Abstract
This review will focus on the impact of molecular genetic approaches on elucidating the bacterial etiology of oral diseases from an historical perspective. Relevant results from the pre- and post-recombinant DNA periods will be highlighted, including the roles of gene cloning, mutagenesis, and nucleotide sequencing in this area of research. Finally, the impact of whole-genome sequencing on deciphering the virulence mechanisms of oral pathogens, along with new approaches to control these organisms, will be discussed.
(A) Introduction
Many students embarking on careers in biology today are required to take courses in molecular biology and develop practical experience using molecular genetic techniques. Furthermore, modern day biology laboratories typically are equipped with multiple computers to “mine genomic databases”, polymerase chain-reaction (PCR) thermal cyclers, and freezers stocked with restriction enzymes, and have access to microarray facilities. These are a few of the indications of the impact that molecular genetics has had in current biological research, and oral microbiology is no exception to this general rule. This review will attempt to place the genetic revolution which has occurred in biology in the historical context of the genomics era in examining the virulence mechanisms of oral pathogens. This is an appropriate time for such a perspective, since this area of research has now begun to exploit genomic approaches for such analyses.
(B) Oral Microbiology in the Pre-genomics Era
Although significant genetic discoveries pre-dated the evolution of recombinant DNA techniques, it was this development in the 1970s (Morrow et al., 1974) which opened the door to the genomics era. Investigators’ ability to isolate and express genes from heterologous organisms, initially in Escherichia coli, together with the development of nucleotide sequencing technologies (Sanger et al., 1977), revolutionized the study of living organisms and provided the foundation for the molecular genetic techniques currently in use. Prior to these major breakthroughs, micro-organisms were principally examined in terms of their physiology. Emphasis was placed on chemical characterization of bacterial end-products or intermediates and isolation of the enzymes involved in their metabolism. At that time, chromatographic columns were as common in microbiology laboratories as PCR thermal cyclers are today.
Twenty-five years ago, oral microbiology was focused primarily on characterization of the organisms present in dental plaque and their potential roles in dental caries, gingivitis, and periodontitis–a quest still in progress (Socransky, 1977; van Winkelhoff, 1998; Kleinberg, 2002). A variety of approaches (Loesche, 1986) had provided evidence that Streptococcus mutans and S. sobrinus, members of the mutans streptococci, played important roles in human dental caries etiology. In addition, the use of animal models suggested that Actinomyces naeslundii might be involved in the destructive alveolar bone loss characteristic of advanced periodontitis (Socransky et al., 1970). Evidence was also presented in the ’70s that black-pigmented organisms related to Bacteroides strains might be involved in periodontitis (Socransky, 1977). However, many of the organisms now considered to play a role in these diseases had not yet been characterized. Therefore, when the use of recombinant DNA technology became available, S. mutans and S. sobrinus were the major candidates for exploitation of this approach in terms of defining the virulence of oral pathogens.
(C) Genetic Examination of Oral Pathogens Prior to the Introduction of Recombinant DNA Approaches
Applying the then-available technology, investigators suggested the important role of sucrose in the cariogenic properties of S. mutans (Loesche, 1986). These organisms were demonstrated to metabolize sucrose, as well as other sugars, rapidly to lactic acid and to convert a portion of the dietary disaccharide to glucan and fructan polymers. Several laboratories purified and characterized the enzymes involved in the synthesis of these polysaccharides (Mooser, 1992). One of the earliest genetic approaches to characterization of the virulence of an oral pathogen was the demonstration, by Tanzer and colleagues (Freedman et al., 1981), that spontaneous or chemically induced mutants of S. mutans defective in glucan synthesis or intracellular polysaccharide storage were attenuated in virulence when examined in a rat dental caries model. These findings, together with a comparison of the physiological properties of different oral streptococci, as well as clinical studies showing a positive correlation between the presence of S. mutans in specific plaque sites with the probability of caries development, confirmed the important role of these organisms in human dental caries. Furthermore, Hillman (1978) isolated a chemically induced mutant of S. mutans which was defective in lactic dehydrogenase activity and was also attenuated in dental caries induction in the rat model (Johnson et al., 1980). This provided a genetic basis for confirmation of the important role of lactic acid production in caries formation.
