Bioorthogonal Correlative Light-Electron Microscopy of Mycobacterium tuberculosis in Macrophages Reveals the Effect of Antituberculosis Drugs on Subcellular Bacterial Distribution

Abstract

Bioorthogonal correlative light-electron microscopy (B-CLEM) can give a detailed overview of multicomponent biological systems. It can provide information on the ultrastructural context of bioorthogonal handles and other fluorescent signals, as well as information about subcellular organization. We have here applied B-CLEM to the study of the intracellular pathogen Mycobacterium tuberculosis ( Mtb ) by generating a triply labeled Mtb through combined metabolic labeling of the cell wall and the proteome of a DsRed-expressing Mtb strain. Study of this pathogen in a B-CLEM setting was used to provide information about the intracellular distribution of the pathogen, as well as its in situ response to various clinical antibiotics, supported by flow cytometric analysis of the bacteria, after recovery from the host cell ( ex cellula ). The RNA polymerase-targeting drug rifampicin displayed the most prominent effect on subcellular distribution, suggesting the most direct effect on pathogenicity and/or viability, while the cell wall synthesis-targeting drugs isoniazid and ethambutol effectively rescued bacterial division-induced loss of metabolic labels. The three drugs combined did not give a more pronounced effect but rather an intermediate response, whereas gentamicin displayed a surprisingly strong additive effect on subcellular distribution.

Application of a Highly Selective Cathepsin S Two-step Activity-Based Probe in Multicolor Bio-Orthogonal Correlative Light-Electron Microscopy

“…Furthermore, BMDCs were given homopropargylglycine labeled E. coli (Hpg- E. coli ) for 3 h prior to labeling with the aforementioned ABP-cocktail ( Figure 5B ). This would allow us to assess whether probe 5 could also be imaged in a ‘dual-click’ approach with the potential to determine the distribution of Cat S activity in response to, and relative to this bacterial stimulus ( Bakkum et al, 2020 ). We noted a high variance in cathepsin activity between BMDCs, which might follow from the fact that GM-CSF-generated BMDCs form a highly heterogeneous population of monocytic cells ( Helft et al, 2015 ).…”

“…When all slides were collected, fixed cells were washed with PBS and PBS containing 20 mM glycine (PBS/glycine). Membrane permeabilization was achieved by incubating with 0.1% Triton-X100 for 20 min, followed by a PBS wash. Dual copper-catalyzed Huisgen cycloaddition (ccHc) reaction was performed sequentially with intermediate PBS washes as described previously ( Bakkum et al, 2020 ), with click cocktail 1 ((0.1 M HEPES, pH 7.3, 1 mM CuSO 4 , 10 mM sodium ascorbate, 1 mM THPTA ligand, 10 mM amino-guanidine, 5 µM AFDye555-azide (Click Chemistry Tools)) for 30 min to label Hpg- E. coli , followed by click cocktail 2 ((0.1 M HEPES, pH 7.3, 1 mM CuSO 4 , 10 mM sodium ascorbate, 1 mM THPTA ligand, 10 mM amino-guanidine, 5 µM AF488-alkyne (Thermo Fisher)) for 30 min to label Cat S. Cells were washed with PBS and blocked with PBS containing 1% BSA for 30 min to assist in removing non-specifically bound fluorophores. After two more PBS washes, the nuclei were counterstained with 2 μg/ml Hoechst 33342 for 5 min and washed once more with PBS, prior to imaging in glycerol/DABCO solution on an Andor Dragonfly 505 Spinning Disk Confocal (Oxford Instruments), containing an 8-line integrated laser engine.…”

“…The sample preparation for bio-orthogonal correlative light-electron microscopy (B-CLEM) and bio-orthogonal labeling was performed as previously described ( Bakkum et al, 2020 ). Briefly, following infection and post-infection incubation with the cathepsin probes, BMDCs were washed three times with conditioned medium and once with PBS.…”

“…These sections were then imaged on a FEI Tecnai 12 BioTwin TEM System (FEI Technologies) at 120 kV acceleration voltage. Correlation of FM and TEM images was performed in Adobe Photoshop CC 2020 (Adobe Systems) as described previously ( Bakkum et al, 2020 ).…”

Metabolic labeling probes for interrogation of the host–pathogen interaction

“…4 for an example image). 125 Approaches that allow expansion of the genetic code of pathogens have also become powerful tools to study hostpathogen interactions. For example, Ngo et al 126 devised a new BONCAT-like strategy that involved genetically modifying the cell type of interest to enable incorporation of NCAA for this cell type only.…”