Periodontal diseases are bacterial-associated inflammatory diseases of the supporting tissues of the teeth, and one of the more aggressive forms of chronic periodontitis is characterized by the destruction of the tooth's supporting structures. Chronic periodontitis has a large economic burden and is a major public health problem. Three bacterial species (Porphyromonas gingivalis, Treponema denticola, Bacteroides forsythus) have been shown to be closely associated with chronic periodontitis in humans, and a commensal oral bacterium Fusobacterium nucleatum has been implicated in aiding the colonization of the periodontopathic bacteria. P. gingivalis, T. denticola, B. forsythus and F. nucleatum exist as part of a high density mixed species (polymicrobial) biofilm in the gingival crevice of humans. Recent evidence using rodent abdominal lesion models has indicated that co-infection of B. forsythus or T. denticola with P. gingivalis enhances lesion development. This synergistic interaction that enhances lesion severity, and potentially virulence of the bacteria, has not been studied in an animal periodontitis model. Further, outer membrane proteins that are altered during this interaction also have not been studied. The broad objective of this application is to determine the impact of interactions between P. gingivalis, T. denticola, B. forsythus and F. nucleatum on the level of bone loss and host antibody response in the murine periodontitis model and to characterize the major antigenic outer membrane proteins of these bacteria using 2D PAGE and mass spectrometric methods. Using these techniques, we have already identified over thirty P. gingivalis outer membrane proteins. We have also established a murine periodontitis model based on oral infection of P. gingivalis. These methodologies will be applied to determine the impact of bacterial interactions on the expression of major antigenic outer membrane proteins and on colonization, bone loss and host immune response. The significance of this work is that it will increase our knowledge of the role of bacterial interaction on polymicrobial virulence in the murine periodontitis model and may have a broader significance in the study of human mucosal pathogens.
The specific aims of the application are: 1) To determine the extent of alveolar bone loss and level of bacterial colonization in the murine periodontitis model when co-inocula of P. gingivalis, T. denticola, B. forsythus and/or F. nucleatum grown as monocultures are used to orally infect mice compared with mono-inoculation of each species. 2) To determine the extent of alveolar bone loss and bacterial colonization in the murine periodontitis model when polymicrobial cultures of P. gingivalis, T. denticola, B. forsythus and/or F. nucleatum are used to orally infect mice compared with mono- and co-inocula of the bacteria grown as monocultures as in (1) above. 3) To determine the host antibody isotype response to each of the bacteria in the murine periodontitis model when co-inocula or polymicrobial cultures of P. gingivalis, T. denticola, B. forsythus and/or F. nucleatum are used to orally infect mice. 4) To identify the major outer membrane and antigenic proteins of P. gingivalis, T. denticola, B. forsythus and F. nucleatum grown in monoculture and as polymicrobial cultures using 2D-gel electrophoresis, mass spectrometry and Western blotting.
