Human gut Faecalibacterium prausnitzii deploy a highly efficient conserved system to cross-feed on β-mannan-derived oligosaccharides

β-Mannans are hemicelluloses that are abundant in modern diets as components in seed endosperms and common additives in processed food. Currently, the collective understanding of β-mannan saccharification in the human colon is limited to a few keystone species, which presumably liberate low-molecular-weight mannooligosaccharide fragments that become directly available to the surrounding microbial community. Here we show that a dominant butyrate-producer in the human gut, Faecalibacterium prausnitzii, is able to acquire and degrade various β-mannooligosaccharides (β-MOS), which are derived by the primary mannanolytic activity of neighboring gut microbiota. Detailed biochemical analyses of selected protein components from their two β-mannooligosaccharides (β-MOS) utilization loci (FpMULs) supported a concerted model whereby the imported β-MOS are stepwise disassembled intracellularly by highly adapted enzymes. Coculturing experiments of F. prausnitzii with the primary degrader Bacteroides ovatus on polymeric β-mannan resulted in syntrophic growth and production of butyrate, thus confirming the high efficiency of the FpMULs’ uptake system. Genomic comparison with human F. prausnitzii strains and analyses of 2441 public human metagenomes revealed that FpMULs are highly conserved and distributed worldwide. Together, our results provide a significant advance in the knowledge of β-mannans metabolism and the degree to which its degradation is mediated by cross-feeding interactions between prominent beneficial microbes in the human gut. Importance Commensal butyrate-producing bacteria belonging to the Firmicutes phylum are abundant in the human gut and are crucial for maintaining health. Currently, insight is lacking into how they target otherwise indigestible dietary fibers and into the trophic interactions they establish with other glycan degraders in the competitive gut environment. By combining cultivation, genomic and detailed biochemical analyses this work reveals the mechanism enabling F. prausnitzii, as a model clostridial cluster IV Firmicute, to cross-feed and access β-mannan-derived oligosaccharides released in the gut ecosystem by the action of primary degraders. A comprehensive survey of human gut metagenomes shows that FpMULs are ubiquitous in human populations globally, highlighting the importance of microbial metabolism of β-mannans/β-MOS as a common dietary component. Our findings provide a mechanistic understanding of the β-MOS utilization capability by F. prausnitzii that may be exploited to select dietary formulations specifically boosting this beneficial symbiont, thus butyrate production, in the gut.


INTRODUCTION
The human distal gut supports a densely populated microbial community that 60 extends the metabolic capabilities lacking in the hosts genome (1). In particular,

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We recently reported the characterization of a novel β-mannan utilization locus 119 conferring Roseburia intestinalis, a model for the clostridial cluster XIVa Firmicutes, 120 with the ability to ferment this fiber through to butyrate via a selfish mechanism (7). 121 β-mannan degradation was proven to be initiated by an endo-acting multi-modular 122 GH26 enzyme localized on the cell surface; the resulting oligosaccharides are 123 imported intracellularly through a highly-specific ABC-transporter, and completely de-124 polymerized to their component monosaccharides by an enzymatic cocktail 125 containing carbohydrate esterases, β-glucosidases and phosphorylases (7).

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Although F. prausnitzii has been described as an efficient degrader of host-derived 127 and plant glycans (23), the ability of this important butyrate-producing microbe to 128 utilize dietary β-mannans has received little attention. In a previous study, we 129 reported that wood-derived acetylated galactoglucomannan stimulates the 130 proliferation of F. prausnitzii populations in a pH-controlled batch culture 131 fermentation system inoculated with healthy adult human feces (24). However, the 132 7 molecular mechanism underlining β-mannan utilization by F. prausnitzii in the human 133 gut has not been explored to date.

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In this study, we describe and biochemically characterize components of two loci that 135 mediate acquisition and catabolism of β-mannooligosaccharides (β-MOS) by F. 136 prausnitzii SL3/3. Together, these data allowed us to outline a pathway for dietary β-137 MOS deconstruction and saccharification to monosaccharides through cross-feeding 138 with Bacteroides species, which contributes to the ecology of β-mannan utilization in 139 the gut ecosystem. Remarkably, we show that the binding proteins that confers β-140 MOS capture in F. prausnitzii targeted ligands with stronger affinity than that of 141 Bacteroides species, thus providing F. prausnitzii with the ability to cross-feed on the 142 β-MOS available in the environment with high efficiency.

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Genes encoding enzymatic activities required to catabolize mannans were identified RiGH3B from the previously characterized β-mannan utilization system in R. 155 intestinalis (7), genes encoding two predicted GH3 β-glucosidases were identified 156 (FpGH3A and FpGH3B). These two genes are located in a different locus in the genome, hereafter referred to as FpMULS, and are likely to be involved in 158 (galacto)glucomannan turnover. Based on known activities within GH families, the β-159 1,4-mannan backbone is predicted to be hydrolyzed by extracellular GH26, GH5 160 and/or GH134 enzymes (see www.cazy.org). However, no gene coding for such 161 enzyme was identified in the genome of F. prausnitzii SL3/3. In addition, endo-β-1,4-162 mannanase activity was originally reported for two GH113 (see www.cazy.org) 163 although we demonstrated that a GH113 within the mannan utilization locus of R. 164 intestinalis is a reducing end mannose-releasing exo-oligomannosidase. A gene 165 encoding a GH113 was detected in the FpMULL (Fig. 1a). Based on a genomic     FpGH113 is a reducing end mannose-releasing exo-oligomannosidase.

