Study On The Surface Glycans Of Gut Microbiota

Carbohydrate exists widely in nature and is an important part of the earth's biomass.At the same time,for organisms,carbohydrate is an indispensable part,which has very important functions.In microorganisms,carbohydrate plays a role in energy source,structural support,infection,and colonization.Surface polysaccharide is an important part of the outer wall of bacteria.Bacterial surface polysaccharides can be divided into peptidoglycan,teichoic acid,lipopolysaccharide and capsule polysaccharide.Although their composition varies,they both support bacteria and protect them from harsh environments.And some bacteria mimic their surface polysaccharides as the host's own,evading the host's immune system and causing diseases.There are more than 1000 kinds of microorganisms in our human gut,with the number reaching 1014 and the weight reaching 0.2 kg.Microbiota in the gut are so diverse and important,but little is known about their surface polysaccharides.This is mainly because there are few detection methods for surface polysaccharides of gut microbiota,which hinders our analysis of surface polysaccharides on gut microbiota.The content of this paper is divided into three parts.The first part is to study the relationship between gut microbiota and blood types,by labeling the surface blood type antigens of gut microbiota with antibodies and identifying gut microbiota containing blood type antigens(Chapter 2).The second part is to label the end monosaccharides of gut microbiota and identify the gut microbiota with specific end monosaccharides(Chapter 3).The third part is the chemoenzymatic labeling of Neu5Ac?2-3Gal on the surface of gut microbiota and the high-throughput identification of gut microbiota containing Neu5Ac?2-3Gal(Chapter 4).Part 1:Firstly,we analyzed the relationship between gut microbiota and blood types by 16S rRNA gene sequencing.And we found that there was no significant difference in the composition of gut microbiota among Chinese population with different blood types.Then,we labeled the gut microbiota of individuals with four blood types by blood type antibodies,then was tested with flow cytometry.It was found that the gut microbiota have blood types as human.We further identified the gut microbiota containing A blood group antigen from the intestinal tract of A blood group people and the gut microbiota containing B blood group antigen from the intestinal tract of B blood group people by the combination of antibody,immunomagnetic bead and 16S rRNA gene sequencing technology.After the analysis of their composition and abundance,we found that there were significant differences in OTU,Beta diversity and phylum compositions between the two groups,which indicated that the gut microbiota containing A blood group antigen in the intestinal tract of A blood group individuals was different from that of B blood group individuals.Our findings suggest that blood type is an independent genetic factor that influences gut microbiota,and gut microbiota,as an organ,can express blood type antigens as well as other organs.Part 2:In this section,we use Griffonia Simplicifolia Lectin ?(GSL ?)Lectin as an example that can specifically identify ?-Gal and ?-GalNAc to achieve the specific labeling of E.coli O86:H2 containing ?-Gal.We also use six lectins to label the terminal monosaccharides on the surface of human gut microbiota,and find that the coverage of?-Fucose on human gut microbiota surface is lowest.The abundance of ?-Mannose and?-Galactose is the highest on the surface of gut microbiota.Then,we use GSL ? and UEA ? lectins to screen gut microbiota with terminal ?-Gal/?-GalNAc and ?-Fuc,then identify their species.It is found that the main species of gut microbiota screened by these two lectins is similar,but the proportion of specific composition is very different.Our findings suggest that lectins can be used to rapidly and comprehensively label and identify gut microbiota with monosaccharides without culturation in vitro.Part 3:In this section,Streptococcus agalactiae containing Neu5Ac?2-3Gal is specifically labeled by the chemoenzymatic method,which is developed by Wen et al.,and Neu5Ac?2-3Gal on the surface of gut microbiota from healthy people is also labeled by this method.Next,we combine chemoenzymatic method and magnetic beads to screen the gut microbiota containing Neu5Ac?2-3Gal,and then identify the gut microbiota with Neu5Ac?2-3Gal by 16S rRNA gene sequencing.We find that gut microbiota containing Neu5Ac?2-3Gal mainly focus on genera Faecalibacterium Pseudomonas,Psychrobacter and Chryseobacterium.After comparing Bacterial Carbohydrate Structure DataBase(BCSDB),we find these four genera have not been reported to express Neu5Ac?2-3Gal.Our findings suggest that chemoenzymatic method can be used to rapidly and comprehensively label and identify gut microbiota with specific glycans in vitro.In conclusion,we use the antibody,lectin and chemoenzymatic method to label the carbohydrate structures on the surface of human gut microbiota.Furthermore,each labeling method is combined with magnetic beads and 16S rRNA sequencing to identify the gut microbiota containing specific carbohydrate.We establish a new method for the study of gut microbiota and surface carbohydrate.