Identification of sialic acid-utilising bacteria in a piglet caecal community using RNA-SIP

Wayne Young, AgResearch Limited Grasslands Research Centre, Palmerston North, New Zealand

Wayne Young1, Markus Egert2, Shalome Bassett1, Nicole Roy1,3, and Rodrigo Bibiloni1
1. AgResearch Ltd., Food Nutrition & Health Team, Food & Bio-based Products Group, Grasslands Research Centre, Palmerston North, New Zealand;
2. Furtwangen University, Faculty of Medical and Life Sciences, Institute of Precision Medicine, Microbiology & Hygiene Group, Villingen-Schwenningen, Germany;
3. Riddet Institute, Massey University, Palmerston North, New Zealand; Arla Foods, Denmark

Sialic acids are monosaccharides that can be found on the end of sugar chains expressed on cell surfaces. Milk contains high concentrations of sialic acids attached to the terminal end of oligo-saccharides, glycolipids and glycoproteins. Sialic acids are also a key components of neural tissue. The highest concentrations of sialic acid in the body are found in the brain where they form an essential part of ganglioside structures and are therefore critical in neural transmission and synaptogenesis. Animal studies have shown a link between learning ability and levels of sialic acid in brain gangliosides and glycoproteins. Milk oligosaccharides are resistant to mammalian digestive enzymes and are instead degraded by the large bowel microbiota. Intestinal bacteria have also been shown to produce glycosidases capable of desialylating milk gangliosides. However, little is known about the fate of ingested sialic acid and sialic acid-containing nutrients in the large bowel.

The objective was to identify bacteria that utilise free sialic acid in a complex intestinal community of piglet origin, we used an established RNA-stable isotope probing (SIP) approach and fully labelled 13C-sialic acid as a model substrate.

Caecal contents from three male 15-day-old piglets were anaerobically cultured at 37oC for 24 hours in minimal media with or without 13C-labelled sialic acid (2 mg/mL), which was the only added carbon source. Total RNA was extracted from the cultures and separated on caesium trifluoroacetate density gradients by ultracentrifugation at 130,000 x g for 65 hours. After centrifugation, 15 fractions of decreasing densities (1.82-1.73 g/ml) were collected from each gradient for analysis. To investigate the capacity of the microbiota to utilise a sialic acid-conjugated substrate, separate caecal culture incubations were also carried out using the ganglioside GD3, a glycosphingolipid with sialic acid linked to a sugar chain.

Higher-density RNA, from bacteria incorporating heavier 13C into their nucleic acids by metabolising 13C sialic acid, was enriched in fractions with densities between 1.78 and 1.80 g/mL, while lighter unlabeled RNA was predominantly found in fractions with densities between 1.72 and 1.78 g/mL. Pyrotag sequencing of 16S rRNA amplicons showed that the bacterial composition profile of heavy fractions differed from that of the lighter fractions in 13C-sialic acid-fed cultures. In addition, corresponding profiles from caecal communities cultured without 13C-sialic acid were different to those cultured with 13C-sialic acid. Bacteria enriched in the heavier fractions, and therefore the likely users of free sialic acid, included Roseomonas, Prevotella, and Sphingomonas. However, the addition of GD3 as a substrate did not alter the microbial community in caecal content incubations, suggesting GD3 was not readily consumed.

While sialic acid was utilised by many members of the cultured microbial community, our results suggest that fewer taxa are capable of utilizing sialic acid-conjugated milk components. Given the importance of sialic acid in nutrition, further RNA-SIP based studies investing the microbial utilization and assimilation of labelled sialic acid-conjugated milk glycolipids and oligosaccharides is warranted.

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