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Report

Teaser, summary, work performed and final results

Periodic Reporting for period 1 - Pectin (The microbial degradation and utilization of complex pectins by Bacteroides in the human intestine)

Teaser

Summary

The human large bowel is colonized by a community of microbes, the microbiota, which has a significant impact on human health and nutrition. Thus, around 10% of calories we consume are derived from the short chain fatty acids (SCFAs) generated by the microbiota, while these SCFAs also have health benefits exemplified by butyrate, which protects against colon cancer. The human gut microbiota (HGM) also has an impact on type 2 diabetes and cardiovascular disease. It is evident, therefore, that the HGM plays a pivotal role in maintaining human health and nutrition. Complex carbohydrates are the major nutrients available to the microbiota. Given the importance of glycans (complex carbohydrates) to the HGMa, dietary and nutraceutical strategies, based on complex carbohydrates, can, potentially, be deployed to ensure that the structure of this ecosystem maximizes human health. This approach, however, is greatly restricted by a lack of understanding of the mechanisms by which glycans are metabolized by the microbiota. Dr Venditto, under the supervision of Professor Gilbert, moved to Newcastle to dissect the mechanisms of complex glycan utilization by the HGM. The PECTIN project focused on how these abundant glycans in the human diet are metabolized by prominent members of the HGM, exploiting the bacterium Bacteroides thetaiotaomicron (B. theta) as the model organism. The PECTIN project: i) generated models for how B. theta and other members of the HGM metabolized the five polysaccharides that comprise the pectin component of the human diet; ii) determined the extent to which there is functional cross-talk between different members of the HGM during pectin metabolism. Furthermore, the MSC fellow Dr Venditto expanded her professional horizon by obtaining new skills in detailed enzyme characterization, anaerobic microbiology, bioinformatics, in vivo bacterial genetic manipulation and the design of microbial ecological experiments. Furthermore, she has actively disseminated his research to public, industrial and academic beneficiaries to maximize the impact of the project.

Work performed

RGII degradation: transcriptomic data identified target proteins upregulated by RGII, the mechanism by which RGII is degraded by B. theta was investigated using a range of enzyme assaysThe data demonstrate that single Bacteroides species are able to fully utilize RGII cleaving 21 of the 22 linkages present in the complex carbohydrate. This is in sharp contrast to previous hypotheses, which proposed that, because of the complexity of RGII, only complex microbial consortia would be able to degrade the glycan. The RGII degradative apparatus revealed enzymes with novel specificities and eight new glycanase families. Each linkage is cleaved by a specific enzyme. This stringent enzymatic specificity likely reflects an apparatus that is tuned to maximise the rate of the degradative process, offsetting the energy required to synthesise such an elaborate catabolic system. The generic relevance of genes in the locus (RGII PUL1) encoding the major RGII degrading enzymes was confirmed by bioinformatic analysis.

Other pectin degrading systems: Models for the degradation of the pectins rhamnogalacturonan I (RGI), homogalacturonan (HG), arabinan and galactan were determined. Initial degradation of the pectins by B. theta is mediated by surface endo-acting glycoside hydrolases (GHs) and polysaccharide lyases (PLs). These enzymes are essential for pectin utilization as they generate oligosaccharides that can be transported into the periplasm, where they were degraded by mainly exo-acting GHs and PLs. RGI released from the pectin network contains remnants of side chains. Prior to RGI backbone depolymerisation these accessory structures must be removed, explaining why RGI-PUL is so complex. Six enzymes encoded by this locus removed these side chain remnants and eight enzymes depolymerised the rather simple disaccharide repeat that comprises the RGI backbone.
The ligands that activate the pectin degradative system were explored. The data showed that the enzymes required to generate these activating ligands had much lower activity than the enzymes that degraded these glycans. The biological rationale for this difference in catalytic competence, may reflect the need to protect the inducing ligand, ensuring that there is continuous production of the activating molecules throughout growth on the respective glycan.
The work carried out by Dr Venditto provides a model for how the pectic network is metabolized by a Bacteroides species in the HGM. A salient feature of pectin utilization is the elaboration of enzymes in the RGI-PUL, reflecting the requirement to remove remnants from other pectic glycans and the extraordinary number of enzymes deployed in depolymerizing the disaccharide backbone. Dissecting the mechanism of pectin degradation contributes to our understanding of the foodweb within the HGM.

Cross-feeding experiments: A key question presented by these data is the source of oligosaccharides available to those organisms that could grow on oligosaccharides but not the cognate polysaccharide. To address this important question mutants of B. theta lacking surface endo-glycanases were generated. The mutants failed to grow on polysaccharides but utilised appropriate oligosaccharides. These mutants grew on the cognate polysaccharide when co-cultured with wild type B. theta. These data show that wild type B. thetaiotaomicron released polysaccharide breakdown products (PBP) into culture media, which were available to other organisms. To summarize, these data illustrate how glycans are made available to the general community by primary degraders. Possible non-Bacteroides beneficiaries of pectin-derived cross-feeding within the HGM are Bifidobacterium species, which generally utilize PBPs rather than the polysaccharide. Contrasting oligosaccharide utilisation profiles observed among Bacteroides spp. may allow for co-existence of species within the same niche targeting different components of the same glycans without competition.

Final results

Research:
The data obtained from the project provided models for the enzymatic basis by which the major pectins are utilised. Dr Venditto has communicated these findings at two major international conferences and has published three high impact papers on her work. The project underpins the development of novel pre- and pro-biotic strategies to manipulate the HGM to maximise its impact on human health.

Training of Fellow:
The PECTIN project allowed Dr Venditto to acquire training in anaerobic microbiology, genetic manipulation of these organisms and the bioinformatics analysis of genes encoding glycan degrading systems. Dr Venditto also developed her teaching skills by supervising several undergraduate final year projects and through presenting seminars to the lab.

Host institute:
Prof Gilbertâ€™s group benefited from Dr Vendittoâ€™s tenure in Newcastle, as her experience in high throughput protein expression and characterization revolutionised the ability of the lab. to rapidly characterise recombinant proteins.