Mechanism of 2′-fucosyllactose degradation by human-associated Akkermansia

Abstract

Among the first microorganisms to colonize the human gut of breastfed infants are bacteria capable of fermenting human milk oligosaccharides (HMOs). One of the most abundant HMOs, 2′-fucosyllactose (2′-FL), may specifically drive bacterial colonization of the intestine. Recently, differential growth has been observed across multiple species of Akkermansia on various HMOs including 2′-FL. In culture, we found growth of two species, A. muciniphila Muc T and A. biwaensis CSUN-19,on HMOs corresponded to a decrease in the levels of 2′-FL and an increase in lactose, indicating that the first step in 2′-FL catabolism is the cleavage of fucose. Using phylogenetic analysis and transcriptional profiling, we found that the number and expression of fucosidase genes from two glycoside hydrolase (GH) families, GH29 and GH95, vary between these two species. During the mid-log phase of growth, the expression of several GH29 genes was increased by 2′-FL in both species, whereas the GH95 genes were induced only in A. muciniphila . We further show that one putative fucosidase and a β-galactosidase from A. biwaensis are involved in the breakdown of 2′-FL. Our findings indicate that the plasticity of GHs of human-associated Akkermansia sp. enables access to additional growth substrates present in HMOs, including 2′-FL. Our work highlights the potential for Akkermansia to influence the development of the gut microbiota early in life and expands the known metabolic capabilities of this important human symbiont. IMPORTANCE Akkermansia are mucin-degrading specialists widely distributed in the human population. Akkermansia biwaensis has recently been observed to have enhanced growth relative to other human-associated Akkermansia on multiple human milk oligosaccharides (HMOs). However, the mechanisms for enhanced growth are not understood. Here, we characterized the phylogenetic diversity and function of select genes involved in the growth of A. biwaensis on 2′-fucosyllactose (2′-FL), a dominant HMO. Specifically, we demonstrate that two genes in a genomic locus, a putative β-galactosidase and α-fucosidase, are likely responsible for the enhanced growth on 2′-FL. The functional characterization of A. biwaensis growth on 2′-FL delineates the significance of a single genomic locus that may facilitate enhanced colonization and functional activity of select Akkermansia early in life.

Pangenomic analysis identifies correlations between Akkermansia species and subspecies and human health outcomes

“…The average nucleotide identity (ANI) between two pairs of genomes has become the gold standard for identifying the genetic boundary of species from genome sequencing data [61,62] . Our pairwise analysis ANI between Akkermansia genomes reinforced the previously established phylogroup structure [19,20,22] and added a new phylogroup [Figure 1A and Supplementary Table 2]. This phylogroup, which we termed AmVI, is represented by Akkermansia isolated from a patient undergoing cancer immunotherapy and a patient with amyotrophic lateral sclerosis.…”

“…Whole-genome analyses revealed potential phylogroup-specific metabolic differences. These were confirmed through the characterization of representative isolates demonstrating variations in growth rates in mucin medium, aerotolerance, ability to metabolize human milk oligosaccharides, vitamin B 12 production, and activation of toll-like receptors (TLR) [20][21][22][23] . Based on the comparison of additional genomes from newly isolated human strains and near complete metagenome-assembled genomes (MAGs), there is an emerging consensus that genomes that were originally referred to as A. muciniphila should be sub-divided into several distinct species based on whole-genome nucleotide identity or conserved gene alignment [24][25][26][27][28] .…”

“…In addition to these, we added 13 new Akkermansia strains that we isolated from human and animal fecal samples. The final collection included 207 genomes from human Akkermansia isolates, 87 of which had been previously phylotyped as AmIa, AmIb, AmII, AmIII, AmIV, and AmV [19][20][21][22]60] , and 26 genomes derived from non-human sources.…”

“…The resulting KOs were filtered to include only those annotated as present or absent in at least three genomes in each phylogroup [Figure 2B]. As previously reported, gene clusters involved in assimilatory sulfate reduction are absent in A. massiliensis and A. biwaensis [20] , and gene clusters involved in cobalamin biosynthesis are unique to A. massiliensis [22] . Additionally, A. biwaensis is enriched with genes involved in formaldehyde assimilation but lacks lipoic acid biosynthetic genes.…”

Carbohydrate-active enzymes fromAkkermansia muciniphilabreakdown mucin O-glycans to completion

“…Between them, the GH29 and GH95 enzymes from A. muciniphila ATCC BAA-835 can degrade a wide variety of fucose-rich substrates (Supplementary Figure 2-3) 13,21 . These enzymes cannot access the fucose in blood group A and B (BGA and B) structures, so these glycan epitopes are first partially broken down by other CAZymes encoded in the A. muciniphila ATCC BAA-835 genome to produce blood group H structures first.…”