Biochemical Characterization Of Glycosaminoglycans-Degrading Polysaccharide Lyases Of Human Gut Firmicutes And Bacteroides

Glycosaminoglycans(GAGs)are consistently present in the human colon both in free forms and as part of proteoglycans.Their utilization is critical for the colonization and proliferation of gut bacteria and therefore the health of hosts.Whereas,dietary and medicinal administration of GAGs has been reported to impact the composition of the human gut microbiota(HGM)that exerts beneficial effects,excessive foraging of host-derived GAGs has been shown to be detrimental on host health.It is believed that a subtly balanced host GAG utilization by HGM is necessary for a healthy gut.Deciphering the precise mechanism of GAG utilization by gut bacteria will augment our understanding of their effects on human health.The complete degradation of GAGs is achieved by a concerted,step-wise action of various proteins,including GAGs polysaccharide lyases(PL),glycoside hydrolase 88(GH88),sulfatases,substrate-binding proteins,carbohydrate transporters,and transcriptional regulators etc.In general,the genes encoding these proteins are physically linked in a carbohydrate active enzyme(CAZyme)gene cluster(CGC),also called polysaccharide utilization locus(PUL),which provides a finely tuned regulation of the GAG degradation pathway.GAG-CGCs have been systematically characterized in the Bacteroidetes phylum of the human gut microbiota,except for a hyaluronate(HA)-specific CGC.However,details of GAG-CGCs belonging to other phyla of the human gut bacteria are not characterized in detail yet.Given the importance of HGM’s GAG degradation for human health,it is essential to characterize the involved components and the underlying mechanisms,especially GAGs PLs,of various members of gut microbiota for GAG degradation.The dissertation has been thus formulated in an attempt to determine the GAGdegradation potential of various phyla of the gut microbiota,to investigate their GAG catabolism machinery,and to reveal their mechanistic differences compared to the GAG catabolism in Bacteroidetes.Furthermore,the dissertation includes the characterization of a HA-specific CGC from Bacteroidetes.The results of the dissertation are as follows:1.In silico analysis of the GAG-degradation potential of various non-Bacteroidetes phyla of the human gut microbiota.The prevalence of putative GAG-specific CAZymes and the associated GAG-CGCs in various non-Bacteroidetes phyla of the intestinal microbiota genomes was analyzed in silico.Our analyses revealed that Firmicutes,especially the genus Hungatella,contains a significant number of putative GAG-CAZymes and GAG-CGCs.While PL8 family proteins of Bacteroidetes that have been shown to be a major GAG polysaccharide lyase are generally periplasmic,the majority of PL8 proteins in Firmicutes possessed a secretory signal peptide,suggesting a possible extracellular degradation of the polysaccharide.In contrast to Firmicutes,two other major phyla of the HGM,Actinobacteria and Proteobacteria,do not seem to show a high prevalence of GAGCAZymes.Nonetheless,4 strains of the genus Collinsella of Actinobacteria were found to possess putative PL12 family proteins.Our analyses indicate that the PL12-CGC of Collinsella tanakaei TF08-14 could represent the first candidate GAG-degrading CGC in colonic Actinobacteria.Remarkably,although the phylum Fusobacteria was previously shown to be a non-GAG degrader,GAG-CGCs were revealed in multiple Fusobacteria strains.Of note,the identified non-Bacteroidetes GAG-CGCs frequently include transcription factors and transporter genes that are different from those of Bacteroidetes CGCs,indicting a potential mechanistic difference in the GAG utilization pathway.2.Isolation of Hungatella hathewayi,an efficient GAG-degrading Firmicutes from human gut and characterization of its chondroitin ABC exolyase with high activity and broad substrate specificity.Our bioinformatic analyses of fecal Firmicutes in previous chapter revealed that their genomes,especially those of Hungatella hathewayi strains,are an abundant source of putative GAG-specific catabolic enzymes.Subsequently,we isolated a Firmicutes strain,H.hathewayi N2-326,that can catabolize various GAGs.While H.hathewayi N2-326 was as efficient in utilizing chondroitin sulfate A(CSA)and dermatan sulfate as Bacteroides thetaiotaomicron.a well-characterized GAG degrader,it outperformed B.thetaiotaomicron in assimilating hyaluronic acid.Unlike