Investigating Microbial-host Interaction In The Rumen Of Cows Using Single-Cell Sequencing And Simplified Microbiome Analysis

The rumen microbiome of cows has a complex microbial community structure,which coexists with the host for a long time and degrades plant polysaccharides to provide energy for the host.The interaction between rumen microbes and the host is not limited to energy supply,and rumen microbes have different degrees of influence on the host’s health status,methane production,and even development status.Up to now,more than 20,000metagenome-assembled genomes(MAGs)of rumen microbes have been constructed,and 18 cell subtypes have been identified in cow rumen tissues.Due to the complexity and diversity of rumen microbes and the diverse cell subpopulations in the rumen,the interaction between the rumen and the microbes has posed a challenge.To date,the interaction between rumen microbes and host rumen cells has not been accurately explained.Single-cell(nucleus)sequencing(sc RNA-seq,snRNA-seq)and metagenomic binning allow us to have a more detailed understanding of the composition of ruminal cells and the structure of rumen bacteria.By combined with artificial rumen systems,metaproteome and other technologies,combining artificial rumen systems,metaproteome and other technologies,we can have a multi-dimensional analysis of the key microbial structure and the functional diversity of ruminal cells.On the other hand,simplifying the complex microbial community structure(e.g.,the simplified human intestinal microbiota,SIHUMIx)is one of the effective ways to study Microbiome-Host Interactions(MHI).In this study,we performed metagenome sequencing and metaproteome under five different in vitro ruminal fermentation conditions,and combined with the results of rumen sc RNA-seq,we constructed a microbial genome set containing 2459 MAGs during in vitro ruminal fermentation and a rumen single-cell data containing 12 cell types.We finally constructed Protein-Protein Interaction Networks(PPI)between key rumen microbes and hosts.The roles of spinous cells and basal cells in maintaining the homeostasis of the rumen microenvironment were further elucidated.The main results obtained in this study are as follows:1.The core microbial community and crude feed-degrading bacterial community constitute a simplified rumen microbial community.First,a metagenomic dataset of the rumen under in vitro fermentation conditions was constructed,containing 2459 non-redundant MAGs,among which the percentages of Firmicutes＿A and Bacteroidota members reached 53.15% and 23.02%,respectively.By changing the proportion of crude feed(50% and 100%)in the rumen simulation technique system(RUSITEC),82 crude feed-degrading MAGs were finally enriched,and 298 MAGs of the core rumen microbiome(core MAG)were obtained through 5 in vitro fermentation conditions.Further,it was found that the occurrence frequency of core MAGs was higher than that of other microbial community under different individuals and different diets.Through annotation of these MAGs,the preferences of these MAGs on degrading plant polysaccharides were obtained.Thus,the simplified ruminal microbiota(SIRM)for subsequent analysis was obtained.2.Single-cell clustering and gene expression profiling in the cattle rumenAnalysis of snRNA-seq data from the rumen tissues of dairy cows revealed that the rumen tissue contained 9 different cell types were found in the rumen tissue,divided into 11 cell subtypes,including 2 clusters of endothelial cell types,5 clusters of epithelial cell types,1 cluster of immune cells,1 cluster of fibroblasts,and 2 clusters of unannotated unknown cell types.By cell similarity analysis and proposed temporal sequence analysis,mitotic cells were identified as complementary to epithelial cells,and two clusters of unknown cell types were found to potentially perform functions related to smooth muscle cells and basal cells,respectively.On the other hand,the high expression of lysozyme 1(LYZ1)by rumen epithelial cells,especially spinous cells,suggested that rumen spinous cells are involved in regulating the structure of the rumen microbiota.These results elucidate the gene expression and functional characteristics of rumen spinous cells and basal cells,and determine the potential interactions between rumen cells and microbes.3.Ruminal spinous cells and basal cells have a role in uptake and metabolism of volatile fatty acidsThe expression of SLC family,gene set of short chain fatty acid(SCFA)metabolism,ketone body production and Toll-like receptor in each rumen cells cluster was statistically analyzed using rumen snRNA-seq data.It was found that the genes related to SLC family during SCFA transport produced by microorganisms were highly expressed in both spinous cells and basal cells,especially SLC14A1 and SLC16A14,and basal cells scored higher in these gene sets than other cell types on SCFA metabolism and ketone body conversion.Further elucidating the division of labor between spinous cells and basal cells in transporting SCFAs produced by microbial metabolism,other cell types of the rumen also expressed genes related to SCFA transport and uptake simultaneously.In addition,Toll-like receptors were not expressed in any of the rumen cell types,suggesting that these cells may be less involved in receptor recognition by bacteria or viruses.Basal and spinous cells are the main members of the transport and metabolism of SCFAs,and other cell types of the rumen are involved in this process at different degrees.4.Ruminal microbes secrete a variety of extracellular proteins and participate in communication with the hostBy analyzing the rumen microbial extracellular proteomic data,it was determined that the rumen environment contains a large number of extracellular proteins derived from SIRM members,including carbohydrases,intracellular/surface moonlighting protein(ISMP),outer membrane proteins,etc.Firmicutes＿A and Bacteroidota contributed 86.6% of the extracellular proteins and five ISMPs accounted for 36.36% of all proteins.Extensive protein interactions between ISMP members PGK and host proteins were found by MHI prediction.In addition,these genes had the highest expression score in the rumen spinous cells cluster.This suggests that rumen microbes derive extracellular proteins to the environment and interact with the host,with the main site of interactions being within rumen spinous cells.In summary,we constructed a simplified rumen microbial genome dataset and rumen single-cell atlas by analyzing metagenome,secretory proteome and rumen snRNA-seq data to reconstruct the processes of microbial and host rumen cells in SCFAs production uptake and metabolism.This study has revealed that rumen microbial extracellular proteins are involved in substrate metabolism and interaction with host proteins,and has determined the division of labor and interaction mechanism of different rumen cell types in the process of communication with microbes.This study provides new insights into the communication between rumen microbes and hosts.