Single cell fluorescence imaging of glycan uptake by intestinal bacteria

Microbes in the intestines of mammals degrade dietary glycans for energy and growth. The pathways required for polysaccharide utilization are functionally diverse; moreover, they are unequally dispersed between bacterial genomes. Hence, assigning metabolic phenotypes to genotypes remains a challenge in microbiome research. Here we demonstrate that glycan uptake in gut bacteria can be visualized with fluorescent glycan conjugates (FGCs) using epifluorescence microscopy. Yeast α-mannan and rhamnogalacturonan-II, two structurally distinct glycans from the cell walls of yeast and plants, respectively, were fluorescently labeled and fed to Bacteroides thetaiotaomicron VPI-5482. Wild-type cells rapidly consumed the FGCs and became fluorescent; whereas, strains that had deleted pathways for glycan degradation and transport were non-fluorescent. Uptake of FGCs, therefore, is direct evidence of genetic function and provides a direct method to assess specific glycan metabolism in intestinal bacteria at the single cell level.

the host intestine, improved research methods to establish metabolic abilities present within the microbiome and to explore how microbes respond to dietary interventions are urgently needed. More specifically, although biochemical characterization of PULs from cultivable isolates has made remarkable progress in defining the molecular basis of PUL function and the broad scale diversity of PULs can be assessed with metagenomics, the field is lacking methods that rapidly assign metabolic phenotypes to genotypes on the single cell level within a microbial community.
Marine bacteria have recently been shown to selectively import fluorescently labeled polysaccharides into their periplasm [8]. In these experiments substrate-based staining was combined with single cell identification by fluorescence in situ hybridization. Here we chose the gut bacterium Bacteroides thetaiotaomicron VPI-5482 (hereafter B. theta) as a model organism to ascertain whether glycans found within human diets can be fluorescently labeled and used to visualize selective glycan metabolism. B. theta consumes yeast αmannan (YM) and rhamnogalacturonan-II (RGII) (Fig. 1a, b) with proteins that are encoded by specifically adapted PULs (SI Fig. 1) [9,10]. Remarkably, despite RGII's extensive structural complexity (SI Fig. 1D) B. theta was shown to cleave all but one of the 21 distinct linkages and utilize all but four of the liberated monosaccharides for energy [10]. These complex glycans are utilized by B. theta through a "selfish mechanism" [9], a feeding strategy that is thought to limit the distribution of "public goods" [3,11] to other members of the community. A hallmark of the selfish uptake mechanism is that complex products generated by extracellular CAZymes are selectively imported and the majority of saccharification occurs within the confines of the periplasm. This feeding strategy underpins that Bacteroidetes SusC/D-like TonBdependent transporters can accommodate large, energy-rich polysaccharides for cellular metabolism.
To determine if selfish metabolism in B. theta could be leveraged for imaging, YM and RGII were conjugated at free diols with 6-aminofluorescein (FLA) using a cyanogenbromide activation chemistry [12,13]. The products of these reaction are fluorescent glycan conjugates (FGCs) FLA-YM and FLA-RGII. B. theta was cultured on unlabeled YM and RGII to metabolically activate the cells, incubated with FLA-YM or FLA-RGII, and then visualized by epifluorescence and super-resolution microscopy. Cells treated with FGCs displayed intracellular accumulation of fluorescent signal (Fig. 1c, d); whereas, cells incubated with unlabeled glycan did not display any fluorescence (SI Fig. 2). We observed that FGC uptake was time-dependent, with some B. theta cells showing rapid incorporation of fluorescent signal (after minutes) and peaking at 24 and 72 h for FLA-YM and FLA-RGII, respectively (SI Figs. 2 and 3). These observations highlight that uptake rates of FGCs can differentiate between PULs, which may reflect the structural complexity of the imported glycan; and FGCs represent potent tools to detect and study differential mechanisms of glycan uptake and metabolism by Bacteroidetes. The differences in uptake between FLA-YM and FLA-RGII were somewhat surprising as the cells were not actively dividing at this stage of the growth, and previously, xylan and laminarin displayed similar uptake kinetics in marine Bacteroidetes [8]. Therefore, this effect may result from the unique, highly branched structures of YM and RGII (Fig. 1b, d) (SI Fig. 1B, D). Alternatively, stochastic additions of FLA conjugations may result in distinct labeling density and positional chemistries, which could effect transport efficiency [12].
Although experiments were carried out with pure cultures, we observed heterogeneity in the extent of cell fluorescence for both FLA-YM and FLA-RGII (Fig. 1c, d). Some cells were highly fluorescent while others did not show any signal. This heterogeneity might indicate that uptake efficiency differs between cells within a population, which has been frequently observed for microbes [14]. In this light, FGCs appear uniquely suited to query heterogeneity of carbohydrate metabolism between individual cells. Overlays of the fluorescent signals for cellular DNA and the FGCs revealed that FGCs concentrated around the proximity of the cell forming a halo. Additional staining with Nile Red, a dye specific for the membrane lipid bilayer, demonstrated that green and red fluorescence colocalized suggesting that the glycan accumulates within the periplasm (Fig. 2a, b). Previously, accumulation of FGCs in the periplasm was reported in marine Bacteroidetes [8] and is also supported by stimulated emission depletion (STED) microscopy ( Fig. 1e) and enzyme protection assays (SI Fig. 4). These patterns of FLA-YM visualization are consistent with the selfish mode of glycan metabolism previously described for YM consumption by B. theta (Fig. 1f, [9]).
Next, we investigated to what extent FGC uptake relies on the presence of specific PULs. Mutant B. theta strains with targeted PUL deletions for YM (BtΔMAN1/2/3) [9] and the major RGII PUL (BtΔRGII) [10] were incubated with FLA-YM and FLA-RGII, respectively (SI Fig. 2). In both cases, PUL removal ablated FGC uptake and fluorescence. This could also be verified, for YM, by FLA signal quantification, using flow cytometry (Fig. 2c), which showed that there was a significant difference in signal intensity between the mutant and WT strains. These results confirmed that glycans are modified and/or transported by proteins encoded within the PULs [7,15]. In the BtΔRGII strain, there was low-level residual staining for RGII substrates (SI Fig. 2B), which suggests the intact orphan SusC/ D-like wine-RGII-specific transporter, BT1682/BT1683, can still import FLA-RGII at basal levels and that it accommodates minimally processed forms of RGII (SI Fig. 1B [10]). Therefore, FGCs can also be used to study preferential PUL hierarchy for engineered systems with multiple loci.

