Illuminating vital surface molecules of symbionts in health and disease

Abstract

The immunomodulatory surface molecules of commensal and pathogenic bacteria are critical to microorganisms' survival and the host's response^1,2. Recent studies have highlighted the unique and important responses elicited by commensal-derived surface macromolecules^3-5. However, the technology available to track these molecules in host cells and tissues remains primitive. We report, here, an interdisciplinary approach that uses metabolic labelling combined with bioorthogonal click chemistry (that is, reactions performed in living organisms)⁶ to specifically tag up to three prominent surface immunomodulatory macromolecules-peptidoglycan, lipopolysaccharide and capsular polysaccharide-either simultaneously or individually in live anaerobic commensal bacteria. Importantly, the peptidoglycan labelling enables, for the first time, the specific labelling of live endogenous, anaerobic bacteria within the mammalian host. This approach has allowed us to image and track the path of labelled surface molecules from live, luminal bacteria into specific intestinal immune cells in the living murine host during health and disease. The chemical labelling of three specific macromolecules within a live organism offers the potential for in-depth visualization of host-pathogen interactions.

Figure 1. Fluorescent D-amino acid labels PGN in commensal bacteria

(a) Schematic of metabolic labeling of the peptidoglycan of commensal bacteria with fluorescent D-amino acid derivatives. The addition of the fluorescent coumarin moiety is highlighted in blue. (b) Images of various commensal bacteria grown overnight in medium with a fluorescent amino acid: HADA or (as a negative control) HALA. Scale bars, 10 μm. (c) Confocal image and expanded region (right) of frozen, fixed tissue slice of mouse colon 1 h after injection of HADA-labeled E. faecalis. AcOr, acridine orange. Scale bar: left, 50 μm; right, 10 μm. (d) Confocal images of Carnoy’s-fixed, paraffin-embedded tissue slices from mice 2 h (small intestine [SI], left) or 4 h (colon, right) after oral gavage with HADA or (as a negative control) HALA. Scale bar, 20 μm. (e) Flow cytometry histogram plots of luminal contents from mice given HADA or HALA orally demonstrate decay of fluorescent signal over time. (f) Confocal images of Carnoy’s-fixed, paraffin-embedded tissue slices from mice given HADA orally. The mice were sacrificed, and tissue was fixed at the specified times after oral gavage. The images show significant retention of fluorescent signal over 24 h. MUC2, mucin 2. Scale bar, 50 μm. Data in c–f are representative of at least three independent experiments.

Figure 2. Use of HADA-labeled PGN to track live commensals in the host

(a) Representative live images from intravital two-photon microscopy of the murine colon 16 h after administration of HADA. Mice were anesthetized and intestinal loops were surgically extracted and mounted for imaging. Purple and green represent tissue autofluorescence (see Supplementary Videos 1 and 2). Scale bar, 10 μm. (b) Numbers of colonic lymphocytes bearing a HADA⁺ signal 4 h after oral gavage of HADA. Data are mean ± s.e.m. values for biological replicates of n=5 mice (P values calculated by paired Student’s t-test). (c) Representative confocal images of live CD11c⁺ (top) or CD11c⁺CX₃CR1⁺ (bottom) mononuclear phagocytes (sorted for a HADA⁺ signal) bearing HADA-labeled bacteria/PGN. Scale bar, 5 μm. (d) Representative images of Carnoy’s-fixed, paraffin-embedded tissue slices obtained 4 h after oral gavage of HADA from mice given normal drinking water (left) or drinking water containing 3% DSS (right) for the preceding 4 days. The damaged mucosal layer and breach with endogenous bacteria are evident in the DSS-induced colitic colons. Bars show mucus thickness is roughly 50 μm in healthy mice while arrows point at bacteria at the epithelial layer in DSS-treated mouse colons. Scale bar, 20 μm. (e) Percentages of the parent subpopulation of colonic lymphocytes bearing a HADA⁺ signal 4 h after oral gavage with HADA in healthy mice and mice with DSS-induced colitis. Data are mean ± s.e.m. values for biological replicates of n=7 mice (P values calculated by paired Student’s t-test). Data in a–e are representative of at least three independent experiments.

Figure 3. Simultaneous labeling of three cell-surface molecules in commensal bacteria

(a) Schematic of the metabolic labeling approach using the non-natural metabolites KDOAz (LPS), GalCCP (CPS), and HADA (PGN) to simultaneously label three cell-surface macromolecules in the commensal bacterium B. vulgatus. The reactive or fluorescent moieties are highlighted in blue. DIBAC-TAMRA, dibenzo-aza-cyclooctyne-carboxytetramethylrhodamine; Cy5, cyanine-5. (b) Images of B. vulgatus grown overnight in medium with KDOAz, GalCCP, and HADA (left) or, as a negative control, with GalNAc and HALA (right) and reacted with DIBAC-TAMRA and tetrazine-Cy5. Scale bar, 10 μm. (c) Confocal images of fixed bone marrow–derived macrophages 1 h after inoculation of three-component-labeled B. vulgatus. Rhod, rhodamine green; DRAQ5, red DNA dye. Scale bar, 5 μm. (d) Zoomed confocal image of bone marrow–derived macrophages bearing three-component-labeled B. vulgatus. Scale bar, 5 μm. (e) Representative images of J775A.1 murine macrophages 4 h and 24 h after inoculation of three-component-labeled B. vulgatus. Arrows highlight where PGN is seen separate from colocalized CPS/LPS. Scale bar, 10 μm. Data in b–e are representative of at least three independent experiments with n=3 technical replicates.

Figure 4. Simultaneous labeling of three cell-surface molecules in commensal bacteria

(a) Confocal image of Carnoy’s-fixed, paraffin-embedded tissue slices obtained from mice 1 h after intestinal injection of labeled B. vulgatus. Zoomed image (lower left) is shown to highlight interaction with the host epithelial layer. Scale bar, 10 μm. (b) Representative live images from intravital two-photon microscopy of the murine small intestine after direct inoculation with three-component-labeled B. vulgatus. Purple represents lectin staining of the epithelium with WGA-633 (see Supplementary Videos 3 and 4). Scale bar, 10 μm. Data are representative of at least three independent experiments with n = 3 mice per experiment.