Harvesting of Prebiotic Fructooligosaccharides by Nonbeneficial Human Gut Bacteria

Abstract

Prebiotic oligosaccharides, such as fructooligosaccharides, are increasingly being used to modulate the composition and activity of the gut microbiota. However, carbohydrate utilization analyses and metagenomic studies recently revealed the ability of deleterious and uncultured human gut bacterial species to metabolize these functional foods. Moreover, because of the difficulties of functionally profiling transmembrane proteins, only a few prebiotic transporters have been biochemically characterized to date, while carbohydrate binding and transport are the first and thus crucial steps in their metabolization. Here, we describe the molecular mechanism of a phosphotransferase system, highlighted as a dietary and pathology biomarker in the human gut microbiome. This transporter is encoded by a metagenomic locus that is highly conserved in several human gut Firmicutes, including Dorea species. We developed a generic strategy to deeply analyze, in vitro and in cellulo, the specificity and functionality of recombinant transporters in Escherichia coli, combining carbohydrate utilization locus and host genome engineering and quantification of the binding, transport, and growth rates with analysis of phosphorylated carbohydrates by mass spectrometry. We demonstrated that the Dorea fructooligosaccharide transporter is specific for kestose, whether for binding, transport, or phosphorylation. This constitutes the biochemical proof of effective phosphorylation of glycosides with a degree of polymerization of more than 2, extending the known functional diversity of phosphotransferase systems. Based on these new findings, we revisited the classification of these carbohydrate transporters.IMPORTANCE Prebiotics are increasingly used as food supplements, especially in infant formulas, to modify the functioning and composition of the microbiota. However, little is currently known about the mechanisms of prebiotic recognition and transport by gut bacteria, while these steps are crucial in their metabolism. In this study, we established a new strategy to profile the specificity of oligosaccharide transporters, combining microbiomics, genetic locus and strain engineering, and state-of-the art metabolomics. We revisited the transporter classification database and proposed a new way to classify these membrane proteins based on their structural and mechanistic similarities. Based on these developments, we identified and characterized, at the molecular level, a fructooligosaccharide transporting phosphotransferase system, which constitutes a biomarker of diet and gut pathology. The deciphering of this prebiotic metabolization mechanism by a nonbeneficial bacterium highlights the controversial use of prebiotics, especially in the context of chronic gut diseases.

Investigating host-microbiome interactions by droplet based microfluidics

“…2, S2 and S3), despite a low yield of recombinant protein production. In such E. coli fosmid clones, only some genes of the cloned loci are expressed spuriously, likely due to the presence within the metagenomic sequence of σ70 promoter sequences for E. coli [35,46,47]. In these metagenomic sequences, one CAZyme was found every 13 kb, which is 3.5 times more frequent than in the randomly sequenced human gut metagenome [48].…”

The gut microbiome in differential diagnosis of diabetic kidney disease and membranous nephropathy

“…ABC transporters mediate the transport of hydrophilic compounds and promote the growth of various bacteria, such as Mycobacterium tuberculosis [ 52 ]. The PTS is involved in the phosphorylation of sugars [ 53 ], and the interconversion of pentose and glucoronate provides substrates for the synthesis of nucleic acids and maintains the reducing state of glutathione. Higher enrichment of these three KEGG pathways was observed in MN patients, and their roles in MN development remain to be determined.…”

Identification of Glycoside Transporters From the Human Gut Microbiome

“…Secondly, for some multi-domains transporters, several genes of the metagenomic locus have to be concomitantly expressed at similar levels. Finally, to be functional, the recombinant transporters must be properly addressed and folded into the cell membranes of the host strain, and, for some of them, functionally complemented by E. coli components, such as TonB for SusCD systems or the phosphoenolpyruvate dependent glycoside phosphorylation machinery for PTSs (Wang et al, 2020).…”

“…MFS, ABC, and PTS systems are all used by native E. coli strains for nutrient uptake (Saier, 2000). In addition, several PTS involved in oligosaccharide utilization by Gram-positive bacteria were previously successfully expressed in E. coli (Lai and Ingram, 1993;Lai et al, 1997;Wang et al, 2020), despite the difference in cell membrane organization. The localization in E. coli of the different components of the transporters identified here from Gram-positive bacteria will need to be explored.…”