| Metaproteomics is the large-scale characterization of the entire protein complement of environmental microbiota at a given point in time.Compared to metagenomics,metaproteomics has the advantage to characterize not only the composition of the environmental microbiome,but also their functional dynamics.The oral cavity is one of the five major human body sites(intestine tract,oral cavity,skin,nasal cavity,and genitourinary tract)that are included in the Human Microbiome Project(HMP).The imbalance of oral micro-ecology can not only induce a variety of oral diseases,but are also closely related to systemic diseases such as tumors,diabetes,rheumatoid arthritis,cardiovascular disease,and preterm birth,showing significant impacts on human health.Compositional and functional characterization of the human oral microbiota by using the state-of-the-art oral metaproteomic technologies could greatly benefit the development of diagnostic test and treatment of these diseases.Oral metaproteomic study has obtained some progress recently,but its data analysis workflows seems not satisfied at present.The Human Oral Microbiome Database(HOMD)is the most frequently used database in oral metaproteomic analysis.However,it does not include a large number of microorganisms that have been identified by recent metagenomic studies.The incompleteness of the microbial proteome database may lead to incorrect metaproteome identification.In addition,there is still a lack of user-friendly metaproteomics data analysis workflows.Therefore,in order to obtain a more comprehensive and accurate oral metaproteomic results,we tried to construction of the human oral metaproteome plus database(HOMPPD)and establish the oral metaproteomic data analysis workflow based on HOMPPD.Periodontitis is one of the common oral diseases that can cause inflammation of the tissues surrounding the teeth.In severe cases,the gums may fall off the teeth or the teeth may loosen and fall off directly.Gingival crevicular fluid is a kind of oral body fluids.As serum exudate or inflammatory exudate,it is capable to reflect the host's response to microbial plaque attacks.Therefore,the host and microbial proteomes of gingival crevicular fluid could provide information for the diagnosis of periodontal and systemic diseases.Metaproteomic analysis has been applied to identify periodontitis-related microbial changes in saliva and plague.However,to the best of our knowledge,no such study has been performed on periodontitis gingival crevicular fluid.This study is composed of two main parts.The first part is the establishment of an optimized oral metaproteomic data analysis workflow.It mainly includes the following research contents:(1)Construction of Human Oral MetaProteome Plus Database(HOMPPD);(2)Establishment of the oral metaproteomic data analysis workflow based on HOMPPD;(3)Application of the HOMPPD and its workflow in the re-analysis of public saliva proteome data.The second part is the study of alternations in the gingival crevicular fluid proteome in periodontitis.It mainly includes the following research contents:(1)Comparison of the composition of human and microbial proteomes between periodontitis and normal groups;(2)Identification of differentially expressed proteins in periodontitis;(3)Investigation of microbial flora changes in periodontitis.The study obtained the following findings: 1.The Human Oral MetaProteome Plus Database(HOMPPD)was constructed by integrating HOMD and the proteome sequences from 95 genera reported by two published metagenomic studies.HOMPPD includes a total of protein sequences from 184 genera and 2793 species.It is available for downloading at ftp://111.198.139.72:4000//pub//metaproteomics//homppd.fasta.2.An oral metaproteomic data analysis workflow was established baded on HOMPPD with a two-step searching strategy,microbial taxonomy identification and downstream bioinformatic analysis.The HOMPPD is the mainly innovational achievement in this workflow.3.HOMPPD were tested by the public saliva proteome raw dataset,results showed that HOMPPD could confidentially identified an extra of 23.98% peptides,39 genera and 124 species that are not included in HOMD.This result indicates that using HOMPPD instead of HOMD could achieve a more comprehensive characterization of the oral microbiome.4.The usefulness of the established HOMPPD and workflow was tested by Re-analysis of the same public saliva proteome raw dataset.It was observed that the saliva microbiota was mainly distributed in the FCB group and four phyla(Proteobacteria,Fusobacteria,Firmicutes,Actinobacteria).Additionally,a high level of individual difference in saliva microbial composition was observed.It also shows that the workflow established above has good practicability,at the same time.5.The human and microbial proteomes in the gingival crevicular fluid from eight patients with periodontitis and healthy controls were qualitatively and quantitatively analyzed,respectively.It was found that in both periodontitis and normal groups,the bacterial proteins accounted for approximately one-third of all identified proteins.And a total of 166 differentially expressed human proteins were identified.Among them,50 proteins that were highly expressed in the periodontitis group are mainly related to immune functions and the inflammatory responses.A total of 67 differentially expressed bacterial proteins were identified.Among them,31 that were highly expressed in the periodontitis group are mainly related to the metabolism of amino acid,carbohydrate and lipid transport.6.The result of bacterial taxonomy identification exhibited decreased total abundance but increased the diversity of bacterial genera in the periodontitis group,in which five genera were uniquely identified.A phylogenetic tree of genera identified in both groups was constructed.Bacteria identified in both group mainly belong to four phyla(Fusobacteria,Proteobacteria,Actinobacteria,and Firmicutes)and one other taxon,FCB group,while the five genera that were uniquely identified in the periodontitis group belong to Firmicutes.Individuals in the two groups could be well separated by the principal component analysis that was performed based on the abundances of commonly identified genera in both groups.The above results suggest that both qualitative and quantitative changes occurred in the periodontitis patients.Differential analysis identified 10 genera that were highly expressed in the periodontitis group.The dominant bacteria genus in the two groups was different;it was Treponema in the periodontitis group and Neisseria in the normal group.In summary,this study constructed a HOMPPD and a complete metaproteomic data analysis workflow from peptide identification to microbial taxonomy identification.In this workflow,HOMPPD was constructed to collect more comprehensive protein sequence information of oral microbiota.The use of HOMPPD in database searching could offer more comprehensive identification of oral microbiome composition.This study then applied this workflow to study the gingival crevicular fluid proteome of patients with periodontitis and normal subjects.Alternations in human and bacterial protein abundances,as well as bacterial compositions in the periodontitis group,were identified,providing valuable information for the studies of disease pathogenesis and biomarker discovery of this disease. |
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