ML was purified from the Malaysian human isolate M. ulcerans 1,615, then quantified by spectrophotometry (λmax = 362 nm; log ε = 4.29) (Spangenberg and Kishi, 2010) on a V-650 spectrophotometer (JASCO). Bodipy-mycolactone (Bdpy-ML) (λex = 496 nm; λex = 503 nm) was synthesized as previously described (Colucci-Guyon et al., 2020). Stock solutions were prepared in ethanol and diluted at least 1,000x for biophysics or cellular assays. In all cases, controls corresponding to the same volume of vehicle were included.

Taylor dispersion were acquired at 20°C with the Viscosizer TD (Malvern Panalytical Ltd., Worcestershire, United Kingdom). Samples were passed through an uncoated capillary (Malvern Panalytical Ltd., Worcestershire, United Kingdom) with internal and outside diameters of 75 and 360 μm, respectively. UV absorption was monitored at 280 nm. Before sample injection, a stray light correction measurement with L-tryptophan at 11 mg/ml was performed on the system. A sizing measurement with caffeine used as a reference at 1 mg/ml was performed during samples measurements to ensure the good quality of the capillary throughout the experiment.

The presence/absence of precipitates in solution was confirmed by DLS with the DynaPro Plate Reader (Wyatt Technology, United States). This DLS instrument combines an 830 nm laser, a detector positioned at 158°C to collect the signal, and a multi-tau hardware correlator to analyze this signal. A volume of 20 µL of each sample was loaded onto a 384-well (Corning) covered with a transparent film to avoid any evaporation. The plates were centrifuged for 1 min at 3,000 rpm to remove bubbles. Three replicates were performed per well and measurements were conducted at 20°C. A number of 20 acquisitions and 10 s of acquisition time were set up in the Dynamics software. The autocorrelation function was processed using the Dynamics software version 7.1.9 (Wyatt Technology, United States) and converted into size-distribution for analysis.

Human serum (10/100 v/v), human albumin (5 mg/ml), human HDL (0.5 mg/ml) or human LDL (0.5 mg/ml) diluted in PBS solution and the subsequent complexes with mycolactone (20 μM) were centrifuged at 42,000 rpm in an Optima AUC analytical ultracentrifuge (Beckman Coulter), at 20°C in an eight-hole AN 50–Ti rotor equipped with 12-mm double-sector aluminum epoxy centerpieces. The concentrations of the individual macromolecules were chosen to be in the range of serum levels and to be detectable by the AUC equipment without saturating the detectors. Detection of the biomolecule concentration as a function of radial position and time was performed by absorbance measurements at 360 nm and by interference detection. At 360 nm, a specific absorbance can be observed for mycolactone in absence of macromolecules’ absorption (Supplementary Figure S1). Ultracentrifugation experiments were performed in PBS (Gibco). Sedimentation velocity data analysis was performed by continuous size distribution analysis c(s) using Sedfit 15.0 software (Brown and Schuck, 2006). All the c(s) distributions were calculated with a fitted fractional ratio f/f0 and a maximum entropy regularization procedure with a confidence level of 0.95. Buffer viscosity and density were calculated from the Sednterp software (www.jphilo.mailway.com/sednterp.htm). Partial specific volumes were also determined with Sednterp.

Fluorescence spectra were recorded on human and mouse plasma at room temperature on a JASCO FP-6300 spectrofluorometer equipped with a right angle 10 mm cuvette. The fluorescence was monitored with an excitation wavelength of 490 nm and a maximum emission wavelength between 495 and 550 nm. For human blood measurments, Bdpy-ML was added at 50 or 500 ng/ml to total human blood collected in heparinized tubes, then incubated overnight at 37°C to allow equilibration without inducing cell lysis. A fraction of total blood was used to perform flow cytometry analyses on blood cells. The other fraction was centrifuged 10 min at 2,000 g at RT to collect plasma. To estimate concentrations in samples, serial range dilutions of Bdpy-ML (0.015–1 μg/ml) have been done in human and mouse plasma; areas under the curves obtained from fluorescence data were calculated and a linear regression curve was built between areas and concentrations using Prism software (Supplementary Figure S2).