Conclusion
Glycan utilization by intestinal Bacteroidetes is critical for digestion of dietary carbohydrates. This host-symbiont interaction is facilitated by cognate PUL systems that endow bacterial strains for consumption of specific glycans. Despite the remarkable progress that has been made in cataloging microbiome composition from diverse sources [16] and defining the molecular basis of PUL function [2], the field still is lacking methods to rapidly assign metabolic phenotypes to genotypes in microbial communities at the single cell level. FGCs provide tools for rapid and selective study of glycanmicrobe interactions, and will assist in deciphering the mechanisms driving microbiome responses to dietary interventions [17]. and stimulated emission depletion microscopy image (STED, right). f The illustration represents the selfish mode of yeast mannan metabolism by B. theta, adapted from [9]. Following limited cleavage by surface exposed GH76 enzymes, product uptake is mediated by SusC/ D-like proteins. The majority of saccharification occurs in the periplasm and is mediated by carbohydrate active enzymes spedific for the hydrolysis of α-mannosyl linkages (e.g. GH92, GH76, and GH125). During this process FLA is transported into the periplasm, where it accumulates and can be visualized by super-resolution microscopy. Scale bar represents 1 µm

Generation of FGCs
Mannan from Saccharomyces cerevisiae (YM) prepared by alkaline extraction was purchased from Sigma (St. Louis, MO, USA; M7504). Wine RGII, purified as described previously [18]. Activation and labeling of YM and RGII was carried out as described in ref. [

