Bioorthogonal Metabolic Labeling of the Virulence Factor Phenolic Glycolipid in Mycobacteria

Surface lipids on pathogenic mycobacteria modulate infection outcomes by regulating host immune responses. Phenolic glycolipid (PGL) is a host-modulating surface lipid that varies among clinical Mycobacterium tuberculosis strains. PGL is also found in Mycobacterium marinum, where it promotes infection of zebrafish through effects on the innate immune system. Given the important role this lipid plays in the host–pathogen relationship, tools for profiling its abundance, spatial distribution, and dynamics are needed. Here, we report a strategy for imaging PGL in live mycobacteria using bioorthogonal metabolic labeling. We functionalized the PGL precursor p-hydroxybenzoic acid (pHB) with an azide group (3-azido pHB). When fed to mycobacteria, 3-azido pHB was incorporated into the cell surface, which could then be visualized via the bioorthogonal conjugation of a fluorescent probe. We confirmed that 3-azido pHB incorporates into PGL using mass spectrometry methods and demonstrated selectivity for PGL-producing M. marinum and M. tuberculosis strains. Finally, we applied this metabolic labeling strategy to study the dynamics of PGL within the mycobacterial membrane. This new tool enables visualization of PGL that may facilitate studies of mycobacterial pathogenesis.


Figure S2
. TLC plates of crude lipids extracted from M. marinum cultured with 2-or 3azido pHB.200 µg of crude lipids and 20 µg of purified PGL were loaded onto TLC plates and developed using 8:2 toluene:acetone.Plates were stained in 0.2% anthrone in H2SO4.The anthrone stain is used to identify compounds with sugars which will be revealed as a royal blue color.The iodine stain is a general stain used for total lipid identification.TLCs were imaged on a ChemiDoc MP imaging system using 590 nm wavelength.

Supplementary methods
General methods for synthesis.Materials and reagents were obtained from commercial sources without further purification unless otherwise noted.Analytical TLC was performed on Millipore Sigma glass-backed Silica gel 60 F254 plates.Prep TLC was performed on Millipore Sigma glass-backed 2 mm thick Silica gel 60 F254 plates with concentration zone.TLC was analyzed using iodine adhered to silica and 0.2% anthrone in H2SO4 stain.
General procedure for synthesis of 3-and 2-azido pHB.WARNING: the synthesis of azide compounds is dangerous and can result in explosions and/or production of toxic gases.It is recommended to synthesize these molecules at small scale (i.e. 1 mmol) behind a blast shield.Proceed with caution.Aniline (2-or 3-amino pHB) starting materials (0.150 g, 1 mmol) were suspended in 2.5 mL of water in a round bottom flask.The round bottom flask was submerged in an ice bath and conc.HCl (0.25 mL) was added slowly.The flask was stirred for 10 minutes.Next, NaNO2 (0.076 g, 1 mmol) was added to the flask and stirred for 10 minutes.Then NaN3 (0.085 g, 1.3 mmol) was added to the flask and stirred for 30 minutes.After 30 minutes the flask was removed from the ice bath and warmed to room temperature.The reaction was monitored by normal-phase TLC using a 1:1 acetone:hexanes mobile phase.After ~2 hrs when the starting material was consumed, an additional 10 mL of water was added to the flask and the product was extracted with 3 x 10 mL of ethyl acetate.The organic layer was then washed with brine (saturated NaCl solution) and dried using magnesium sulfate.The crude product was purified by silica gel chromatography using a gradient solvent system of acetone:hexanes.The product was achieved as an off-white/yellow solid.Yield of 2-azido pHB: 110 mg, 65%.Yield of 3azido pHB: 0.131 g, 75%.
FigureS3.M. marinum cultured in various concentrations of pHB followed by treatment with DBCO-647 and analyzed by flow cytometry.General procedure for metabolic labeling was followed.Flow cytometry analysis represents three independent replicates.Relative MFI is normalized to DMSO control.Statistical analysis was performed using a one-way analysis of variance (ANOVA) followed by a Dunnett's multiple comparisons test.Significance is represented as follows: *p £ 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, and ns (not significant) for p > 0.05.

Figure S4 .Figure S5 .
Figure S4.Crude lipid extracts from PGL-deficient M. smegmatis, PGL-producing WT M. marinum, and PGL-deficient M. marinum mutants indicate presence or absence of PGL.Purified PGL from M. marinum was used as a standard.Crude lipid extracts (100 µg) and purified PGL-M.marinum (5 µg) were loaded onto a silica gel 60 TLC plate and developed in 8:2 toluene:acetone.Compounds with sugars are visualized as a blue color in the anthrone stain.The iodine stain is a general stain used to visualize total lipids.TLCs were imaged on a ChemiDoc MP imaging system using 590 nm wavelength.
1 H and 13 C NMR spectra were obtained using a Brucker Neo-500 MHz instrument with chemical shifts in ppm (d) referenced to solvent peaks.Flow cytometry analysis of M. marinum was performed on a Novocyte Penteon flow cytometer at the Stanford shared FACS facility.Flow cytometry analysis of M. tuberculosis BD FACSCalibur cytometer at UT Southwestern.High resolution mass spectrometry characterization of azide compounds was performed on an Thermo Orbitrap Fusion nano LC/MS instrument at the Stanford Mass Spectrometry facility.FRAP was performed at the Stanford microscopy facility on an inverted Zeiss 780 multiphoton laser scanning confocal microscope.Lipidomics analysis of crude extracts was performed on a reversed-phase Agilent 1260 series HPLC system and an Agilent 6546 Accurate-Mass Q-TOF mass spectrometer.