Jurkat T cells (E6.1 clone, ECACC#88042803) and THP-1 cells (ECACC#88081201) were cultured in RPMI GlutaMAXTM (Life Technologies), supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Invitrogen), penicillin (100 U/ml) and streptomycin (100 μg/ml). Human primary macrophages were obtained from peripheral blood-derived monocytes isolated by adhesion to tissue culture plastic-ware and cultured with 10 ng/ml human GM-CSF (Peprotech) for 7–12 days. For uptake assays, cells were rinsed in serum-free medium and resuspended in presence of Bdpy-ML (2.5–250 nM) diluted in medium containing either 0.5% or 10% of human serum (Sigma-Aldrich), 0.5% human serum albumin (Sigma-Aldrich), or 0.5 mg/ml human HDL (Sigma-Aldrich) then incubated at 37°C. For release experiments, cells were loaded with Bdpy-ML diluted in medium containing 10% human serum during 1 h at 37°C. Cells were then rinsed twice in serum-free medium before being resuspended in culture medium containing either 0.5% or 10% of human serum (HS), 0.5% human serum albumin (HSA), or 0.5 mg/ml human HDL. In both uptake and release experiments, intracellular content of Bdpy-ML was measured by flow cytometry (FACS) at different time points following addition or removal of Bdpy-ML. Block lipid transfer (BLT)-1, BLT-4, and BLT-5 (Sigma) were used as SR-B1 inhibitors in uptake assays at non-toxic and biologically active concentrations. Zosuquidar and Valspodar (Sigma) were used as MDR1 inhibitors in efflux assays.

Uptake of natural mycolactone was evaluated by measuring the release of IL-2 and TNF-α in Jurkat T cells and THP-1 cells, respectively. Briefly, cells were incubated for 1 h in the presence of 75 ng/ml mycolactone in RPMI medium or in RPMI supplemented with 10% HS, 0.5% HSA (Sigma-Aldrich), or 0.5 mg/ml human HDL, then activated 16 h by addition of PMA (100 ng/ml) and calcimycin (2.5 μg/ml). The release of IL-2 and TNF-α in the culture medium was measured by ELISA using the human IL-2 and TNF-α ELISA Max kits (BioLegend).

Sec61 wt or R66G Sec61 mutant sequences were cloned upstream of an internal ribosome entry site (IRES) of the pRetroX–IRES-DsRedExpress retroviral vector (Addgene) for simultaneous translation of Sec61α and DsRed in mouse primary T cells as described in (Baron et al., 2016). Platinum-E ecotropic packaging cells (platE, Biolabs) were transduced with pRetroX–IRES-DsRedExpress plasmids (Addgene) containing Sec61α sequences to produce retroviral particles. Mouse CD3⁺ primary T cells were isolated from spleens and lymph nodes by negative selection using the Pan T cell Isolation kit (Miltenyi Biotec) then activated with Dynabeads^® Mouse T-activator CD3/CD28 (Miltenyi Biotec) prior to retroviral transduction as described in (Baron et al., 2016). For uptake assay of Bdpy-ML, transduced cells were incubated with different levels of CellTrace (ThermoFisher) to distinguish populations then mixed in equivalent proportion in culture medium before addition of Bdpy-ML for 24 h.

Cells lysates were prepared in lysis buffer (pH 7.5) containing 20 mM Tris-acetate, 150 mM NaCl, 50 mM NaF, 1 mM EDTA, 1% Triton-X100 and 0.1% protease inhibitors (Sigma-Aldrich). Equal amounts of protein samples were loaded on NuPAGE Bis-Tris gels and transferred to nitrocellulose membranes (Life Technologies). Protein detection was done with anti-actin (#3700, Cell Signaling Technology) and anti-Sec61α (NB120-15575, Novus Biologicals) and revealed with the ECL Prime detection reagent (Cytiva) and chemiluminescence reading on a Fuji LAS-4000 Luminescent Image Analyzer.

siRNA transfections were performed in THP-1 cells using 50 nM of SR-B1 siRNA (On-Target plus human SCARB1, Horizon Discovery) and 1 μl of Lipofectamine RNAimax reagent (Thermofischer) in 500 μl of antibiotic-free medium, following the manufacturer’s instructions. SR-B1 expression was controlled by flow cytometry after 48 h.

Flow cytometry analysis of THP-1 cells and total blood was performed using the following antibodies. Conjugated anti-human SR-B1 APC (Miltenyi Biotec 130-111-237), CD235a APC Cy7 (BioLegend 3,49,115), CD45 BUV805 (BD Biosciences 6,12,892), CD56 BUV 737 (BD Biosciences 6,12,767), CD11c PeCy7 (BioLegend 3,37,216), CD11b PE (eBioscience 12-0118-41), CD3 PerCP Cy5.5 (BD Biosciences 5,60,835), CD19 BV786 (BioLegend 3,02,239), HLA-DR BUV 395 (BD Biosciences 7,40,302), CD14 BV605 (BioLegend 3,01,834). Labeling of mouse blood was done using the following conjugated antibodies: CD45.2 PE (eBioscience 12-0454–82), CD115 PerCPeF710 (eBioscience 46-1,152-82), CD11b BV650 (eBioscience 48-0112–20), Ly6G BV510 (BD Biosciences 5,63,402), CD3 APC-eF780 (BioLegend 47-0032–82). Fc receptors were blocked using FCR blocking reagent (Myltenyi Biotec). Surface membrane staining was performed in PBS FBS 2%. The Zombie UV fixable viability dye (BioLegend 4,23,107) was used to exclude dead cells. Samples were acquired on a Cytoflex (Beckman Coulter) and analyzed with FlowJo 10 (BD Biosciences).

