The mucin-degradation strategy of Ruminococcus gnavus: The importance of intramolecular trans-sialidases

Abstract

We previously identified and characterized an intramolecular trans-sialidase (IT-sialidase) in the gut symbiont Ruminococcus gnavus ATCC 29149, which is associated to the ability of the strain to grow on mucins. In this work we have obtained and analyzed the draft genome sequence of another R. gnavus mucin-degrader, ATCC 35913, isolated from a healthy individual. Transcriptomics analyses of both ATCC 29149 and ATCC 35913 strains confirmed that the strategy utilized by R. gnavus for mucin-degradation is focused on the utilization of terminal mucin glycans. R. gnavus ATCC 35913 also encodes a predicted IT-sialidase and harbors a Nan cluster dedicated to sialic acid utilization. We showed that the Nan cluster was upregulated when the strains were grown in presence of mucin. In addition we demonstrated that both R. gnavus strains were able to grow on 2,7-anyhydro-Neu5Ac, the IT-sialidase transglycosylation product, as a sole carbon source. Taken together these data further support the hypothesis that IT-sialidase expressing gut microbes, provide commensal bacteria such as R. gnavus with a nutritional competitive advantage, by accessing and transforming a source of nutrient to their own benefit.

Keywords: Ruminococcus gnavus; glycoside hydrolase; gut bacteria; intestinal mucin; intramolecular trans-sialidase; mucin glycans; sialic acid.

Figures

Figure 1.

Growth curves of R. gnavus ATCC 29149 and ATCC 35913 on pPGM. The growth curves represent the average growth, measured at OD600nm, of at least 3 biological replicates.

Figure 2.

Comparison of the distribution of glycoside hydrolases (GHs) between R. gnavus strains. GHs are represented by light gray boxes for R. gnavus ATCC 35913, striped boxed for R. gnavus ATCC 29149 and dark gray boxes for R. gnavus E1.

Figure 3.

Relative level of transcription of GH genes in R. gnavus ATCC 29149 (A) and ATCC 35913 (B). The transcriptomic analysis has been performed by RNASeq from R. gnavus grown in presence of pPGM and compared to Glc as sole carbon source. The relative level of transcription was expressed as the Log2 of the fold change in gene transcription and the figures showed averages of 4 biological replicates for the GH genes that exhibited increased transcription (Log2 fold change > 1). Data were analyzed by DESeq2. The significance of differential expression was determined by the Benjamini-Hochberg corrected p-values of the Wald test of the negative binomial test per each set of two conditions. The transcription level was considered significantly increased when p < 0.05 and a Log2 (fold change) >1 and significant results were labeled with *. Error bars were plotted as the standard error of the Log2 fold change.

Figure 4.

The Nan locus in R. gnavus ATCC 29149 and ATCC 35913. (A) Schematic representation of the nan genetic organization in ATCC 35913. RGNV35913_01299 encodes a putative GDSL-like protein. RGNV35913_01298encodes a putative sugar isomerase involved in sialic acid catabolism. RGNV35913_01297 encodes a protein with homology with transcriptional regulators of the AraC family. The following 3 genes code for a predicted solute-binding protein (RGNV35913_01296) and two putative permeases (RGNV35913_01295 and RGNV35913_01294), components of a sugar ABC transporter. The following gene has homology with oxidoreductases from the Gfo/Idh/MocA family. The sialidase gene nanH (RGNV35913_01292) predicted to encode the GH33 enzyme comes next. Then nanE (RGNV35913_01291), which encodes a predicted ManNAc-6-P epimerase is followed by nanA (RGNV35913_01290) encoding a putative Neu5Ac lyase. nanK (RGNV35913_01289) is the last gene of the cluster, coding for a predicted ManNAc kinase. The previously described R. gnavus ATCC 29149 nan cluster shares 99.9% identity with the one present in ATCC 35913. Level of transcription of nan genes in R. gnavus ATCC 29149 (B) or ATCC 35913 (C). R. gnavus was grown in basal YCFA medium supplemented with either glucose (Glc) or mucin (pPGM) as sole carbon source. Cells were collected during the exponential phase of growth; RNA was extracted from 4 biological replicates for each carbon sources. The level of transcription of each gene was determined by RNASeq. The transcription of each gene was compared when the bacterium grew with pPGM vs. Glc using the R package DESeqx; it was considered significantly increased when the transcript was present at least twice more frequently, with a padj value (p-value adjusted for multiple testing) <=0.05 (* padj<=0.05).

Figure 5.

Proposed pathways for the catabolism of sialic acid in R. gnavus ATCC 29149 and ATCC 35913. RgNanH releases 2,7-anhydro-Neu5Ac from α2–3 linked sialylated substrates. (A) It can be hypothesized that 2,7-anhydro-Neu5Ac is transported inside the bacterium via a 2,7-anhydro-Neu5Ac-specific ABC transporter composed of a solute-binding protein (RUMGNA_02698 in ATCC 29149;RGNV35913_01296 in ATCC 35913) and two putative permeases (RUMGNA_02697 and RUMGNA_02696 in ATCC 29149; RGNV35913_01295 and RGNV35913_01294 in ATCC 35913) and then hydrolyzed into Neu5Ac, possibly by the action of RUMGNA_02701 or RGNV35913_01299, before being catabolized into GlcNAc-6-P following the traditional pathway by the successive action of NanA (Neu5Ac lyase), NanK (ManNAc kinase) and NanE (ManNAc-6-P epimerase). (B) Alternatively, both 2,7-anhydro-Neu5Ac and Neu5Ac could enter the cells via the ABC transporter but NanA would either be inactive or specific for 2,7-anhydro-Neu5Ac, explaining the absence of growth of the bacteria on sialic acid.