B.thetaiotaomicron,H.hathewayi N2-326 could not utilize heparin.The chondroitin lyase activity of H.hathewayi N2-326 was found to be present predominantly in the culture supernatant.Genome sequence analysis revealed 3 putative GAG lyases,but only the HH-chondroitin ABC lyase was upregulated in the presence of CSA.In addition,five CAZyme gene clusters containing GAG metabolism genes were significantly upregulated when grown on CSA.Further characterization of the recombinant HH-chondroitin ABC lyase revealed that it cleaves GAGs predominantly in an exo-mode to produce unsaturated disaccharides as the primary hydrolytic product while exhibiting a higher specific activity than reported chondroitin ABC lyases.HH-chondroitin ABC lyase represents the first characterized chondroitin lyase from intestinal Firmicutes and offers a viable commercial option for the production of GAG oligosaccharides,and also for potential medical applications.3.Biochemical characterization of a Hyaluronate lyase from Enterococcus faecalis,an intestinal Firmicutes and a common opportunistic pathogen.Enterococcus faecalis is one of the most common Enterococcal commensals and also one of the most frequent opportunistic pathogens in human.Despite HA lyases being one of the most important virulent factors,E.faecalis HA lyase has not been biochemically characterized yet.In this part,bioinformatic analysis was first performed to reveal that HA lyases are widespread in the E.faecalis strains of the human gut microbiota.An HA lyase from E.faecalis(EFPL8)was identified then and characterized.Biochemical characterization showed that EFPL8 displayed an exclusive susbtrate specificity and a high activity on HA acting in an exomode with the unsaturated tetra-and disaccharide as the major hydrolytic products.Moreover,the importance of the aromatic patch of TrpTrp-Phe in the active site cleft for the activity of EFPL8 was demonstrated through homology modeling and mutational analysis.Ascorbic acid,a known aromatic patch inhibitor of Streptococcal HA lyases,was also found to inhibit the activity of EFPL8.Hence,the HA lyase of virulent E.faecalis strains can be targeted by ascorbic acid or its derivatives to provide a secondary line of defense against Enterococcal infections.While the high HA degrading activity of EFPL8 may give the bacterium advantage over other commensals in the competitive microbiotic environment,the identified enzyme can also provide an alternate commercial HA lyase candidate for medical and R&D applications.4.Biochemical characterization of two highly active PL35 hyaluronate lyases in hyaluronate-specific PUL of Bacteroides xylanisolvens.Despite the presence of an exhaustive knowledge of GAG degradation in Bacteroides,the information on a HA-specific PUL is lacking.In this chapter,bioinformatic analysis revealed that Bacteroides xylanisolvens contains 21 putative CAZymes specific for GAG,including 15 putative GAG lyases.Notably,B.xylanisolvens has two PL35 proteins,which are not present in B.thetaiotaomicron VPI-5482.These two PL35 proteins(BxPL35-1,and BxPL35-Ⅱ)encoding genes were found to be present in a PUL of Bacteroies xylanisolvens CNGBCC1 801 192 along with genes encoding a putative GH88,an HTCS regulator,SusC and SusD homologs,and a carbohydrate esterase family 20 protein.Further analysis reavealed that the PL35-PUL is also widespread in B.ovatus strains.Growth of B.xylanisolvens on HA was then examined,revealing that the bacterium showed a better growth on high-molecular weight HA(HAH)than lowmolecular weight HA(HAL).Biochemical characterization of the two PL35 family proteins showed their exclusive activity towards HA,displaying a significantly higher activity on HAH than previously characterized Bacteroides enzymes.In addition,similar to the substrate preference for the growth of the bacterium,both enzymes showed higher catalytic efficiency on HAH than HAL,with BxPL35-Ⅱ showing a substrate inhibition in the presence of HAL.Product analysis showed that while both PL35 enzymes seemed to degrade HA in an endolytic mode with relatively longer HA oligosaccharides being released in the initial phase of hydrolysis,BxPL35-Ⅰ accumulated much shorter oligosaccharides(tetra-and disaccharides)than BxPL35-Ⅱ.Altogether these results implicating a slightly different product specificity between these two enzymes.The characterization of HA-specific PL35 enzymes from Bacteroides xylanisolvens thus contributes to our understanding of HA degradation mechanism by Bacteroides strains.