Eight-week-old female mice (C57BL/6JRj) were housed and bred under pathogen-free conditions with food and water ad libitum. Mice were injected intravenously with 2.5 mg/kg Bdpy-ML first concentrated in ethanol then diluted in physiologic solution (final ethanol concentration 5%). Mice were sacrificed 4, 24, 48, 72, 96 h post-injection (hpi) then 7 days post-injection.

Statistical analyses and graphical representations were performed using GraphPad Prism software (8.3.1, La Jolla, CA). Unpaired non-parametric Mann-Whitney tests or two-way ANOVA were used as statistical tests.

By measuring critical micelle concentrations, Kubicek-Sutherland et al. showed that in the 30–60 µM range, mycolactone forms water-insoluble aggregates (Kubicek-Sutherland et al., 2019). Due to this hydrophobic property, mycolactone is highly soluble in ethanol. Indeed, Taylor dispersion analysis showed that solubilized mycolactone has a hydrodynamic radius of 0.48 nm (Supplementary Figure S3A), as expected for well solubilized small components. Dynamic light scattering (DLS) analysis confirmed that mycolactone does not form aggregates in ethanol but forms major aggregates in aqueous solution such as PBS at concentrations >15 nM (Supplementary Figures S3A,B). At a concentration as low as 15 nM in PBS, mycolactone formed particles with radii greater than those of solubilized mycolactone (Supplementary Figure S3). It is therefore likely that mycolactone needs hydrophobic component to be miscible in aqueous buffers. The soluble macromolecules contained in serum that possess hydrophobic patches on their surface or apolar core such as albumin and lipoproteins (Haskard and Li-Chan, 1998; Wasan et al., 2008) represent good candidates to solubilize mycolactone in serum-containing culture media or blood-circulating mycolactone.

In order to identify the biological macromolecules serving as mycolactone carriers in serum, we took advantage of the high separative capacity of analytical ultracentrifugation (AUC). AUC allows the separation of molecular species in solution, based on their shape, size and density and direct analysis through a dual UV/visible and interference detection system, with a resolution that is superior to that offered by other separative technique (Gandhi et al., 2017). Human serum samples spiked with mycolactone and subjected to AUC were analyzed by interference and absorbance, reflecting the sedimentation of proteins and mycolactone, respectively. In the absence of mycolactone, serum contained three main species with sedimentation peaks at 3.2, 4.5 and 6.7 S (Figure 1A). Addition of mycolactone altered the serum sedimentation profile, indicating that molecular interactions occur between mycolactone and serum proteins. Mycolactone detection by absorbance at 360 nm (Spangenberg and Kishi, 2010) revealed that it associates with serum proteins with sedimentation peaks ranging from 3 S to 10 S, particularly at 4.8 S (Figure 1A). We then analyzed the behavior of mycolactone in PBS solutions containing physiological concentrations of macromolecules present in serum: human albumin, HDL or LDL (Figures 1B–D). Interferometry measures showed that mycolactone slightly perturbs HDL sedimentation (Figure 1B), and strongly increases the heterogeneity of albumin species, as illustrated by the broadening of peaks at 4.5 and 6.6 S corresponding to albumin monomer and dimer, respectively, (Figure 1C). Mycolactone detection by absorbance confirmed that it interacts with HDL (4.8 S), as well as albumin monomers and dimers (Figures 1B,C, respectively). When incubated with LDL in PBS, mycolactone induced multiple sedimentation peaks ranging from 3 to 9 S with a main peak at 6 S (Figure 1D), suggesting that it greatly perturbs LDL structural integrity, probably by integrating the apolar core of the LDL. Our data confirm that HDL and LDL improve mycolactone solubility in aqueous solutions (Kubicek-Sutherland et al., 2019), and identify albumin as an additional carrier protein able to solubilize mycolactone in serum.

