Visualizing germination of microbiota endospores in the mammalian gut

Abstract

Transmission of bacterial endospores between the environment and people and the following germination in vivo play critical roles in both the deadly infections of some bacterial pathogens and the stabilization of the commensal microbiotas in humans. Our knowledge about the germination process of different bacteria in the mammalian gut, however, is still very limited due to the lack of suitable tools to visually monitor this process. We proposed a two-step labeling strategy that can image and quantify the endospores' germination in the recipient's intestines. Endospores collected from donor's gut microbiota were first labeled with fluorescein isothiocyanate and transplanted to mice via gavage. The recipient mice were then administered with Cyanine5-tagged D-amino acid to label all the viable bacteria, including the germinated endospores, in their intestines in situ. The germinated donor endospores could be distinguished by presenting two types of fluorescent signals simultaneously. The integrative use of cell-sorting, 16S rDNA sequencing, and fluorescence in situ hybridization (FISH) staining of the two-colored bacteria unveiled the taxonomic information of the donor endospores that germinated in the recipient's gut. Using this strategy, we investigated effects of different germinants and pre-treatment interventions on their germination, and found that germination of different commensal bacterial genera was distinctly affected by various types of germinants. This two-color labeling strategy shows its potential as a versatile tool for visually monitoring endospore germination in the hosts and screening for new interventions to improve endospore-based therapeutics.

Keywords: Bacterial endospore; FDAA; germination; in vivo metabolic labeling; two-color fluorescence imaging.

Figure 1.

Schematic illustration of the two-step labeling strategy for monitoring endospores’ germination in the mammalian gut. Donor endospores collected from mice cecal microbiotas, which had been treated with 75% ethanol and heating, were labeled with FITC and then given to the recipient mouse by gavage. Cy5ADA probe was simultaneously administered by intraperitoneal injection for gut microbiota labeling. The recipient’s gut microbiota was collected, and analyzed by confocal fluorescence microscopy and flow cytometry. The bacteria co-labeled by FITC and Cy5ADA probes were the germinated endospores, which were then sorted and analyzed by 16S rDNA sequencing. Visual validation of the germinated endospores was conducted by FISH-staining.

Figure 2.

Fluorescence analyses of the bacterial endospores labeled by FADA, FITC and 5-FAM SE. (a) Confocal fluorescence imaging of B. subtilis endospores (indicated by arrow) labeled by FADA in vitro. DIC, differential interference contrast. Scale bar, 5 μm. (b) Confocal fluorescence imaging of cecal endospores labeled by FADA in vivo. Scale bar, 5 μm. Dashed lines in florescence channel indicate the endospores’ positions. (c) Confocal fluorescence imaging of B. subtilis endospores (indicated by arrow) stained by FITC (two graphs on the left) and 5-FAM SE (two graphs on the right) in vitro. Scale bar, 5 μm. (d) Statistical analysis of the labeling coverage for B. subtilis endospores, together with bacteria stained by FITC and 5-FAM SE in vitro, Mean ± s.d. are presented for n = 3. (e) Confocal fluorescence imaging of cecal endospores (indicated by arrow) stained by FITC (two graphs on the left) and 5-FAM SE (two graphs on the right) in vitro. Scale bar, 5 μm. (f) Statistical analysis of the labeling coverage for cecal endospores, together microbiota stained by FITC and 5-FAM SE in vitro, respectively. Mean ± s.d. are presented for n = 3. Representative images of germinated endospores from at least three independent experiments are presented.

Figure 3.

Imaging of bacterial sporulation in the gut recorded via sequential labeling. (a) Confocal fluorescence imaging of the cecal endospores (arrows) sequentially labeled with FITC and TRITC in vivo. Scale bar, 5 μm. (b) Zoomed views of the endospores indicated from the merged image above. Scale bars, 2 μm. (c) Two-color fluorescence imaging of a sub-terminal endospore. Scale bar, 2 μm. Representative images of germinated endospores from at least three independent experiments are presented.

Figure 4.

Evaluation of endospores’ germination in vitro by two-step tagging with FITC and Cy5ADA probes. (a) Schematic illustration of the assessment of endospores’ germination in vitro. (b) Two-color fluorescence imaging of the B. subtilis endospore (arrows) germination in vitro. Scale bar, 5 μm. (c) Statistical analysis of the labeling coverage for germinated B. subtilis endospores. Mean ± s.d. are presented for n = 3. (d) Two-color fluorescence imaging of the cecal endospore (arrows) germination in vitro. Scale bar, 5 μm. (e) Flow cytometry analysis of the germinated cecal endospores. The germinated endospores having both FITC and Cy5ADA labeling signals were sorted by FACS. (f) Statistical analysis of the ratios for germinated cecal endospores. Mean ± s.d. are presented for n = 3. (g) 16S rDNA sequencing analysis of the cecal endospores before and after sorting uncovered that Bacillus was the major germinated bacteria after 12 h of incubation. Representative images of germinated endospores from at least three independent experiments are presented.

Figure 5.

Analyses of endospores’ germination in the gut. (a) Schematic illustration of the procedures for assessing endospores’ germination in the gut. (b) Flow cytometry analysis of the germinated B. subtilis endospores from the recipient mice. The upper right sector indicates the in vivo germinated endospores, which accounts for 7.0% of the recipient mice’s microbiota. (c) Confocal fluorescence imaging of the in vivo germinated B. subtilis endospores. Scale bar, 2 μm. (d) Flow cytometry analysis of the germinated cecal endospores from the recipient mice. Cells distributed at the upper right corner indicates the in vivo germinated endospores, which accounts for 2.73% of the recipient mice’s microbiota. (e) Confocal fluorescence and DIC imaging of the germinated endospores (arrows) in vivo. Scale bar, 5 μm. (f) Representative fluorescence images of in vivo germinated endospores having different morphologies. Scale bars, 2 μm. (g) Confocal fluorescence imaging of germinated SFB endospores (arrow) in the gut. Scale bar, 2 μm. Representative images of germinated endospores from at least three independent experiments are presented.

Figure 6.

Evaluation of the effects of germinants and aging-time on cecal endospores’ germination in the gut. (a) Statistical analysis of the ratios of germinated cecal endospores in the gut supplied with different germinants. Mean ± s.d. are presented for n = 4. (b) Statistical analysis of the ratios of germinated cecal endospores in the gut after aging for 12, 24 and 48 h. Mean ± s.d. are presented for n = 4. (c) Heat map showing the enrichment of different bacteria after cell sorting. The post- to pre-sorting ratios (in log₂ scale) of different genera’s relative abundances were shown; blank spots mean no enrichments. (d) Confocal fluorescence imaging of the dually labeled and FISH-stained spores belonging to four genera. The germinated cecal spores in mice received two-step labeling of FITC and Cy5ADA were stained by different FISH probes (blue) targeting corresponding genera. Scale bars, 2 µm. Representative images of germinated endospores from at least three independent experiments are presented.