Study On The Molecular Mechanism Of The Biofilm Formation Of Clostridium Perfringens

In this study,optical microscope and scanning electron microscope were used to observe the surfaces of Clostridium perfringens biofilm after 12,24,48,and 72 h of incubation.RNA-seq and iTRAQ were selected to identify the differentially expressed genes and proteins of C.perfringens biofilm.The results will provide a theoretical basis for the prevention and treatment of C.perfringens and the development of new antibacterial agents.The results are as follows:(1)By optical microscope observation,it's found that the number of bacteria attached to the surface of glass slides gradually increased with the incubation time.After 24 h of incubation,C.perfringens formed micro colony,the area of local aggregation increased after 48 h,and a mature biofilm was formed after 72 h.By scanning electron microscopy observation,it's found that the bacteria connected to form a network structure after 12 h,and a mature biofilm was formed after 48 h,the biofilm cells encased in a dense,self-secreted matrix material.(2)We carried out comparative transcriptomic analysis between biofilm and planktonic cells using RNA-sequencing.In total,1162 genes were significantly differentially expressed,with 413 being up-regulated and 749 down-regulated.The GO enrichment results showed that the up-regulated genes were most in the metabolic process and binding,and the down-regulated genes were most in the heterocyclic metabolic processes,cell membrane and transferase activity.The KEGG enrichment results showed that up-regulated genes involved in oxidative phosphorylation metabolism,glycolysis/gluconeogenesis,butyric acid metabolism and so on.Besides,C.perfringens biofilm was regulated by two component systems,including OmpR family,NtrC family and spore forming family.In addition,the Agr quorum sensing system also regulated the formation of C.perfringens biofilm.To validate the RNA sequencing results,qRT-PCR analysis was performed using thirteen selected differentially expressed genes,the result showed that 6 genes were up-regulated and 7 genes were down-regulated,which was in accordance with the RNA sequencing results.(3)The proteomic analysis of C.perfringens biofilm was carried out by iTRAQ.Result showed that there were 153 significantly differentially expressed proteins between biofilm and planktonic cells,with 48 being up-regulated and 105 down-regulated.Differentially expressed proteins were enriched in 94 GO functional categories,biological process included cell redox homeostasis,de novo pyrimidine nucleobase biosynthetic process,metal ion transport and so on,and molecular function included phospholipase C activity,oxidoreductase activity,protein disulfide oxidoreductase activity and so on.The differentially expressed proteins were enriched in 52 pathways,such as pyrimidine metabolism,longevity regulating pathway and FoxO signaling pathway.(4)We analyzed the correlation between transcriptome and proteome,the result showed that there were 77 differentially expressed genes(proteins)identified by transcriptome and proteome.Up-regulated genes which expression level was consistent at both mRNA and protein level were mainly involved in pyruvate metabolism and pyrimidine metabolism,and down-regulated genes which expression level was consistent at both mRNA and protein level were mainly involved in pyrimidine metabolism,starch and sucrose metabolism,carbon metabolism and thiamine metabolism.