We made the hypothesis that mycolactone binding to serum lipid carriers might condition its partition between extra- and intracellular compartments. To examine the potential impact of serum lipid carriers on cellular import and export of mycolactone, we used Bodipy-mycolactone (Bdpy-ML), a previously described fluorescent derivative of mycolactone retaining its biological activity (Chany et al., 2011; Guenin-Macé et al., 2013; Colucci-Guyon et al., 2020). Bdpy-ML associated with serum lipid carriers comparably to natural mycolactone in AUC (Supplementary Figure S4). Using non-differentiated THP-1 and Jurkat cells as models for human monocytes and T cells respectively, we next assessed by flow cytometry the effects of serum lipid carriers on cellular incorporation and release of Bdpy-ML. In both cell types, Bdpy-ML diffused intracellularly in a time-dependent manner, reaching a plateau after 24 h (Figure 2A). Adding 10% serum to the cell culture medium induced a transient delay Bdpy-ML incorporation of by THP-1 cells (Figures 2A,B), an effect that was mimicked by incubation of the cells with albumin at physiological serum concentration (5 mg/ml) (Figure 2B). Notably, physiological concentrations of HDL (0.5 mg/ml) also induced a marked decrease in the early uptake of Bdpy-ML, but in this case, the decrease in Bdpy-ML incorporation persisted after 24 h (Figure 2B). These experiments using Bdpy-ML suggested that HDL, and albumin to a lower extent, may retain mycolactone in the extracellular compartment.

Production of IL-2 by activated T cells, and production of TNF- α by THP-1 cells, correlate with the amount of intracellular mycolactone (Supplementary Figure S5). We used this read-out to determine whether extracellular retention of ML by lipid carriers limits its intracellular diffusion. Figure 2C shows that intracellular incorporation of mycolactone, as estimated by its suppressive effect on cytokine production, was decreased by addition of serum and purified lipid carriers, particularly HDL, to the cell culture medium (Figure 2C).

We next examined the impact of serum lipid carriers on the release of intracellular mycolactone in the extracellular medium. To this end, THP-I cells were loaded with Bdpy-ML then rinsed before supplementation of the medium with serum, albumin or HDL. The presence of lipid carriers in the extracellular medium promoted the release of Bdpy-ML from THP-1 cells (Figure 2D). Compared to albumin and serum, HDL displayed a stronger stimulatory effect on Bdpy-ML export by pre-loaded cells (Figure 2D).

Altogether, these data suggested that mycolactone distributes in both the intra-and extracellular compartments, the partition depending on the presence and nature of lipid carriers in the extracellular environment. They supported the view that serum lipid carriers contribute to mycolactone diffusion and persistence in vivo.

We previously observed that bacterially-produced mycolactone gains access to serum (Sarfo et al., 2011) and peripheral blood cells (Hong et al., 2008) of infected organisms. Having shown that it associates to lipid carrier proteins in vitro, we next asked how mycolactone partitions between cells and serum in human blood, and whether it displays a particular tropism for certain cell subsets. To address this question, Bdpy-ML was added to total human blood before measuring fluorescence levels in cells and plasma (Figure 3). In agreement with our previous studies using radiolabeled mycolactone and mouse blood (Hong et al., 2008), flow cytometric analyses of human blood cell populations revealed efficient internalization of Bdpy-ML by leukocytes (CD45⁺ cells), but not erythrocytes (CD235a+ cells) (Figure 3A). Interestingly, we noticed a differential accumulation of Bdpy-ML across the different CD45⁺ cell subsets, which was independent of the cell size. Monocytes were the most efficient at internalizing Bdpy-ML, followed by B cells, NK cells and T cells (Figure 3B). To quantify Bdpy-ML in plasma, we used the linear relationship between fluorescence emission signals between 495 and 550 nm (λex 490 nm) and Bdpy-ML concentration in the 8–500 ng/ml range (Supplementary Figure S2). In blood incubated with 50 or 500 ng/ml Bdpy-ML, we estimated that 50.4% (±11.5%) of added Bdpy-ML distributed to plasma fractions (Figure 3C). To verify that Bdpy-ML distribution into plasma fractions depended on its mycolactone moiety, we incubated blood samples with 500 ng/ml Bdpy-ML and the same concentration of natural mycolactone. Recovery of Bdpy-ML in plasma decreased by 25% suggesting a competition between Bdpy-ML and mycolactone for association with lipid carriers (Figure 3C). We concluded that mycolactone distributes to both plasma and human blood cells, with a preference for monocytes.

