1. Molecular Techniques in Oral Microbial Taxonomy, Identification and Typing

Sirkka Asikainen and Maribasappa Karched

The rapid development of molecular techniques during the past decade has revolutionized the field of microbiology. Two issues are of profound importance. First, the discovery of phylogenetically informative DNA sequences, such as the 16S rRNA gene, radically changed the concept of bacterial relatedness and provided a universal system for bacterial identification and categorization. Second, it became possible to detect, identify, and type bacteria independent of their cultivability and, by these new means, to elucidate the diversity and spatial organization of complex oral bacterial communities. Of considerable benefit has been the fact that the same nucleic acid-based molecular approaches can be applied in all microbial environments, ranging from the oral cavity, to the surfaces of historical monuments, to the depths of open oceans. This has led to development of versatile PCR- and hybridization-based techniques that allow a rapid and convenient analysis of the bacterial contents of oral samples and offer previously unattainable possibilities for expanding studies on bacterial epidemiology and characterization. In particular, the emerging new microarray technology will facilitate great strides in understanding the structure and dynamics of oral bacterial communities and bacteria-host interactions, and will form the basis for developing novel diagnostics for oral infections.

2. Applied Genomics of Some Oral Bacteria

Stuart G. Dashper, Paul D. Veith, Ching Seng Ang, Peter S. Zilm and Eric C. Reynolds

The simultaneous and inter-related rapid advances in genome sequencing, mass spectrometry and computing power have provided the tools for examining oral bacteria and their interactions with each other and the host at a global molecular level. In this chapter we focus on the development and usage of applied genomics techniques, mainly proteomics, for the study of Gram-negative anaerobic bacterial species found primarily in subgingival plaque. Proteomic analyses have already been applied to the discovery and characterization of proteins that comprise the proteome of Porphyromonas gingivalis and are now being applied to aid our understanding of the effects of environmental cues on the proteomes of oral bacteria, including Fusobacterium nucleatum, Tannerella forsythia and Treponema denticola. These species share the same environment and have been shown to interact with each other on a number of levels. We examine the range of currently-available proteomic methodologies that can be used to investigate the elaborate interactions between cells in the complex microbial biofilm communities found in the oral cavity and the interaction of these bacteria with the host.

3. Horizontal gene transfer in Oral bacteria

Alex Mira

Analyses of gene phylogenies and DNA-composition reveal that an important source of genetic innovation in prokaryotes is the transfer of sequences from other bacteria. The advent of genomic data now allows determination of the importance of horizontally-transferred genes on a genomic scale. By comparative analysis of fully- sequenced oral bacteria and between them and non-oral relatives, it is clear that horizontal gene transfer (HGT) is extremely frequent in oral pathogens and that this process probably also takes place also between bacteria and oral fungi. It is likely that HGT is facilitated, in oral biofilms such as dental plaque, by the close physical contact between phylogenetically distant bacteria. . In some cases, certain sets of genes are shared between more than two species co-inhabiting the mouth ecosystem, supporting the idea that there is an environment-associated gene pool formed by sequences of special adaptive importance. This habitat gene reservoir is also present in other natural ecosystems. A survey of HGT in oral bacteria is presented, as well as the evolutionary and biological consequences of this process.From the applied viewpoint, some of these genes could be potential vaccine candidates, targeting more than one potentially pathogenic species.

4. Cell-cell Communication in Oral Microbial Communities

Alex H. Rickard, Gilad Bachrach and David G. Davies

Most of the bacterial species found in the mouth belong to microbial communities, called "biofilms", a feature of which is inter-bacterial communication, mediated by two distinct phenomena. The first is through direct cell-cell contact, which is mediated by specific protein "adhesins" and often, as in the case of inter-species aggregation, by complementary polysaccharide receptors.Intra (auto-) and inter-species (co-) aggregation both promote ordered successional integration of species into the biofilm.The second method of communication invoves cell-cell signalling molecules, which are of two classes; those used for intra-species and those used for inter-species signalling. An example of the former is "quorum sensing", a process in which acyl-homoserine lactones (AHLs) induce members of the same population to produce and release specific enzymes or to initiate biofilm formation. As yet, no oral bacteria have been shown to produce AHLs. However, they can produce small peptides, such as "competence stimulating peptides", which have been shown to mediate intra-species signalling and to promote single-species biofilm formation. A common form of inter-species signalling is mediated by 4, 5-dihydroxy-2, 3-pentanedione (DPD). This spontaneously forms a family of inter-convertible compounds, collectively called "Autoinducer-2" (Al-2). With respect to oral biofilm communities, the present review will focus on the molecular basis of communication and the effects of cell-cell contact and signal molecules on gene expression. A model relating inter-species cell-cell communication and biofilm development is proposed.

5. The Molecular Biology of Cariogenic Bacteria

Roy R. B. Russell

Molecular biology techniques have made a major contribution to our understanding of dental plaque bacteria, helping to clarify the taxonomic relationships of the various species of oral bacteria and their association with dental caries. Methods developed for gene cloning and targeted gene knockout have allowed analysis of the function of individual genes and their contribution to aspects of cellular adhesion and physiology that may be important for cariogenicity. The availability of the Streptococcus mutans genome sequence has given access to new tools for discovering novel genes and exploring their regulation, as well means by which diversity within the species can be explored.