All of the authors in the studies cited above, utilizing spontaneous or chemically induced mutants of S. mutans, recognized the limitations of this approach, i.e., that it was not possible to prove unequivocally that the phenotypes observed resulted from the mutation in question. Such a confirmation required the application of recombinant DNA techniques, as was subsequently carried out several years later. Nevertheless, it should be recognized that these “pre-recombinant DNA” genetic approaches were successful in the identification of several important virulence properties of S. mutans.
In contrast to the focus on identifying virulence factors in cariogenic streptococci, periodontal microbiology prior to the 1980s was focused primarily on identifying the organisms which were associated with periodontitis. The earliest molecular studies were focused on Bacteroides melaninogenicus (now named Porphyromonas gingivalis) and its interactions with Gram-positive bacteria (Slots and Gibbons, 1978). One of the earliest genetic studies with these organisms suggested a correlation between the black pigmentation (iron accumulation) of these organisms with virulence in a mouse abscess model (Marsh et al., 1989). However, in the absence of specific mutagenesis approaches, the significance of these earlier observations was not clear. More recent genetic analyses have suggested a possible molecular explanation for the relationship between pigmentation and virulence in these organisms (Genco et al., 1995; Chen et al., 2000a). Furthermore, genetic approaches for examining the potential virulence properties of other organisms now recognized as periodontopathogens– Treponema denticola, Tannerella forsythensis (previously designated as Bacteroides forsythus), Actinobacillus actinomycetemcomitans, etc.–were not initiated until relatively recently, principally with the use of recombinant DNA techniques.
(D) Application of Recombinant DNA Approaches for Defining the Virulence of Oral Pathogens
(a ) The mutans streptococci
The first application of recombinant DNA technology to the investigation of oral bacteria was carried out in the Curtiss laboratory (Univ. of Alabama, Birmingham) (Table 1). This group initially isolated the S. mutans UAB62 gene encoding aspartic semi-aldehyde dehydrogenase in E. coli (Jagusztyn-Krynicka et al., 1982). Because of the safety concerns then prevailing regarding this new technology, these earlier studies were carried out under what was then called “P2 containment conditions”. The gene coding for the S. mutans (now S. sobrinus) 6715 SpaA surface protein was also isolated and characterized in the Curtiss laboratory, with cosmid cloning vectors (Holt et al., 1982). An important utilization of this technology relative to the potential virulence factors of S. mutans was the isolation, by Russell and colleagues, of two distinct glucosyltransferase (gtf) genes from S. mutans (now S. downei) Mfe28, utilizing lambda phage cloning vectors (Gilpin et al., 1985). This provided direct confirmation of the expression of multiple Gtfs from mutans streptococci, as was suggested by earlier enzymatic approaches (Loesche, 1986). However, the later approaches could not unequivocally demonstrate that these multiple Gtfs were distinct enzymes or derived from each other. Subsequent application of these techniques has demonstrated the existence of three gtf genes in S. mutans and at least four genes in S. sobrinus (Kuramitsu, 2000). Because of the important role of sucrose in caries etiology, several genes of these organisms involved in sucrose metabolism were subsequently isolated and characterized (Macrina et al., 1990). In addition, genes involved in intracellular polysaccharide storage, attachment to tooth surfaces, and sugar transport, as well as other physiological properties, have now been isolated and characterized from the mutans streptococci (Kuramitsu, 2000).
After the introduction of recombinant DNA technology, it became clear that it would be possible to utilize these techniques to construct monospecific mutants in an organism (Table 1). This would then provide a relatively straightforward approach for defining the virulence of an organism when used in conjunction with in vitro or in vivo virulence assays. In fact, this approach has become the standard for defining virulence factors in pathogenic micro-organisms. However, for such mutagenesis to be carried out, gene transfer systems for an organism must be available. An early study (LeBlanc et al., 1978) suggested the possibility that gene transfer could occur between oral streptococci. However, this information could not then be exploited for the development of gene inactivation systems in these organisms. Perry, who had previous experience transforming S. sanguis strains, was able to demonstrate that some, but not all, strains of S. mutans were naturally transformable (Perry and Kuramitsu, 1981). In addition to chromosomal markers, streptococcal plasmid vectors were also demonstrated to be transformable into these strains (Kuramitsu and Long, 1982). This then provided the foundation for the genetic manipulation of S. mutans which has now become a standard approach for characterizing genes in these organisms (Macrina et al., 1990). In addition, some strains of these organisms which are not naturally transformable can now be transformed by electroporation (McLaughlin and Ferretti, 1995). The construction of specific mutants of S. mutans defective in Gtfs, SpaP, or intracellular polysaccharide accumulation (Kuramitsu, 2000) has helped define these gene products as important virulence factors in these organisms. Interestingly, it has not yet been possible to construct monospecific mutants of S. sobrinus by transformation, but a conjugation strategy has been successfully utilized in this regard (Buckley et al., 1995).
