Abstract
The non-digestible oligosaccharide fraction of maternal milk represents an important of carbohydrate and energy source for saccharolytic bifidobacteria in the gastrointestinal tract during early life. However, not all neonatal bifidobacteria isolates can directly metabolise the complex sialylated, fucosylated, sulphated and/or N-acetylglucosamine-containing oligosaccharide structures present in mothers milk. For some bifidobacterial strains, efficient carbohydrate syntrophy or crossfeeding is key to their establishment in the gut. In this study, we have adopted advanced functional genomic approaches to create single and double in-frame deletions of the N-acetyl glucosamine 6-phosphate deacetylase encoding genes, nagA1 and nagA2, of B. breve UCC2003. In vitro phenotypic analysis followed by in vivo studies on co-colonisation, mother to infant transmission, and evaluation of the relative co-establishment of B. bifidum and B. breve UCC2003 or UCC2003ΔnagA1ΔnagA2 in dam-reared neonatal mice demonstrates the importance of crossfeeding on sialic acid, fucose and N-acetylglucosamine-containing oligosaccharides for the establishment of B. breve UCC2003 in the neonatal gut. Furthermore, transcriptomic analysis of in vivo gene expression shows upregulation of genes associated with the utilisation of lactose, sialic acid, GlcNAc-6-S and fucose in B. breve UCC2003, while for UCC2003ΔnagA1ΔnagA2 only genes for lactose metabolism were upregulated.
Highlights
Bifidobacteria are among the earliest and most abundant bacterial colonisers of the neonatal gut where their presence is associated with a myriad of benefits to the host intestinal, metabolic and immune health[1]
B. breve UCC2003 cannot directly utilise the most abundant human milk oligosaccharides (HMOs) in mother’s milk, fucosyllactose (2′FL or 3′FL), but can crossfeed on monosaccharides, including fucose, that are released during co-culture with B. bifidum PRL2010 in mMRS medium supplemented with porcine mucin[9]
Expression of nagA1is significantly upregulated during growth of B. breve UCC2003 in mMRS medium supplemented with the host derived sulphated carbohydrate N-acetyl glucosamine-6-sulphate (GlcNAc-6-S) or Lactosamine-HCl, while expression of nagA2 is significantly upregulated during growth of B. breve UCC2003 in mMRS medium supplemented with sialic acid, LNT, LNnT, GlcNAc-6-S and when B. breve UCC2003 is grown in co-culture with B. bifidum PRL2010 in medium supplemented with mucin[8,9,10,11]
Summary
Bifidobacteria are among the earliest and most abundant bacterial colonisers of the neonatal gut where their presence is associated with a myriad of benefits to the host intestinal, metabolic and immune health[1]. Infantis and B. bifidum have been studied extensively for their ability to utilise host derived carbohydrates and have been found to harbour dedicated, yet distinct, metabolic capabilities for the utilisation of HMOs4,5 while more recently specific strains of B. longum subsp. We have previously shown that B. breve UCC2003, a nursling infant stool isolate, can efficiently utilise sialic acid, but not host derived 3′ sialyllactose, as sole carbohydrate source[8]. B. breve UCC2003 can crossfeed on released sialic acid derived from the extracellular metabolism of 3′ sialyllactose by B. bifidum PRL2010 during an in vitro sequential co-culture experiment[8]. B. breve UCC2003 cannot directly utilise the most abundant HMO in mother’s milk, fucosyllactose (2′FL or 3′FL), but can crossfeed on monosaccharides, including fucose, that are released during co-culture with B. bifidum PRL2010 in mMRS medium supplemented with porcine mucin[9]. The B. breve strains were examined for their cross-feeding capability and ability to establish, in the presence of B. bifidum, in the gut of dam fed neonatal mice
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