Cell fixation
Twenty microliters of each growth cultures were aliquoted into sterile 2 mL screw cap tubes from the cells and resuspended in 2 mL 2× MM (0 h). FLA-labeled and unlabeled substrates were then added to the activated cells. 40 µL samples of each treatment were taken at three time points: 1 min, 24, and 72 h. The samples were centrifuged (rcf: 1500 × g) for 10 min, and the supernatants removed. To fix the cells, pellets were resuspended in 1 mL formaldehyde (Sigma; product no. F8775), diluted to 1% with 1× phosphate-buffered saline (PBS; pH 7.4): 137 mM NaCl, 2.7 mM KCl, 10 mM Na 2 HPO 4 , 1.8 mM KH 2 PO 4 , and incubated overnight at 4°C. The next day, cells were centrifuged (rcf: 1500 × g for 10 min), pellets washed in 1× PBS, and centrifuged a final time. The supernatant was removed and the pelleted cells were stored at 4°C until further analysis.

Epifluorescence microscopy
For epifluorescence microscopy fixed B. theta cells were filtered onto 25 mm polycarbonate filters (0.2 µm pore size) using a gentle vacuum (<200 mbar). The cells were then counterstained with DAPI and mounted using a Citiflour (Electron Microscopy Sciences, USA) and Vector Shield (Vector Laboratories, Germany) mounting solution (4:1). The cells were visualized on a Zeiss Axioskop 2 motplus fluorescent microscope using the Axiovision software (Zeiss, Germany).

Super-resolution microscopy
For visualization, 30 μL of fixed cell culture was heat fixed at 40°C to poly-D-lysine-coated coverslips (#1.5, thickness 0.17 mm). Subsequently, additional salts from the medium are removed by washing the coverslip in MQ.
For STED and confocal laser scanning microscopy (CLSM) cells were mounted using Prolong Diamond (Thermo Fischer, Germany). The cells were visualized on an Abberior Instrument (Aberrior Instruments GmbH, Germany) using, for FLA-YM detection, a 485 nm excitation laser, 525/50 nm detector and 595 nm STED laser were used, with a pinhole of 70 µm, a pixel size of 20 nm, dwell time 20 µs and nine line accumulation.

Flow cytometry and fluorescence quantification
Cell fluorescence due to FLA-substrate uptake was quantified in all growth cultures using an Accuri C6 flow cytometer (BD Accuri Cytometers) as described previously [8]. The 8-peak and 6-peak validation bead suspensions (Spherotech, Lake Forest, IL, USA) were used as internal references. The FCM output was analyzed using FlowJo v10.4.2 (Tree Star, USA). The FCM files were imported into FlowJo, and the main population (representing single cells) was gated (green) in the FL1-H and SSC-H view. The sample statistics (counts, mean and standard deviation) and raw FL1-H results for each event in the gate (n = 10,000) were exported and analyzed using the R software. Statistical differences were calculated by Welch's t-test in R. Additionally the percentage of total events which were above (Q2) or below (Q1) the minimum FL1-H values, gated according to controls events FL1-H was calculated.
Growth profiling of B. theta strains Five milliliters of BHI media was inoculated from frozen stocks of B. theta, BtΔMAN1/2/3, and BtΔRGII. During exponential phase, cells were centrifuged (rcf: 4700 × g for 5 min) and washed with 2× MM. The cells were diluted to OD 600nm~0 .15. Falcon 300 µL flat-bottomed 96-well microtiter plates were used for growth curves. Each well was filled with 100 µL filter sterilized substrate (1% glucose, 1% mannose, 1% YM, 1% RG-II), as well as 100 µL of inoculum (n = 6), to get a final concentration of 0.5% substrate and 1× MM. 100 µL of sterile water was added to wells with 100 µL culture as negative controls. Positive controls consisted of 180 µL of BHI and 20 µL culture. Breathe-Easy gas-permeable polyurethane membranes (Sigma-Aldrich Z380059) were used to seal the multiwell plates. An Eon microplate reader (Biotek) with Gen5 software (BioTek) was used to measure and record absorbance (600 nm) every 30 min for 48 h. Data was analyzed using GraphPad Prism software.