The other advance which has had a marked impact on the study of biological mechanisms was the development of nucleotide sequencing techniques during the same time period when recombinant DNA procedures were introduced (Sanger et al., 1977). Investigators’ ability to both isolate and determine the nucleotide sequence (and the deduced amino acid sequence) of individual genes markedly accelerated our understanding of cellular physiology and virulence. For example, a comparison of the amino acid sequences of the Gtfs from S. mutans with those from S. sobrinus revealed the existence of distinct functional domains involved in sucrose breakdown and glucan binding (Janacek et al., 2000). In addition, nucleotide sequencing upstream or downstream of a targeted gene often revealed the existence of other related genes or novel genes. This added bonus of sequencing approaches is not as significant now because of the current availability of whole genome sequences. Furthermore, a comparison of nucleotide sequences immediately upstream of a gene frequently revealed information (operon structure, promoters, regulatory “boxes”) regarding potential regulatory mechanisms involved in gene expression. The availability of whole genome sequences facilitates these studies. More recently, nucleotide sequences have been utilized to assess the evolutionary relationships between and among different organisms (Ferretti et al., 2001). In addition, such information can be used to evaluate the possibility of gene transfer between different organisms. This may be especially important in dental plaque, where bacteria are close to one another.
(b ) Other Gram-positive oral bacteria
Genetic investigations of other Gram-positive oral bacteria were initiated several years after the first gene was isolated from S. mutans. Gtf activities were demonstrated in several non-mutans streptococci (Hamada and Slade, 1980), and the corresponding genes were isolated from S. gordonii (Sulavik et al., 1992) and S. salivarius (Simpson et al., 1995). Since these organisms are not considered to be virulent, gene inactivation studies were not carried out early on in conjunction with animal models. Nevertheless, the tools for constructing such mutants are basically the same as those used for S. mutans. However, the more recent recognition that non-mutans streptococci can also play a role in dental caries (van Houte, 1994) and could influence the cariogenic potential of plaque (Kleinberg, 2002) may lead to the use of relevant non-mutans streptococcal mutants in animal caries models (Tanzer et al., 2001). Furthermore, the more recently recognized roles of oral streptococci in systemic diseases such as bacterial endocarditis have stimulated renewed interest in these organisms (Herzberg et al., 1997). Using the genetic tools mentioned above, several laboratories have constructed monospecific mutants of oral streptococci in conjunction with animal models to identify potential virulence factors involved in endocarditis (Munro and Macrina, 1993; Burnette-Curley et al., 1995).
Because of its presence in dental plaque and the demonstration that some strains can produce alveolar bone loss in rodents (Socransky et al., 1970), A. naeslundii was an early subject of genetic studies. The gene for the type 2 fimbrial subunit of A. naeslundii WVU45 was isolated by Cisar and colleagues (Donkersloot et al., 1985). Subsequently, Yeung and her co-workers isolated the type 1 fimbrial gene from these species (Yeung, 1992). However, further genetic analysis of A. viscosus was hampered by the inability to transfer genes into these organisms, until Yeung (1999) developed such a system. Despite the fact that A. naeslundii strains are not currently considered to be periodontopathogens, their high numbers in supragingival plaque suggest that they may play an important role in plaque formation (Ellen and Burne, 1996).
(c ) Periodontal bacteria
During the early 1980s, when the genetic analysis of S. mutans was being initiated, a consensus was being reached by researchers regarding the identity of some of the subgingival plaque organisms associated with periodontitis (Slots and Genco, 1984). Particular attention was devoted to P. gingivalis and A. actinomycetemcomitans, since many clinical studies positively associated these organisms with periodontitis. The first report of the isolation of a gene from P. gingivalis was made in 1988 (Dickinson et al.) and involved the fimA gene encoding the major fimbrial subunit of these organisms (Table 2).
