Correlative light electron ion microscopy reveals in vivo localisation of bedaquiline in Mycobacterium tuberculosis-infected lungs

Abstract

Correlative light, electron, and ion microscopy (CLEIM) offers huge potential to track the intracellular fate of antibiotics, with organelle-level resolution. However, a correlative approach that enables subcellular antibiotic visualisation in pathogen-infected tissue is lacking. Here, we developed correlative light, electron, and ion microscopy in tissue (CLEIMiT) and used it to identify the cell type-specific accumulation of an antibiotic in lung lesions of mice infected with Mycobacterium tuberculosis. Using CLEIMiT, we found that the anti-tuberculosis (TB) drug bedaquiline (BDQ) is localised not only in foamy macrophages in the lungs during infection but also accumulate in polymorphonuclear (PMN) cells.

Fig 1. The CLEIMiT workflow and correlative imaging of BDQ in M. tuberculosis–infected foamy macrophages within lung tissue.

(A) Diagram illustrating the in vivo experimental setting. C3HeB/FeJ mice were infected with M. tuberculosis H37Rv expressing E2-Crimson (Mtb-E2Crimson) by aerosol. After 21 days, infected mice were treated with 25 mg/kg of BDQ or vehicle daily for 5 days via oral gavage. Lungs were removed, fixed with 10% formalin, contrasted, and embedded in LMA then imaged by μCT for sequential vibratome sectioning. (B) Fluorescence microscopy: a tile scan of a tissue section (approximately 100-μm thickness) stained with DAPI (blue) and BODIPY (green), granulomatous lesions are marked with a solid white line to indicate the boundary (scale bar = 1,000 μm). (C) Light microscopy of an ROI: (i) zoomed fluorescence image (white box from Fig 1B), “landmarks” used for downstream location recognition, are indicated by the solid white boundary lines (scale bar = 500 μm). White rectangle shows the ROI for downstream analysis. (ii) A confocal image of the region indicated by the white box above shows an area of strong cellular infiltration and the accumulation of BODIPY (green) positive cells. Cells infected with Mtb-E2Crimson (red) are also visible throughout this region. The same landmarks marked in image (i) are present. White box indicates the selected infected foamy cell. Scale bar = 100 μm. (iii) Zoomed in image showing the selected foamy cell infected with Mtb-E2Crimson (red) for correlative analysis. Scale bar = 5 μm. (D) Electron microscopy of the ROI: (ii) tissue overview (600× magnification) with landmarks present, white box indicates the selected infected foamy cell. Scale bar = 100 μm. (iii) Zoomed in image showing the selected foamy cell infected with Mtb (15,000× magnification). Scale bar = 5 μm. (E) Ion microscopy of the selected cell: Panel shows the individual nanoSIMS images for the following ion signals from left to right; ¹²C¹⁴N^–, ³¹P^–, ³²S^–, and ⁷⁹Br^–. (F) CLEIMiT: Left, a correlated image overlaying fluorescent signal from BODIPY (green) and Mtb-E2Crimson (red) against the SEM image. Right, a correlated image overlaying the ⁷⁹Br⁻and ³¹P^– signals with the SEM image. Center, the corresponding SEM image of the infected foamy cell. Scale bar = 5 μm. BDQ, bedaquiline; CLEIMiT, correlative light, electron, and ion microscopy in tissue; LMA, low melting point agarose; nanoSIMS, nanoscale secondary ion mass spectrometry; ROI, region of interest; SEM, scanning electron microscopy; μCT, micro-computed tomography.

Fig 2. Quantitative distribution of BDQ reveals intracellular distribution of BDQ in foamy macrophages and PMN within granulomatous lesions.

(A) Left: a tiled SEM image of a region of granulomatous lung tissue indicating the zoomed areas. Scale bar = 40 μm. Right: a mosaic of 49 individual ion micrographs showing the quantitative distribution of ion signals in the respective area of tissue. Sulphur ³²S^– is shown in green, bromine ⁷⁹Br⁻in red, and phosphorus ³¹P^– in blue. (B) PMN cells are recruited to the foamy macrophage rich lesions. SEM of zoom 1 (left) and the distribution of secondary ions ³¹P^–, ⁷⁹Br^–, and ³²S^– (right). Overlaid image between SEM (left) and secondary ion (right) is shown in the center. PMN are demarcated by the white boundary line. Scale bar = 5 μm. (C) An infected PMN from panel B showing strong accumulation of BDQ. Overlay between the SEM and secondary ion signals for ⁷⁹Br⁻(red) and ³¹P^– (blue) is depicted in the center. White arrowheads indicate intracellular bacteria. Scale bar = 5 μm. (D) BDQ strongly enriched in PMN that are not only associated with the foamy macrophage-enriched areas. SEM of zoom 2 (left) and the distribution of secondary ions ³¹P^–, ⁷⁹Br^–, and ³²S^– (right). Overlaid image of SEM and secondary ions is depicted at the center. Demarcation of PMN is outlined by the white boundary line. Scale bar = 5 μm. (E) Left panel: quantitative analysis of BDQ associated with Mtb in BDQ treated and untreated mice. Center panel: quantitative analysis of BDQ associated with LD in BDQ treated and untreated mice. Right panel: BDQ association to Mtb inside or outside LD in untreated vs BDQ-treated mice. Data show mean ± standard deviation. t test adjusted for multiple comparisons. ns, nonsignificant; p-value is as shown. At least 140 objects were counted from each treatment condition. Data can be found in S1 Data. AU, arbitrary units; BDQ, bedaquiline; LD, lipid droplet; nanoSIMS, nanoscale secondary ion mass spectrometry; PMN, polymorphonuclear; SEM, scanning electron microscopy.