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When combined, using 25 nM of each esterase, the deacetylation rate, k cat and 287 specific activity were approximately 2-fold higher compared to the values from 288 treatments with FpCE17 and 2-fold lower compared to the values from treatments 289 with the FpCE2 when used on its own, respectively ( Table 1).The reduced resulting 290 rate of deacetylation suggests that the esterases are not acting synergistically but 291 may rather be competing for the substrate, a behavior previously reported in 292 cocktails of multiple enzymes for lignocellulose hydrolysis (29).

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Melting curves for both enzymes in buffers at pH 5.0-8.0 were obtained using a 294 protein thermal shift assay ( Fig. 4g-h). Both FpCE2 and FpCE17 displayed an 295 irreversible thermal unfolding transition, which is consistent with their multi-domain 296 structure (28, 30). FpCE17 was stable up to 73 °C, with the highest observed melting 297 temperature at pH 6.0; its lowest observed melting temperature was 58 °C at pH 5.

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For FpCE2 the unfolding took place at higher temperature, with a melting point of 62 299 °C at pH 6.0 and a highest melting point of 73 °C at pH 7.0 and 8.0 (Fig. 4g-h) (Fig 4i), thus confirming the manno-oligosaccharide specificity of FpCE17 and 311 FpCE2.  (Fig. 5a). The optical densities obtained when 321 F. prausnitzii was grown in monoculture in the no-carbon source control (Fig. 5b)

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were similar to those obtained in 0.2% (w/v) KGM, suggesting that the microbe is not 323 able to utilize this glycan on its own. Notably, the maximum OD 650 of the co-culture 324 (OD 650 = 0.45) appeared higher in the β-mannan polymer than those observed by B. 325 ovatus in single culture (OD 650 = 0.37), indicating that syntrophic growth exists 326 between these two populations in these conditions (Fig. 5a). F. prausnitzii is a 327 butyrate producer while carbohydrate fermentation by B. ovatus results in the 328 production of propionate (5). Therefore, comparing differences in butyrate levels 329 between the single F. prausnitzii culture and co-culture may provide evidence as to 330 whether cross-feeding of β-mannan breakdown products by F. prausnitzii occurred.

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Butyrate concentrations were significantly increased (p = 0.004) in the co-culture 332 compared to the mono-culture in KGM (Fig. 5d) or the co-culture in minimal medium 333 (Fig. 5e), which suggests that F. prausnitzii can effectively compete for β-MOS 334 generated by the cell-surface exposed endo-mannanase BoMan26B from B. ovatus 335 (31). This effect required the presence of living B. ovatus cells, as no evidence of an 336 increase of butyrate levels was detected when F. prausnitzii was co-grown with a 337 heat-treated B. ovatus culture (Fig. S3). When F. prausnitzii was co-cultured with R. 338 intestinalis in 0.2% KGM or in the absence of a carbon source, the growth curves 339 appeared very similar to when R. intestinalis was cultured on its own (Fig. 5g-h). As   Members of the dominant Bacteroides genus, such as B. ovatus, and Roseburia 371 species that possess GH26 endo-mannanases have been described as the keystone 372 bacteria for mannan degradation in the gut (7, 21, 31). In contrast, F. prausnitzii may 373 only access oligosaccharides, released by these primary degraders, which can be 374 imported without the need for extracellular enzymatic cleavage. In this context, we 375 demonstrate that β-MOS are indeed released into the culture medium by B. ovatus 376 during co-growth on KGM, and that F. prausnitzii is capable to efficiently compete for 377 and utilize these oligosaccharides ( Fig. 5a and 5d). However, we observed no 378 explicit evidence of cooperative growth between R. intestinalis and F. prausnitzii 379 ( Fig. 5g and 5j). We recently demonstrated the competitiveness of R. intestinalis on 380 β-mannan when in co-culture with B. ovatus during growth on AcGGM (7), and 381 highlighted a pivotal role of a transport protein (RiMnBP) within the uptake system,  (Fig. 1c). Indeed, we did not observe correlation with any particular Bacteroides. This is in line with the fact that, when in co-culture, F. prausnitzii 423 showed cross-feeding behaviors with B. ovatus, whose own β-MOS uptake requires 424 a SusD-like protein that binds oligosaccharides with about 10-fold lower affinity than 425 FpMOBP. Furthermore, this study in conjunction with a previous report (7)   by PCR, using appropriate primers (Table S1). All primers were designed to 474 amplify constructs to exclude predicted signal peptides (predicted by the SignalP 475 v4.1 server (41)). PCR products were generated using the Q5 High-Fidelity DNA  in other publicly available F. prausnitzii genomes were conducted using a similar 594 strategy as described previously (7). Briefly, the identification of similar MULs in 595 strains other than F. prausnitzii SL3/3 was done using BLASTN and the Gene