Although several genes from P. gingivalis were subsequently isolated and characterized, it was not until 1994 that this information could be used to construct monospecific mutants (Malek et al., 1994; Hamada et al., 1994; Nakayama, 1994). Influenced principally by the work with Bacteroides fragilis (Salyers et al., 1987), investigators accomplished genetic manipulation of P. gingivalis using strategies successfully utilized with the former organisms. Both conjugation (Malek et al., 1994) and gene transfer following electroporation (Fletcher et al., 1995) have been used to construct monospecific mutants in these organisms. The isolation of several distinct mutants coding for suspected virulence factors in these organisms, together with the development of animal models for virulence, indicated that several genes of P. gingivalis, including the fimA and rgp genes, likely play significant roles in periodontitis (Malek et al., 1994; Fletcher et al., 1995). However, since mutants of both genes display pleiotropic effects (Malek et al., 1994; Nakayama et al., 1995), it is still not clear precisely how each gene product is involved in these diseases. One factor somewhat limiting the genetic analysis of P. gingivalis is the unavailability of stable shuttle vectors for use in common strains of these organisms.
The recognition that A. actinomycetemcomitans is associated with several forms of periodontitis stimulated interest in the use of genetic approaches to the investigation of these periodontopathogens. Since the ability of these organisms to secrete leukotoxin could be correlated with the apparent virulence of some strains, genetic approaches were applied to characterize the leukotoxin gene from these organisms (Kolodrubetz et al., 1989; Lally et al., 1989). The leukotoxin gene has been defined and leukotoxin negative mutants constructed (Kolodrubetz et al., 1995). In addition, it has been proposed that trans-activating regulatory elements may be involved in regulating the various levels of leukotoxin expressed by different strains of these organisms (Kolodubretz et al., 1996). Interestingly, some strains which did not secrete this putative virulence factor still harbored a leukotoxin gene with a modified promoter region (Kolodrubetz et al., 1996).
Despite the fact that a gene inactivation system for A. actinomycetemcomitans was initially described in 1995 (Kolodrubetz et al.), relatively few mutants of these organisms were constructed following that report. However, very recently, Chen and co-workers (Y Wang et al., 2002) have described a method based upon the natural transformability of some strains of A. actinomycetemcomitans which resulted in the construction of several distinct mutants. This may lead to the construction of mutants which will increase our understanding of other additional virulence factors of these organisms.
Principally through the efforts of Fives-Taylor and colleagues (Meyer et al., 1996), it has been suggested that A. actinomycetemcomitans invasion of epithelial cells might be an important virulence property of these organisms. Although the role of cellular invasion by these organisms in vivo has been questioned, the recent demonstration of these organisms, as well as P. gingivalis, in healthy buccal epithelial cells (Rudney et al., 2001) suggests that invasion can occur in the oral cavity. Another interesting aspect of A. actinomycetemcomitans is that the rough colonial adherent strains of these organisms normally isolated from the oral cavity can spontaneously switch to a smooth non-adherent colonial morphology in vitro (Inouye et al., 1990). However, the molecular basis for this property has not yet been determined and is currently under investigation (Haase et al., 1999).
Early microscopic examination of plaque samples isolated from periodontitis sites showed a preponderance of spirochete organisms (Loesche, 1988). However, many of these organisms still cannot be cultivated, and their respective roles in periodontal diseases still remain to be demonstrated. However, the application of novel molecular approaches (Kroes et al., 1999; Sakamoto et al., 2003) should allow for an assessment of the potential role of these non-cultivable organisms in periodontitis. One of the few spirochetes which could be readily cultivated in the laboratory was T. denticola. Several reports suggested that these organisms could be positively correlated with periodontitis (Armitage et al., 1982; Simonson et al., 1988). Therefore, potential virulence factors in these organisms were sought, initially by enzymatic approaches (Grenier et al., 1990; Uitto et al., 1986). Initial gene cloning in these organisms was undertaken in the early 1990s and resulted in the isolation of a surface protein gene (Miyamoto et al., 1991). The first genetically characterized candidate virulence factor for these organisms was the Msp outer sheath protein of strain 35405 (Haapasalo et al., 1992). Subsequent studies have demonstrated that this protein could be important in attachment to host tissue and modification of host cells (Fenno et al., 1998). In addition, the gene for the major surface chymotrypsinlike protease was isolated by Ishihara and colleagues (Ishihara et al., 1996) and given the name “dentilisin”. Since motility appears to be an important virulence property of these organisms, it was not surprising that several of the genes involved in motility and chemotaxis in these organisms were isolated and characterized (Kataoka et al., 1997; Ruby et al., 1997). These genes were recently demonstrated to play important roles in the penetration of these organisms through tissue (Lux et al., 2001).
