included as part of the ESI.† Author contributions Conceptulisation: CSG, EF; methodology: CSG, JAG, JRC, JCMG, CdW, EF; formal analysis: CSG, JAG, JRC, JCMG, JA, EF; investigation: CSG, JAG, JRC, GW, JCMG, CMF, EF; resources: JA; visualisation: CSG, JAG, JRC, JCMG, EF; supervision: GW, JCMG, JA, EF; writing -original dra: CSG , JAG, JRC, JCMG, JA, EF; writing -review & editing: CSG, JAG, JRC, CdW, CMF, GW, JCMG, JA, EF; project administration: JA, EF; funding acquisition: JA, EF. Conflicts of interest There are no conicts to declare. Acknowledgements This work was supported by a Sir Henry Dale Fellowship to EF jointly funded by the Wellcome Trust and Royal Society (104193/ Z/14/Z and 104193/Z/14/B) and a research grant from the Leverhulme Trust (RPG-2019-087). We thank Dr Ben Swarts (Central Michigan University, USA) for providing the TreT construct, Professor Rainer Kalscheuer (HHU Dusseldorf, Germany) for kindly providing the M. bovis BCG mutant strains, Dr Saskia Bakker for help with sample preparation for FIB-SIMS and Dr James Harrison for critical reading of this manuscript. We acknowledge equipment access, training, and support made available by the Research Technology Facility (managed by Dr Sarah Bennett) of the Warwick Integrative Synthetic Biology Centre (WISB), which received funding from EPSRC and BBSRC (BB/M017982/1). JA and JCMG thank support from Ministerio de Ciencia e Innovaci´ on via the grant AEI/10.13039/ 501100011033/PID2022-142879NB-I00, co-funded by the European Regional Development Fund (ERDF) “A way of making Europe”. References 1WHO Global Tuberculosis Report, 2023, https://www.who.int/ tb/publications/global_report/en/. 2 G. B. Migliori, S. Tiberi, A. Zumla, E. Petersen, J. M. Chakaya, C. Wejse, M. 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Published by the Royal Society of Chemistry Chemical Science Edge Article Open Access Article. Published on 12 August 2024. Downloaded on 4/29/2025 10:23:41 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. View Article Online