Biology subjects

Ndeh, D.

Publications and source records attributed to Ndeh, D..

4 recordsLinked to original sources

bioRxiv2026

Towards a comprehensive chemical and genetic tool library for rhamnogalacturonan-II oligosaccharides and exploitation

Rhamnogalacturonan-II (RG-II) is considered the most complex glycan in nature. It forms part of an intricate network of complex glycans in the plant cell wall where it plays a critical role in plant growth, development and defence. It has been identified as an important nutrient source for the human gut microbiota (HGM), a key modulator of human health and disease status. Increasing evidence also suggests that RG-II can modulate plant-microbe interactions. Given its importance and potential, detailed studies of RG-IIs structure-function relationships and metabolism are required to underpin future crop-improvement strategies and to harness its benefits for plant and human health. Progress in this field is however hampered by RG-IIs structural complexity and limited access to enabling tools, in particular chemically defined RG-II-derived oligosaccharide (CDRO) substructures. Achieving targeted, efficient, and scalable production of CDROs remains a significant challenge and is indeed one of the major reasons why RG-II and glycomic research in general, significantly lag behind genomic and proteomic research. Here, we have genetically engineered as well as screened a diverse set of genetic strains, including transposon (Tn) mutants of the prominent model human gut microbe Bacteroides thetaiotaomicron (B. theta) and its gut and plant-associated relatives for new CDRO-generating and/or RG-II-utilising strains. Several CDROs, some of which had never been produced before by any other means (including chemical synthesis), where generated and characterised by a combination of high-resolution mass spectrometry (MS), enzymatic profiling and 2D-NMR. In addition to expanding the CDRO toolbox, we identified key genetic strains that will serve as a base or platform for the production of an unprecedented amount of CDROs covering the complexity and diversity of chemical modifications in RG-II. CDROs were later exploited to gain new insights into the microbial metabolism of RG-II in the human gut, revealing key aspects of its chemical structure that drive or limit its metabolism in B. theta. Notably, we generated new evidence in support of an alternative operational paradigm for polysaccharide utilisation systems that are widespread in the Bacteroidota phylum. We confirmed the presence of pathways for the metabolism of RG-II and/or RG-II core sugars D-apiose (D-Apif), and 3-deoxy-D-manno-2-octulosonic acid (D-Kdo) in aerobic plant-associated microbes including fungi and Flavobacterium spp., highlighting their potential to be exploited as cost-effective alternatives to B. theta for the generation of CDROs.

biochemistry↗

bioRxiv2024

Hybrid xyloglucan utilisation loci are prevalent among plant-associated Bacteroidota

The plant hemicellulose xyloglucan (XyG) is secreted from the roots of numerous plant species, including cereals, and contributes towards soil aggregate formation in terrestrial systems. Whether XyG represents a key nutrient for plant-associated bacteria is unclear. The phylum Bacteroidota are abundant in the plant microbiome and provide several beneficial functions for their host. However, the metabolic and genomic traits underpinning their success remain poorly understood. Here, using proteomics, bacterial genetics, and genomics, we revealed that plant-associated Flavobacterium, a genus within the Bacteroidota, can efficiently utilise XyG through the occurrence of a distinct and conserved gene cluster, referred to as the Xyloglucan Utilisation Loci (XyGUL). Flavobacterium XyGUL is a hybrid of the molecular machinery found in gut Bacteroides spp., Cellvibrio japonicus, and the plant pathogen Xanthomonas. Combining protein biochemistry, computational modelling and phylogenetics, we identified a mutation in the enzyme required for initiating hydrolysis of the XyG polysaccharide, an outer membrane endoxyloglucanase glycoside hydrolase family 5 subfamily 4 (GH5_4), which enhances activity towards XyG. A subclade of GH5_4 homologs carrying this mutation were the dominant form found in soil and plant metagenomes due to their occurrence in Bacteroidota and Proteobacteria. However, only in members of the Bacteroidota spp., particularly Flavobacterium spp. was such a remarkable degree of XyGUL conservation detected. We propose this mechanism enables plant-associated Flavobacterium to specialise in competitive acquisition of XyG exudates and that this hemicellulose may represent an important nutrient source, enabling them to thrive in the plant microbiome, which is typified by intense competition for low molecular weight carbon exudates.

microbiology↗

bioRxiv2024

A platform for the recombinant production of Group A Streptococcus glycoconjugate vaccines

Strep A is a human-exclusive bacterial pathogen killing annually more than 500,000 patients, and no current licensed vaccine exists. Strep A bacteria are highly diverse, but all produce an essential, abundant, and conserved surface carbohydrate, the Group A Carbohydrate, which contains a rhamnose polysaccharide (RhaPS) backbone. RhaPS is a validated universal vaccine candidate in a glycoconjugate prepared by chemical conjugation of the native carbohydrate to a carrier protein. We engineered the Group A Carbohydratte biosynthesis pathway to enable recombinant production using the industry standard route to couple RhaPS to selected carrier proteins within E. coli cells. The structural integrity of the produced recombinant glycoconjugate vaccines was confirmed by NMR spectroscopy and mass spectrometry. Purified RhaPS glycoconjugates elicited carbohydrate-specific antibodies in mice and rabbits and bound to the surface of multiple Strep A strains of diverse M-types, confirming the recombinantly produced RhaPS glycoconjugates as valuable vaccine candidates.

microbiology↗

bioRxiv2024

A genetic locus in the gut microbe Bacteroides thetaiotaomicron encodes activities consistent with mucin-O-glycoprotein processing and plays a critical role in N-acetylgalactosamine metabolism

It is increasingly appreciated that members of the gut microbiota are key modulators of human health and the status of major diseases including cancer, diabetes and inflammatory bowel disease. Central to their survival is the ability to metabolise complex dietary and host-derived glycans including intestinal mucins. The latter are critical components of the gut epithelium glycocalyx and mucus barriers, essential for microbiota-gut homeostasis and protection from infections by pathogens. The prominent and model human gut microbe Bacteroides thetaiotaomicron (B. theta) is a versatile and highly efficient complex glycan degrader thanks to the expansion of gene clusters termed polysaccharide utilisation loci (PULs) in its genome. While the mechanisms for several singular dietary glycan-induced PULs have been elucidated, studies on the 16-18 mucin-induced PULs in B. theta significantly lag behind. A combination of the scale and complexity of B. theta transcriptomic response to mucins and complex glycan configurations of mucins represent major hurdles for the functional characterisation of the mucin induced PULs. As a result, there is very limited knowledge on how mucin metabolism is coordinated in B. theta and what specific PULs, genes and metabolites are critical for mucin-B. theta, and more generally mucin-microbiota interactions and their importance in microbiota-gut homeostasis. Here we show that a mucin inducible PUL BT4240-50, (i) encodes activities consistent with a machinery that couples the processing of mucin-O glycan glycoproteins with the metabolism of N-acetylgalactosamine (GalNAc), an abundant mucin O-glycan sugar; (ii) is important for competitive growth on mucins in-vitro; (iii) encodes a key kinase enzyme (BT4240) that is critical for GalNAc metabolism and (iv) has related PULs encoded by a range of prominent Bacteroides species in the human gut. Furthermore, BT4240 kinase was also critical for glycosaminoglycan metabolism, thus extending the PULs function beyond mucins. Our work advances our understanding of the vital metabolic processes that govern mucosal glycoprotein metabolism and by implication, a key aspect of host-microbiota interactions at mucosal surfaces and highlight GalNAc as a key metabolite targeted for competitive growth.

biochemistry↗