The respective roles of the genes isolated from T. denticola strains in the pathogenicity of these organisms were further suggested with the utilization of monospecific mutants of the spirochetes in model systems. These mutants were first constructed following electroporation (Li et al., 1996) and utilized an erythromycin-resistance cassette originally developed for use in P. gingivalis (Fletcher et al., 1995). More recently, it has also been possible to use shuttle plasmids to complement erythromycin-resistant mutants of T. denticola 33520 (Chi et al., 2002). This plasmid transformation system was also used to express a gene from T. pallidum (Chi et al., 1999), suggesting that T. denticola may be useful as a surrogate organism for the characterization of genes from uncultivable spirochetes.
The third member of the “red complex” (organisms most frequently associated with periodontitis) (Socransky et al., 1998) implicated as an important periodontal pathogen is B. forsythus. Studies on the physiology and genetics of these organisms lag behind similar studies for the other two members of this complex, P. gingivalis and T. denticola. However, several genes, including a protease and putative adhesin from these organisms, have recently been isolated and sequenced (Saito et al., 1997; Sharma et al., 1998). More recently, a gene inactivation system for these organisms, based upon a conjugation strategy, has been successfully developed by Sharma and colleagues (Honma et al., 2001). With this procedure, a mutant attenuated in the expression of the leucine-rich repeat surface protein BspA has also been shown to be defective in producing alveolar bone loss in a rat model system (A. Sharma, personal communication). It is anticipated that additional mutants of B. forsythus will be constructed in the near future for testing in rodent periodontitis models.
The plaque organism Fusobacterium nucleatum, although not considered a primary periodontal pathogen, has attracted interest because of its important role in mediating the attachment of several bacterial species into the plaque biofilm (Kolenbrander et al., 2002) as well as its possible role in pre-term births (Hill, 1998). Although a plasmid transfer system has been reported for this species (Haake et al., 2000), a system for constructing monospecific mutants in these organisms has yet to be reported. In addition, only a few genes (Demuth et al., 1996; Han, unpublished results) have been isolated from these organisms.
Several genes have also been isolated from the putative periodontal pathogen Eikenella corrodens (Rao and Progulske-Fox, 1993; Yumoto et al., 1996; Villar et al., 2001), and mutants of these organisms have been constructed (Villar et al., 2001). Likewise, Kolodrubetz and colleagues (Wang et al., 2002) have recently isolated genes involved in S-layer formation in Campylobacter rectus, another organism associated with periodontitis, and constructed mutants defective in the synthesis of this extracellular polymer. However, animal models for testing the virulence of these mutants have not yet been described.
Therefore, the application of molecular genetic approaches has been important in the rapid advancement of our understanding of the etiology of periodontitis which has taken place in the past decade (Lamont and Jenkinson, 1998).
(E) Application of Whole Genome Screening Techniques Prior to the Development of Microarray Technologies
Prior to the availability of the genome sequences of oral pathogens, our ability to isolate a gene from these organisms depended upon a priori information regarding the properties of the gene or its product (activity or immunogenicity of the product, nucleotide sequence of a homologous gene from another organism) in commonly utilized positive selection strategies. Initial gene cloning from many organisms during the 1980s was primarily dependent upon such information. Strategies for seeking genes without such information became available with the advent of transposon mutagenesis (Gawron-Burke and Clewell, 1984). Theoretically, this strategy would allow for the random mutagenesis of a genome in an organism of interest for the identification of mutants which displayed a readily detectable phenotypic alteration. Using the streptococcal transposon Tn916, investigators isolated and characterized mutants of S. mutans and S. gordonii attenuated in several properties (Loo et al., 2000; Kuramitsu, 2000). However, for this strategy to be maximally effective, the target organism must be transformed at a relatively high frequency with a suicide vector containing Tn916. Subsequently, a strategy using transposon Tn917, which will also work for some poorly transformable strains of S. mutans, was developed, and mutants defective in a variety of characteristics were isolated (Gutierrez et al., 1996).
