A proteomic investigation of Fusobacterium nucleatum alkaline-induced biofilms

Abstract

BackgroundThe Gram negative anaerobe Fusobacterium nucleatum has been implicated in the aetiology of periodontal diseases. Although frequently isolated from healthy dental plaque, its numbers and proportion increase in plaque associated with disease. One of the significant physico-chemical changes in the diseased gingival sulcus is increased environmental pH. When grown under controlled conditions in our laboratory, F. nucleatum subspecies polymorphum formed mono-culture biofilms when cultured at pH 8.2. Biofilm formation is a survival strategy for bacteria, often associated with altered physiology and increased virulence. A proteomic approach was used to understand the phenotypic changes in F. nucleatum cells associated with alkaline induced biofilms. The proteomic based identification of significantly altered proteins was verified where possible using additional methods including quantitative real-time PCR (qRT-PCR), enzyme assay, acidic end-product analysis, intracellular polyglucose assay and Western blotting.ResultsOf 421 proteins detected on two-dimensional electrophoresis gels, spot densities of 54 proteins varied significantly (p < 0.05) in F. nucleatum cultured at pH 8.2 compared to growth at pH 7.4. Proteins that were differentially produced in biofilm cells were associated with the functional classes; metabolic enzymes, transport, stress response and hypothetical proteins. Our results suggest that biofilm cells were more metabolically efficient than planktonic cells as changes to amino acid and glucose metabolism generated additional energy needed for survival in a sub-optimal environment. The intracellular concentration of stress response proteins including heat shock protein GroEL and recombinational protein RecA increased markedly in the alkaline environment. A significant finding was the increased abundance of an adhesin, Fusobacterial outer membrane protein A (FomA). This surface protein is known for its capacity to bind to a vast number of bacterial species and human epithelial cells and its increased abundance was associated with biofilm formation.ConclusionThis investigation identified a number of proteins that were significantly altered by F. nucleatum in response to alkaline conditions similar to those reported in diseased periodontal pockets. The results provide insight into the adaptive mechanisms used by F. nucleatum biofilms in response to pH increase in the host environment.

Environmental Stimuli Shape Biofilm Formation and the Virulence of Periodontal Pathogens

“…F. nucleatum cells appear to change their metabolism in response to high pH, because some enzymes of the glycolytic pathway, as well as glutamic acid and histidine catabolism, are downregulated in planktonic cells grown at an alkaline pH of 7.8 [75]. However, when the pH is further raised to 8.2, which induces biofilm formation, the amounts of glycolytic enzymes do not increase, whereas glucose storage and lactate production increases [77]. In contrast to increased glucose storage, the production of various proteins involved in protein synthesis decrease at high pH values in F. nucleatum biofilm [77].…”

“…However, when the pH is further raised to 8.2, which induces biofilm formation, the amounts of glycolytic enzymes do not increase, whereas glucose storage and lactate production increases [77]. In contrast to increased glucose storage, the production of various proteins involved in protein synthesis decrease at high pH values in F. nucleatum biofilm [77]. At pH 8.2, another change in the expression of metabolic enzymes is the increased production of glutamate dehydrogenase, which might indicate that the bacterium adjusts its metabolism to the increased concentration of glutamate in the gingival crevicular fluid associated with inflamed periodontal tissue [77].…”

“…In contrast to increased glucose storage, the production of various proteins involved in protein synthesis decrease at high pH values in F. nucleatum biofilm [77]. At pH 8.2, another change in the expression of metabolic enzymes is the increased production of glutamate dehydrogenase, which might indicate that the bacterium adjusts its metabolism to the increased concentration of glutamate in the gingival crevicular fluid associated with inflamed periodontal tissue [77]. The cellular stress response in bacteria might also be activated at an alkaline pH because F. nucleatum cells upregulate the expression of peptidyl-prolyl cis -trans isomerase (PPI) and the heat-shock protein GroEL [75,77].…”

“…At pH 8.2, another change in the expression of metabolic enzymes is the increased production of glutamate dehydrogenase, which might indicate that the bacterium adjusts its metabolism to the increased concentration of glutamate in the gingival crevicular fluid associated with inflamed periodontal tissue [77]. The cellular stress response in bacteria might also be activated at an alkaline pH because F. nucleatum cells upregulate the expression of peptidyl-prolyl cis -trans isomerase (PPI) and the heat-shock protein GroEL [75,77]. Of these proteins, GroEL might have a role in the host-bacterium crosstalk because GroEL-like proteins may modulate the host immune response, as shown with F. nucleatum [78] and A. actinomycetemcomitans [79].…”

“…In particular, the proteins that are involved in adhesion to other periodontal pathogens as well as to host cells may play important roles in virulence. When F. nucleatum was grown at an alkaline pH of 8.2, elevated levels of FomA adhesion isoforms were detected [77]. FomA may function as recruiter of other periodontal pathogens, such as P. gingivalis , [81], and has been proven to be a possible vaccine target in a mouse study [82].…”