We used the mouse model to explore further mycolactone distribution and persistence in vivo. Mice were injected intravenously with Bdpy-ML (2.5 mg/kg) and fluorescence levels were measured in blood cells and plasma, as well as splenocytes, at several time points post-injection using the above-described approaches. In agreement with our in vitro data (Figure 3B), Bdpy-ML was relatively better internalized by monocytes than T cells, 4 h post-injection (hpi) (Figures 4A,B). After 24 h, Bdpy-ML was almost cleared from T cells (Figure 4B) while persisting up to 72 h in monocytes (Figure 4A). Profiles of Bdpy-ML incorporation were very similar in cells isolated from blood and spleen after 4 hpi, suggesting an efficient distribution of blood-contained Bdpy-ML to this organ. We estimated plasma concentrations of circulating Bdpy-ML with the approach described in Figure 3, and a standard curve made with serial dilutions of Bdpy-ML in mouse plasma (Supplementary Figure S6). At 4 hpi, plasma levels of Bdpy-ML were highly variable with mean values estimated at 238 ng/ml ± 128 (Figure 4C), which corresponded to 0.78% of injected Bdpy-ML (Mean mouse weight: 18.4 mg ± 0.6, estimated blood volume 77–80 ml/kg (Mitruka and Rawnsley, 1981)). Plasma concentrations of Bdpy-ML then decreased overtime to stabilize at 42 ng/ml ± 13 after 72 h. Notably, Bdpy-ML was still detected 7 days post-injection (Figure 4C). In line with our findings in the zebrafish (Colucci-Guyon et al., 2020), data obtained in intravenously injected mice support a rapid and almost complete diffusion of blood contained mycolactone into body organs. The long persistence of low levels of Bdpy-ML in plasma is consistent with a slow release of tissue-contained mycolactone in the blood compartment, where our in vitro data suggest that it largely localizes in the plasma.

Our in vitro and in vivo data revealed a differential incorporation of Bdpy-ML by monocytes and lymphocytes. We hypothesized that this difference could result from a relatively higher concentration of Sec61 in monocytes. To test this hypothesis, we compared the incorporation of Bdpy-ML in Jurkat T cells transfected with either the wild type (WT Sec61α) or the mycolactone-blind R66G mutant of Sec61α (Baron et al., 2016). We previously demonstrated that over-expression of R66G Sec61α rescues the cytokine responses of mycolactone-treated T cells (Baron et al., 2016). Compared to untransfected cells, transfected cells showed higher and comparable expression of the WT or the mutant form of Sec6α as (Figure 5A). Bdpy-ML uptake was comparable in untransfected cells and cells over-expressing WT or R66G Sec61α (Figure 5B), suggesting that mycolactone uptake is independent of Sec61α level.

Addition of equimolar or higher concentrations of natural mycolactone induced a slight yet significant decrease in Bdpy-ML uptake by THP-1 cells (Supplementary Figure S7), suggesting that mycolactone uptake does not rely exclusively on passive diffusion. The scavenger receptor SR-B1 is an HDL receptor whose primary role is to selectively take up HDL-derived cholesteryl esters into cells and tissues by a non-endocytic mechanism. Since mycolactone associates with HDL, we hypothesized that SR-B1 may contribute to its cellular import. In support of this view, blood cell subsets incorporating the highest levels of Bdpy-ML (Figure 3) were those expressing the highest levels of SR-B1 (Figure 6A). To assess SR-B1 contribution to cellular import of mycolactone, we used a panel of pharmacological inhibitors (BLT-1, BLT-4, and 5) that specifically block SR-B1-dependent lipid transport from HDL (Nieland et al., 2002; Nieland et al., 2008). We measured the uptake of Bdpy-ML into primary human macrophages after 1 h of incubation. In the presence of the inhibitors, Bdpy-ML uptake showed variable and partial inhibition, with BLT-4 having the greatest effect (Figure 6B). BLT-4 also significantly reduced mycolactone uptake by THP-1 cells after 1 h (Figure 6C).

To test the contribution of scavenger receptors SR-B1 to mycolactone uptake by an independent approach, THP-1 cells were transfected with anti-SR-B1 or unrelated siRNAs. Forty-eight hours post-transfection, SR-B1 expression was down-regulated in 72% of cells (Supplementary Figure S8). In these cells, anti-SR-B1 siRNAs reduced SR-B1 expression by 92% (±5%) (Figure 6D). We then compared cells transfected with anti-SR-B1 or unrelated siRNAs for Bdpy-ML incorporation after 1 and 24 h (Figure 6E). Compared to control cells and cells transfected with control siRNAs, cells transfected with anti-SR-B1 siRNAs had a 10% reduction in ability to internalize Bdpy-ML after 1h, which persisted to a lesser extent after 24 h (Figure 6E). Collectively, the data reported in Figure 6 suggest that SR-B1 contributes to the cellular uptake of mycolactone.