Analysis of Interspecies Interactions of Periodontal Bacteria Using Microarrays.

摘要

Periodontitis is an oral bone resorption disease known to be associated with the gingival colonization of numerous bacteria; the most notorious of which is a group of bacteria known as the red complex, composed of Treponema denticola, Tannerella forsythia and Porphyromonas gingivalis. A fourth species, Fusobacterium nucleatum, is commonly associated with both healthy and diseased tissue and is thought to contribute to periodontal disease by providing anchorage for some periodontal pathogens. Animal and human studies indicate that the organisms are usually not individually sufficient for disease progression, suggesting cooperative pathogenesis occurs. However, very little is currently known regarding the interplay between the organisms that contribute to their association and pathogenic cooperation. The goal of this thesis was to provide a first step in the elucidation of the types of genes and processes that are altered upon the combinatorial pairing of periodontal bacteria, using Treponema denticola and Fusobacterium nucleatum as model organisms. The driving hypothesis is that physiologically relevant interactions do occur between species of periodontal bacteria and that the nature of the interactions depends on the pairing of bacteria.;These pilot studies required a powerful tool that would allow comparison of genetic processes between monospecies and dual species combinations. Microarray technology was chosen for its high-throughput capability and adequate sensitivity. To optimize the technology for use in proposed studies, microarray experiments were performed to analyze the stress response capabilities of T. denticola , about which very little is known. This approach allowed the identification of genes involved in heat stress, oxygen stress, osmotic stress and adaptation to blood exposure. A general stress response was identified that unified core elements of each stress response.;Extending the microarray application further, genes and processes involved in interspecies interactions of T. denticola were examined. The gene expression profiles induced by interacting bacteria seemed to correlate with their relationship with T. denticola. Whereas T. denticola responded with similar transcriptional profiles to the periodontal pathogens T. forsythia, P. gingivalis , no such response was observed for the distally associated oral bacterium S. sanguinis. Likewise, the transcriptional response to F. nucleatum shared none of the genes that were altered in response to T. forsythia or P. gingivalis, suggesting different types of relationships exist amongst red and orange complex bacteria. The significant overlap between the responses to the two red complex organisms, especially amongst downregulated genes, might reflect the cooperative relationships between red complex bacteria. In addition, numerous genes were identified that could prove relevant in interspecies communication and cooperation.;A similar methodology was applied to F. nucleatum and its interacting partners. We exploited its propensity to coaggregate and form biofilms with both early and late colonizers, and examined gene expression alterations in dual species biofilms. Similar to the case of T. denticola and its associative partners, similar relationship of interacting partners seemed to correlate with similar responses. For example, significant overlap was observed between the response to the late colonizers T. forsythia and T. denticola. Likewise, much of the response to the early colonizers S. sanguinis and S. gordonii overlapped. However, very little similarity was observed between the responses to early and late colonizers.;Expression of several outer membrane autotransporters were found to be altered by interacting bacteria. Mutant analysis of the autotransporters FN2047 and FN1526 revealed roles in phenotypic and transcriptional response to spent S. mitis medium and S. sanguinis cells, respectively. To our knowledge, this is the first time autotransporters of any kind have been implicated in a transcriptional response.;In conclusion, this thesis highlights: the components and organization of numerous stress responses of T. denticola; the different types of relationships that periodontal bacteria can have; and the novel roles of F. nucleatum autotransporter adhesins in environmental sensation.