Growth pH and Expression of GroEL and DnaK by Fusobacterium

Adult periodontitis is a destructive chronic inflammatory disease of oral connective tissues and bone and has an infective aetiology involving different bacteria. Fusobacterium nucleatum is a Gram-negative anaerobe that has been implicated in the pathogenesis of oral diseases including pulpal infection, alveolar bone abscesses and periodontal diseases. Its synergism with other bacteria in mixed infections is associated with its dominant role in the establishment of plaque biofilms. The physiology and pathogenic potential of (oral) bacteria maybe influenced by changes in environmental conditions leading to the synthesis of specific proteins known as heat shock proteins (HSPs). GroEL (HSP60) and DnaK (HSP70) act as molecular chaperones in the assembly and folding of proteins and are considered to be immunodominant antigens in many human pathogens; the presence of common epitopes in host and microbial HSPs have led to the hypothesis that chronic inflammatory reactions, such as periodontal diseases, may be due to defects in the ability to regulate the anti-self capability. During the onset of periodontal disease the pH of the periodontal pocket increases with its depth and also with the severity of the host inflammatory response. Objectives: To study the effect of growth pH on the expression of GroEL and DnaK. Methods: F. nucleatum ATCC 10953 was grown anaerobically in continuous culture, D=0.07h-1, at 37oC. Cells were harvested following equilibration (10 generations) at pH 7.4, and 6.4 respectively. DnaK and GroEL were identified by mLC tandem Mass spectrometry following 2D-PAGE separation of cytosolic proteins. Expression analysis was performed using PD-Quest imaging software. Results: Expression of both DnaK and GroEL was down-regulated at least 2 fold when grown at pH 6.4 compared to 7.4. Conclusion: Expression of the immunodominant HSPs, GroEL and DnaK by F. nucleatum is influenced by environmental growth pH. Authors gratefully acknowledge support from the Australian Dental Foundation Inc.