A similar strategy based upon conjugation was also developed for P. gingivalis mutagenesis mediated by the transposon Tn4351 (Hoover et al., 1992) and, more recently, Tn4400 (Chen et al., 2000b). Using this strategy, several laboratories have isolated mutants which are defective in potential virulence factors in these organisms (Genco et al., 1995; Watanabe-Kato et al., 1998). It is not possible to predict whether transposon mutagenesis will also prove to be effective in other Gram-negative periodontal pathogens, since no transposons for these organisms are currently available, and/or gene transfer into these organisms occurs at frequencies which do not allow for efficient mutagenesis of their genomes.
A strategy which has been proven useful for identifying genes expressed under specific environmental conditions is the use of genome libraries fused to reporter genes (Lane et al., 1991). When libraries of suicide plasmids constructed in E. coli containing random DNA fragments cloned upstream of a reporter gene are transformed into a target organism, integration of the plasmids into the chromosome results in random gene fusions to the reporter gene (lacZ, green fluorescent protein, etc.). Using this approach, investigators have identified genes of S. mutans which are regulated in the presence of sugars (Lane et al., 1991; Peruzzi et al., 1998). For organisms for which DNA microarrays are not available, this is a viable option for the identification of genes regulated by specific environmental conditions, provided that the organism in question can be genetically manipulated at relatively high frequencies.
More recently, differential display has been utilized to identify genes regulated by specific growth environments. Although originally developed for eukaryotic systems, it has been proven effective for the identification of differentially regulated genes in S. mutans (Chia et al., 2001), T. denticola (Tsai and Shi, 2000), P. gingivalis (Chung et al., 2001), and S. gordonii (Du and Kolenbrander, 2000). This approach has the advantage that the organisms examined do not have to be genetically manipulated. However, the disadvantage of this technique is that only a relatively small number of regulated genes can be readily identified with this approach.
Although not considered to be a genomic approach, 2-D gel electrophoresis has been utilized for the examination of globally regulated genes in micro-organisms. Indirectly, this can be considered as a genetic approach, since the identification of the proteins on the gels is made based upon a comparison with the protein sequences deduced from the genome sequences of an organism. This approach has been utilized in several recent investigations regarding the effects of various environmental factors on S. mutans (Svensater et al., 2000; Lemos and Burne, 2002).
(F) The Genome Era
There is now no doubt that the generation of these databases has had a profound influence on the progress of microbiological research. The first genomes of oral bacteria to be sequenced were those of P. gingivalis and S. mutans (Ajdic et al., 2002), and their respective sequences became available to the scientific community as early as 1998 (Tables 1, 2). With the value of such sequences becoming universally recognized, sequencing of additional genomes such as those of T. denticola, A. actinomycetemcomitans, S. gordonii, S. Sanguis, and F. nucleatum has been completed (Kapatral et al., 2002) or is currently in progess. Although all of these sequences are not currently fully annotated, their availability on the Internet (e.g., www.TIGR.org) has facilitated many aspects of oral microbiological research. Besides serving as the basis for the development of microarrays (discussed below), the resulting databases have provided unique opportunities for researchers working with these organisms to gain insights into the physiology of these organisms which could not otherwise be attainable in such relatively short time periods.
Perhaps the most striking lesson to be learned from gleaning the genome databases of micro-organisms is that we know so little of the role of many of these genes in the respective organisms. For example, more than 30% of the putative open reading frames (ORFs) in each microbial genome identified so far correspond to proteins of unknown function (Ajdic et al., 2002). Some of these appear to be unique to a given organism, and if a small fraction of these are essential genes, they, in turn, could be exploited to design specific antagonists of their protein products and thereby serve as specific growth inhibitors of the organisms.
