Biofilm and planktonic lifestyles of Porphyromonas gingivalis and Fusobacterium nucleatum. Proteomic analysis of bacteria grown as planktonic cells, mono- and dual species biofilm, and characterization of the biofilm extracellular polymeric matrix’
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Periodontitis is one of the most prevalent infectious diseases affecting humans. Periodontitis leads to the destruction of the dental support tissues, which in the terminal stage causes loss of teeth. Periodontitis is biofilm related, a situation where several bacterial species are organized as a community whose resident species differ in many respects from their planktonic (free-living) counterparts. Fusobacterium nucleatum and Porphyromonas gingivalis are among the subgingival bacterial species that play a major role in the dental biofilm formation. F. nucleatum acts as a bridge between early and late colonizers in the dental biofilm and coaggregates with almost all the species that are considered putative periodontal pathogens. P. gingivalis harbors many virulence factors that facilitate colonization and invasion of the periodontal epithelial lining. The main aim of this project was to study in depth and characterize in vitro a dual species biofilm composed of F. nucleatum and P. gingivalis using molecular imaging techniques and proteomics. Furthermore, we wanted to explore the extracellular polymeric substances in the biofilm matrix of the dual and mono-species biofilm, followed by protein identification and analysis of their differential expression.
Our results show that proteins and carbohydrates are the major components of the biofilm matrix, and that extracellular (eDNA) is also present. The matrix components are also shown to vary among the species. Proteinase K enzyme showed no effect on the concentration of the eDNA or carbohydrate isolated from the treated matrices. DNase I and proteinase K enzymes had no significant effect on biofilm formation or on mature biofilms under the conditions studied. In the flow-cell biofilm model, F. nucleatum was able to grow in partially oxygenated conditions while P. gingivalis failed to form a biofilm alone under similar conditions but it can grow with F. nucleatum as a dual species biofilm.
We identified 542, 93 and 280 proteins from the matrices of F. nucleatum, P. gingivalis, and the dual-species biofilms, respectively. Nearly 70% of all matrix proteins in the dual-species biofilm originated from F. nucleatum, and a majority of these were cytoplasmic proteins, suggesting enhanced lysis of F. nucleatum cells. The proteomic analysis also indicated an interaction between the two species: 22 F. nucleatum proteins showed differential levels between the mono and dual-species extracellular polymeric matrices (EPMs), and 11 proteins (8 and 3 from F. nucleatum and P. gingivalis, respectively) were exclusively detected in the dual-species EPM. Oxidoreductases and chaperones were among the most abundant proteins identified in all three EPMs. The biofilm matrices also contained several known and hypothetical virulence proteins, which can mediate adhesion to the host cells and disintegration of the periodontal tissues.
Comparisons between the protein profiles for the two bacterial species grown as a biofilm or in the planktonic state, and when grown as a mono- or dualspecies biofilm, showed significant differences between each setting examined. The most abundant proteins have function such as oxidoreductases, acyltransferases, outer membrane proteins and proteases. Several virulence factors were among the most abundant proteins in both biofilm and planktonic growth conditions. Vitamin B biosynthesis proteins were increased in the biofilm setting compared to the planktonic. When grown in dual species, P. gingivalis showed reduced protein levels in many functions including vitamin biosynthesis, nucleotide biosynthesis, lipid or fatty acid biosynthesis and translation and ribosomal process. These results indicated how growing in a community provides a favorable environment to P. gingivalis and reduces its stress.