Elucidation of a sialic acid metabolism pathway in mucus-foraging Ruminococcus gnavus unravels mechanisms of bacterial adaptation to the gut

Abstract

Sialic acid (N-acetylneuraminic acid (Neu5Ac)) is commonly found in the terminal location of colonic mucin glycans where it is a much-coveted nutrient for gut bacteria, including Ruminococcus gnavus. R. gnavus is part of the healthy gut microbiota in humans, but it is disproportionately represented in diseases. There is therefore a need to understand the molecular mechanisms that underpin the adaptation of R. gnavus to the gut. Previous in vitro research has demonstrated that the mucin-glycan-foraging strategy of R. gnavus is strain dependent and is associated with the expression of an intramolecular trans-sialidase, which releases 2,7-anhydro-Neu5Ac, rather than Neu5Ac, from mucins. Here, we unravelled the metabolism pathway of 2,7-anhydro-Neu5Ac in R. gnavus that is underpinned by the exquisite specificity of the sialic transporter for 2,7-anhydro-Neu5Ac and by the action of an oxidoreductase that converts 2,7-anhydro-Neu5Ac into Neu5Ac, which then becomes a substrate of a Neu5Ac-specific aldolase. Having generated an R. gnavus nan-cluster deletion mutant that lost the ability to grow on sialylated substrates, we showed that-in gnotobiotic mice colonized with R. gnavus wild-type (WT) and mutant strains-the fitness of the nan mutant was significantly impaired, with a reduced ability to colonize the mucus layer. Overall, we revealed a unique sialic acid pathway in bacteria that has important implications for the spatial adaptation of mucin-foraging gut symbionts in health and disease.

Figure 1. R. gnavus ATCC 29149 nan operon

a) Diagram depicting the genomic organisation of the nan operon RUMGNA_02701 (putative sialic acid esterase; tan) RUMGNA_02700 (putative YhcH family protein; dark blue), RUMGNA_02699 (predicted transcriptional regulator; purple), RUMGNA_02698 – 02696 (putative sialic acid ABC transporter of the SAT2 family; green), RUMGNA_02695 (putative oxidoreductase, pink), RUMGNA_02694 (RgNanH (Intramolecular trans sialidase), gold), RUMGNA_02693 (NanE (epimerase), blue), RUMGNA_02692 (NanA (aldolase), dark green), RUMGNA_02691 (kinase) (NanK, red). b) qPCR analysis showing fold changes in expression of nan genes when R. gnavus was grown with 3’SL or 2,7-anhydro-Neu5Ac compared to glucose using ΔΔCt calculation. Error represent standard deviation and are based on three biological replicates analysed in triplicate. Statistical significance was determined using a 1-way ANOVA with a Dunnett’s multiple comparison test. NS – no significant change in expression (p > 0.05), * - p 0.05 – 0.01, ** - p 0.01 – 0.001, *** - p < 0.001.

Figure 2. Steady-state fluorescence analysis of ligand binding to RgSBP.

Fluorescence emission spectrum of 0.5 μM RgSBP excited at 297 nm in the presence or absence of a) 2,7-anhydro-Neu5Ac or b) Neu5Ac. The data shown are representative of triplicate readings. c) Titration of 0.5 μM RgSBP with 2,7-anhydro-Neu5Ac. The data represents the mean of triplicate readings. d) Displacement of Neu5Ac with 2,7-anhydro-Neu5Ac, six sequential additions of 10 μM Neu5Ac to 0.5 μM RgSBP followed by one addition of 10 μM 2,7-anhydro-Neu5Ac, and displacement of 2,7-anhydro-Neu5Ac with Neu5Ac, one addition of 10 μM 2,7-anhydro-Neu5Ac followed by 6 subsequent additions of 10 μM Neu5Ac. The data shown are representative of triplicate experiments, the signal peaks are artefacts attributed to external light during sample addition.

Figure 3. Biophysical analysis of ligand binding to RgSBP.