A comparison of the databases in terms of the deduced amino acid sequences of the genes has also been very valuable to investigators for identifying the functions of many of these genes. Comparisons of the databases of oral bacteria with those from organisms whose physiology and genetics are more well-established (E. coli, Bacillus subtilis, Streptococcus pneumoniae, Staphylococcus aureus) have allowed for the preliminary identification of many genes. For example, by this simple strategy, genes coding for the quorum-sensing regulatory gene luxS have been identified in S. gordonii, A. actinomycetemcomitans, S. mutans, and P. gingivalis (Kolenbrander et al., 2002). Numerous examples of such in silico gene cloning now exist in the literature. In addition, database comparisons can identify the presence or absence of metabolic pathways and help to explain the nutritional requirements of an organism. In this regard, an examination of the P. gingivalis database indicated the absence of several amino acid biosynthesis pathways which is reflected in the elaboration of potent proteinase activities by these organisms (Potempa et al., 1995).
The genome databases have also been exploited for the identification of genes expressing novel phenotypes in an organism. For example, by using the sequences of known two-component signal transduction systems in non-oral bacteria, Burne and colleagues (Bhagwat et al., 2001) have searched the S. mutans UA159 database and have identified six homologous systems in this strain. Utilizing mutants defective in each of these signaling gene pairs, one pair of these genes appears to be involved in regulating biofilm formation by these organisms in vitro. A novel approach with use of the P. gingivalis W83 database has also been used for the identification of genes of the organism which code for potential cell-surface proteins (Ross et al., 2001). Some of these appear to be particularly immunogenic and are potential candidates for the development of anti-P. gingivalis vaccines. Therefore, the choice of appropriate “in silico probes” should allow for the identification of specific classes of genes in each database.
(G) The Microarray Era
The availability of whole genome sequences ushered in the current era of DNA microarray analysis. Each gene (oligonucleotide sequences) could be spotted onto a suitable matrix (glass, paper) for use in identifying mRNA molecules which could be regulated under specific environmental conditions. In addition, the arrays can be utilized to probe differences between bacterial genomes. The earliest bacterial microarrays were constructed only relatively recently (deSaizieu et al., 1998). These arrays have been utilized to identify genes which are regulated during biofilm formation (Whiteley et al., 2001) as well as under other environmental conditions.
At present, microarrays for oral bacteria are not generally available. Concerns regarding the marketability of such microarrays for oral bacteria may limit their commercial availability. However, because of the demonstrated utility of this technology, it is anticipated that microarrays for individual organisms will be developed privately. In this regard, the National Institute of Dental and Craniofacial Research, together with The Institute for Genome Research (TIGR), has funded the development of a limited quantity of P. gingivalis micro-arrays which are being currently tested in six different laboratories, in both the USA and the United Kingdom. Preliminary reports from these laboratories have indicated both the usefulness and the limitations of the microarrays. The reproducibility of the results from the use of the microarrays is primarily dependent upon the fidelity of array construction as well as on the quality of the RNA used for analysis. In addition, the results of such approaches need to be confirmed by direct transcript analysis. Microarrays are currently being used to identify genes which are regulated during colonization in vitro (Chen and Kuramitsu, unpublished observations) and in vivo (Baker, unpublished observations), those involved in glycosylation of proteases (Curtis, unpublished observations), genes regulated under iron-limiting conditions (Lewis and Macrina, unpublished observations), up- and down-regulated genes following interactions with host cells (Progulske-Fox, Duncan, unpublished observations) or heterologous bacteria (Chen et al., unpublished observations), and by various signaling modulators (Progulske-Fox, Chen and Kuramitsu, Duncan, unpublished observations). In addition, the microarrays are being utilized to identify genomic differences among different strains of P. gingivalis (Curtis, Duncan, unpublished observations). It is anticipated that the subsequent publication of these results will prove helpful to the entire oral microbiological community, i.e., some gene changes detected with P. gingivalis may also occur in other organisms under similar environmental conditions. We hope that the exploitation of microarray data for all organisms will be carried out in a collaborative manner, since individual laboratories will not be able to focus on all of the gene changes likely to be detected by the microarrays.
