Stable Isotope Probing of gut bacteria RNA. Wayne Young. Identifying gut bacteria that can use sialic acid using an RNA-SIP approach

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1 Stable Isotope Probing of gut bacteria RNA Identifying gut bacteria that can use sialic acid using an RNA-SIP approach Wayne Young Food Nutrition & Health Team Food & Bio-based Products Group AgResearch Grasslands

2 Sialic acids: localisation and function Family of 9 carbon monosaccharides. Found on cell surfaces and attached to soluble proteins (eg mucin). High levels of sialic acid conjugated to oligosaccharides, glycolipids, and glycoproteins in milk. Critical nutrient for brain development and function. Sialic acids are not only the most interesting molecules in the world, but also the most important.* * Vimr, E. R Microbial sialidases: does bigger always mean better? Trends Microbiol. 2:

3 Sialic acids: two most common forms Most common form is N-Acetylneuraminic acid (Neu5Ac). Humans only have this form of sialic acid N-Glycolylneuraminic acid (Neu5Gc) found in most other mammals including ruminants. Cow, goat and sheep milk contains both Neu5Ac and NeuGc

4 Sialic acids: Piglets fed sialic acid supplemented milk show improved learning and memory Wang et al (2007) Am J Clin Nutr 85:561-9

5 Role of gut microbiota and sialic acids Sialic acid is present in the colon in the form of conjugated host glycans and dietary sources such as sialylated oligosaccharides, proteins and lipids. However, very little is known about the role of the microbiota. Using RNA-SIP, we explored the assimilation of sialic acid by microbiota using cultured piglet caecal contents.

6 Which gut bacteria use sialic acid? Stable Isotope probing of RNA AIM: Identify which gut bacteria can use sialic acid and incorporate its metabolites into their cellular components. METHOD: Culture caecal bacteria from piglets +/- 13 C labelled sialic acid. Independent cultures, 3 + sialic acid, 3 - sialic acid. Fractionate bacterial RNA using cesium tri-fluoroacetate (CsTFA) gradient by ultracentrifugation. Analyse bacterial composition in each fraction by pyrotag sequencing.

7 Which gut bacteria use sialic acid? Stable Isotope probing of RNA Piglet caecal contents

8 Which gut bacteria use sialic acid? Stable Isotope probing of RNA EXPECTED RESULTS: RNA incorporating 13 C is heavier than 12 C RNA and will therefore be enriched in denser CsTFA fractions. Analysis of 13 C enriched RNA will show which bacteria are using labelled sialic acid. 12 C 16S rrna 13 C + 12 C 16S rrna

9 Separate RNA-SIP density gradients after ultracentrifugation Refractory index Fraction density

10 More bacterial cdna in dense fractions from cultures with 13 C-sialic acid Cultures with 13 C sialic acid Control cultures

11 PCoA plot of Unifrac distances: Separation of profiles in heavy and light fractions. Also clustering based on piglet source. PC3 Blue: fractions in control cultures without sialic acid Red: fractions in cultures with 13 C-sialic acid. Green: fractions 4-6 in cultures with 13 C-sialic acid. PC2 PC1

12 Similar bacterial diversity between dense and light fractions

13 RNA-SIP density gradient bacterial profile 13 C Dense 13 C Light Control Light

14 Prevotella and Lactobacillus enriched among sialic acid using bacteria Escherichia/Shigella decreased by adding sialic acid

15 RNA-SIP density gradient predicted metagenome (picrust) Despite large taxonomic differences, the predicted metagenomes are similar

16 Summary Many different bacteria can use free sialic acid. The most prominent users are Prevotella and Lactobacillus. Addition of sialic acid reduced Escherichia/Shigella group. Changes suggest sialic acid reduced bacteria that use complex carbohydrates (Ruminococcus and Eubacterium) and increased those able to degrade mucin (Prevotella and Lactobaccillus). Adding free sialic acid reduced prevalence of potential pathogens Escherichia/Shigella and Klebsiella in our study.

17 Contrast between free and conjugated sialic acid Very few changes when cultured with sialic acid conjugated ganglioside GD3. Few bacteria able to use sialylated GD3 in our culture conditions

18 Other researchers have found feeding sialyllactose to piglets modifies the colon microbiota Jacobi et al (2016) J. Nutr (146)2: Prevotella Ruminococcus Enterobacteriaceae Concentrations of ganglioside sialic acid in the brain. Role in brain function for bacteria with sialidase activity

19 Sialic acid released by commensal bacteria can feed E. coli and drive intestinal inflammation Huang et al (2015) Nature Communications 6:8141 Wild-type mice + DSS leads to outgrowth of E. coli Sialyltransferase (ST) KO mice + DSS do not show increase in E. coli E. coli cannot liberate sialic acid on its own, relying on other member of the community to release it from host glycans and milk.

20 Future research Using RNA-SIP to better understand the dynamics of how the gut microbiota liberates and utilises sialic acid from conjugated sources. Gain a better understanding of the competition for sialic acid between commensal and potentially pathogenic bacteria. Expand model to human microbiota.

21 RNA-SIP study authors Wayne Young Markus Egert Shalome Bassett Rodrigo Bibiloni Acknowledgments Nicole Roy Leigh Ryan Stacey Burton Jason Peters Funding