6. Molecular Windows into the Pathogenic Properties of Aggregatibacter actinomycetemcomitans: A Status Report with a View to the Future

Daniel H. Fine, David Figurski, Scott Kachlany and Jeffrey Kaplan

Three lines of evidence implicate Aa as an oral pathogen; 1) virulence factors established at the genetic level that affect; attachment to soft and hard tissues, and interference with both innate and acquired arms of host defense, 2) a powerful association of Aa with localized aggressive periodontitis in young adolescents, and 3) prospective evidence that Aa initiates bone loss in an animal model. The chapter will review; 1) molecular proofs that have provided evidence of Aa genes that are critical for its pathogenic nature. In addition the chapter will describe new strategies designed to reflect complex bacterial-host interactions. It is suggested that these new approaches may help to develop insights into the role of Aa in disease initiation and progression as well as its role in adaptation to its host. The chapter concludes with a recommendation that our focus on eliminating bacteria might profit from a shift toward exploration of bacterial strategies for adaptation and survival in the environment of the human host. The lessons learned from this paradigm shift might serve to allow us to gain a better understanding of the beneficial outcomes derived from the lives we share with bacteria.

7. The Molecular Biology of Porphyromonas gingivalis

Margaret J. Duncan

Porphyromonas gingivalis is a Gram-negative oral anaerobe strongly associated with chronic adult periodontitis. The bacterium is relatively easy to grow, produces a number of well-characterized virulence factors, and can be manipulated genetically. The availability of the genome sequence will enhance our understanding of the biology of the P. gingivalis, and how it interacts with the environment, other bacteria, and the human host. Beginning with a brief description of the genome, this chapter proceeds with a discussion on strain variability, and the relationship of P. gingivalis to other oral and enteric Bacteroidetes. The genetic methods we use with P. gingivalis are based on those for the enteric Bacteroides, and the development and use of these systems is discussed. Among the most studied P. gingivalis genes are those that code for functions that either interact directly with the human host cells or protect the bacterium from host activities. Several recent studies are presented to illustrate the technologies and the scope of this research. Finally, new studies to determine the molecular mechanisms of gene regulation are described.

8. The Molecular Biology of the Survival and Virulence of Treponema denticola

Marie-Claude Jobin, Mohsen Amin and Richard P. Ellen

The levels of oral spirochetes are elevated in patients with periodontal diseases. Among this group, Treponema denticola is the most studied and is considered as one of the main etiological bacteria of periodontitis. The environment in which this bacterium thrives has shaped it into the motile and highly proteolytic bacterium that we know today. Application of new molecular biology tools and genomic analysis has contributed greatly to the understanding of this fastidious bacterium. Mutagenesis of specific genes and the complete genome sequence of the Type strain, ATCC 35405, have given new insights into its intrinsic survival mechanisms. This chapter presents a review of the latest reports on T. denticola genes and proteins implicated in motility, growth and virulence.

9. The Molecular Basis of Host-Pathogen Interaction in the Oral Cavity

Janina P. Lewis

The interaction between oral microflora and eucaryotic cells is highly complex and involves active processes allowing both types of partners to co-exist. The microbial encounter with the host is initiated by attachment of the organism to the host; and bacterial surface proteins, termed "adhesins", mediate this step. Bacteria also secrete mediators that bind or invade the host. The microbial attachment, as well as the encounter with the secreted proteins, serve as signals that are deciphered through multi-mediator cascades ultimately affecting gene expression in the host. Many receptors recognizing the pathogens and mediating the host response, as well as the variety of microbial molecules triggering the host response, have been demonstrated. Also, the application of high-throughput microarray analyses have revealed that many more players remain to be investigated, as well as providing insight into the complex nature of the host- microbe interaction. The recent advances regarding both the host and microbial players involved in the host-pathogen dialogue, as well as possible mechanisms utilized by pathogenic bacteria to evade the host protective recognition mechanisms, will be discussed. Finally, new directions, with regard to the investigation of the molecular mechanisms of the host-pathogen interaction, are discussed. This includes genomic approaches to investigating host-pathogen interactions on a large scale.

10. The Molecular Basis of Host Defense Mechanisms in Oral Disease

Alex B. Berezow, Lijian Jin and Richard P. Darveau

The innate defense status of clinically healthy periodontal tissue is highly orchestrated. For example, histochemical, immunohistochemical, and in situ studies examining clinically healthy human periodontal tissue have revealed that neutrophils routinely transit through periodontal tissue from the highly vascularized tissue surrounding the tooth root surface to the gingival crevice located outside of periodontal tissue. Several innate defense response mediators including IL-8, ICAM, and E-selectin, have been shown to be expressed in clinically healthy tissue and have been proposed to facilitate neutrophil trafficking. Both host developmental programs and the host protective response to oral bacterial commensal colonization contribute to the highly organized tissue structure and molecular expression of select innate defense components.

11. Molecular Approaches to Vaccination against Oral Infections

George Hajishengallis and Michael W. Russell

Given that both dental caries and periodontitis have an infectious etiology, immunization has been proposed as a means of controlling them. However, the approaches vary according to the nature of the bacteria involved and the mechanisms of pathogenesis for these two very different diseases. For dental caries, surface and secreted proteins involved in colonization of teeth by mutans streptococci , and specific functional domains derived from these, have been identified and demonstrated to serve as targets for antibodies that inhibit the cariogenic process. Strategies have been devised to induce salivary secretory IgA antibodies which are particularly well suited to fulfilling this function. Further progress will depend on applying the results from animal models to human trials. Periodontal vaccines are less well developed, but some antigenic targets of two principal agents, Porphyromonas gingivalis and Aggregatibacter a., have been identified. Experiments in rodent and primate models have shown the ability of fimbrial antigens of either, and gingipains from the former, to elicit serum antibodies that inhibit colonization or the pathogenic mechanisms of these bacteria. However, questions remain about the role of host immune responses in the pathogenesis of periodontal disease, and whether current approaches to immunization can modulate these immunopathological processes.