ITC Isotherms of RgSBP binding to a) 2,7-anhydro-Neu5Ac or b) Neu5Ac, showing both DP – differential power and ΔH – enthalpy change. The data shown are representative of triplicate experiments. c) Saturation Transfer Difference (STD) NMR binding epitope mapping of 2,7-anhydro-Neu5Ac interacting with RgSBP. The initial slopes STD₀(%) were normalized against the highest STD₀, assigned as 100%. The obtained factors were then classified as weak (0-60 %), intermediate (60-80 %), and strong (80-100%) and used to identify the close contacts found at the interface of binding, data is representative of triplicate readings d) Average Differential Epitope Mapping (DEEP) STD factors for 2,7-anhydro-Neu5Ac obtained saturating RgSBP in spectral regions 0.6, 0.78, 1.44 ppm for aliphatic and 7.5, 7.23, 7.27 ppm for aromatic residues. Each differential mapping epitope obtained using different saturation frequencies are combined and the average DEEP STD is calculated resulting in five points for each frequency and a total of fifteen points for each proton receiving saturation. The data reported are the mean ± SEM of a sample of data of fifteen points for each proton receiving saturation.

Figure 4. R. gnavus sialic acid aldolase enzymatic reaction.

a) Change of A_340nm over time using R. gnavus sialic acid aldolase (RgNanA) with Neu5Ac (pink) or 2,7-anhydro-Neu5Ac (orange), or E. coli sialic acid aldolase (EcNanA) with Neu5Ac (black) or 2,7-anhydro-Neu5Ac (green) reactions coupled to lactate dehydrogenase, error bars represent standard error from 3 independent experiments. b) Michaelis-Menten plot of RgNanA rate of reaction with increasing concentration of Neu5Ac, error bars represent standard error. The rate of reaction at each concentration (μM NADH) was determined in triplicate by measuring A_340nm change using a standard curve. The mean value is plotted with standard error of the meaning shown with error bars c) Cartoon representation of the wild type RgNanA crystal structure showing the (β/α8) TIM barrel organisation and Lys167 as yellow sticks. d) The RgNanA K167A active site is shown in orange with bound Neu5Ac in the open-chain ketone form shown in cyan. The green mesh represents the Neu5Ac F_o-F_c difference map at the 3σ level (for a stereo image of Neu5Ac and F_o-F_c difference map see Supplementary Figure 5d). Hydrogen bonding interactions are depicted using black dashed lines. In addition, the unbound RgNanA wt active site is shown in grey.

Figure 5. RUMGNA_02695 catalyses the conversion of 2,7-anhydro-Neu5Ac to Neu5Ac.

a) High Performance Liquid Chromatography (HPLC) expand acronyms number replicates with same outcome analysis of DMB labelled RUMGNA_02695 reactions with 2,7-anhydro-Neu5Ac using different co-factors. NAD (black), NADH (pink), FAD (blue), no co-factor (brown), and a Neu5Ac standard (green), data is representative of five independent experiments. b) Michaelis-Menten plot of the rate of reaction for RUMGNA_02695 with increasing concentration of 2,7-anhydro-Neu5Ac. The rate of reaction (μM NADH) at each concentration was determined in triplicate by measuring A_340nm change and using a standard curve, the mean value is plotted with standard error of the meaning shown with error bars

Figure 6. Colonisation of germ-free C57BL/6J mice with R. gnavus ATCC 29149 wild-type or nan mutant strains.

Mice were monocolonised with a and b sample size (n) and define centre measure mean (a) R. gnavus wild-type (black; n = 4) or nan mutant (red; n = 4) strains individually or (b) in competition (n = 4). Mice were orally gavaged with 1x10⁸ of each strain, faecal samples were analysed at 3,7 and 14 days after inoculation and caecal samples at 14 days after inoculation using qPCR, centre line denotes the mean. (c) Fluorescent in situ hybridisation (FISH) and immunostaining of the colon from R. gnavus monocolonised C57BL/6 mice. R. gnavus ATCC 29149 and R. gnavus nan mutant are shown in red. The mucus layer is shown in green and an outline of the mucus is shown in the first panels. Cell nuclei were counterstained with Sytox blue, shown in blue. Scale bar: 20 μm. Image is representative of 70 total images (d) Quantification of the distance between the leading front of bacteria and the base of the mucus layer. A total of 70 images of stained colon from 8 R. gnavus monocolonised mice were analysed. The asterisks (***) show the significance (P=0.0135, by linear mixed model analysis, including fixed effects of genotype and area and random effects of mouse and each individual image. There was substantial spatial correlation between adjacent observations and so an AR(1) correlation structure was added. The resulting model had no residual autocorrelation as judged by visual inspection of autocorrelation function. The nmle package version 3.1-137 using R version 3.5.3 was used to estimate the model), centre point indicates the mean, box limits, upper and lower quartiles; whiskers, minimum and maximum.