(H) Application of Genetic Approaches to Examination of the Role of Microbial Ecology in Oral Diseases
(a ) Identification of oral pathogens
Despite the fact that individual organisms have been shown to be important in dental caries and periodontitis, it is becoming increasingly apparent that it is the interaction of different plaque bacteria with each other and the host tissues (microbial ecology) which ultimately determines whether disease develops (Marsh, 1994). Therefore, both the identification of the multitude of micro-organisms present in the oral cavity and their interactions with each other are important issues which need to be considered in defining the etiology of dental caries and periodontitis. The recognized limitations of earlier identification of these organisms by culturing–i.e., the fact that a significant fraction of the organisms visualized in plaque samples associated with periodontitis has not yet been cultivated and characterized–opens the possibility that some significant periodontopathogens have still not been recognized. In addition, the growing recognition (Becker et al., 2002) that non-mutans streptococci can also play significant roles in caries etiology suggests that a more thorough examination of the composition of dental plaque associated with dental caries is in order. The recent development of molecular genetic approaches which can obviate the necessity of cultivating the organisms in question should prove to be extremely useful in this regard. For example, using the sequencing of 16S rRNA genes, Relman and colleagues (Kroes et al., 1999) have demonstrated that a significant number of organisms present in subgingival plaque have yet to be characterized. A very recent analysis using similar techniques has also suggested that other organisms, in addition to mutans streptococci, may play important roles in the initiation of dental caries (Becker et al., 2002).
DNA probes based upon the nucleotide sequences of the 16S rRNA genes of these organisms could be incorporated into matrices such as the “checkerboards” developed by Socransky and colleagues (Socransky et al., 1994) to correlate the presence or absence of these organisms with disease. Furthermore, fluorescent derivatives of these probes in conjunction with confocal microscopy could be utilized to localize these organisms in plaque samples (Moter et al., 1998). Likewise, the recent demonstration that the green fluorescent protein can be expressed in several oral bacteria (Saint Girons et al., 2000; Hansen et al., 2001; Yoshida and Kuramitsu, 2002a) suggests that these proteins can be utilized to localize specific organisms in biofilms. In this manner, it may eventually be possible to determine the role of spatial relationships among different bacteria in plaque in modulating disease progression.
(b ) Biofilm formation by oral bacteria
Since both dental caries and periodontitis result from the action of organisms present in physiologically distinct structures termed “biofilms”, renewed interest has been directed toward the process of plaque formation. The application of molecular genetic approaches to define the mechanisms of biofilm formation for several organisms has revealed the complex nature of this process (Burne et al., 1997). For example, following Tn916 mutagenesis, a variety of genes in S. gordonii was demonstrated to play a role in biofilm formation in vitro (Loo et al., 2000). Likewise, several genes of S. mutans were demonstrated to play essential roles in biofilm formation in similar systems (Wen and Burne, 2002; Yoshida and Kuramitsu, 2002b), including the com competence regulon (Li et al., 2002; Yoshida and Kuramitsu, 2002b). Interestingly, this later signaling system was also demonstrated to regulate not only transformation and biofilm formation but also the acid tolerance response (Li et al., 2001b) as well as bacteriocin production (Wang and Kuramitsu, unpublished observations) in some strains of S. mutans.
As mentioned previously, a comparison of the genome databases for a variety of bacteria has revealed the presence of homologs of the luxS gene involved in the synthesis of a furanone signaling molecule (Bassler, 1999). This has prompted the suggestion that, unlike the com- and LuxR-mediated quorum-sensing systems, the LuxS system may play a role in “sensing” the presence of heterologous organisms. Many, but not all, oral bacteria have been demonstrated, either by complementation experiments (Frias et al., 2001) or by database comparisons, to contain LuxS homologs. However, an examination of luxS mutants of P. gingivalis (Chen and Kuramitsu, unpublished observations) and S. mutans (Shi et al., unpublished observations; Yoshida and Kuramitsu, 2002b) has suggested that this gene is not involved in biofilm formation by these two organisms, at least in vitro. However, luxS mutants in these organisms (Chung et al., 2001; Burgess et al., 2002; Shi et al., unpublished results), as well as in A. actinomycetemcomitans (Fong et al., 2001), are attenuated in a variety of other phenotypes and are currently under investigation in several laboratories. Very recently, it has been demonstrated that autoinducer-2 (the product of the activity of LuxS) may be required for the attachment of P. gingivalis to S. gordonii (McNab et al., 2003). Such interactions may be important in the conversion of Gram-positive enriched dental plaque to a predominantly Gram-negative biofilm.
Relatively little information is currently available regarding the genetic basis for biofilm formation by periodontal pathogens. However, a recent study has demonstrated that the polyphosphate kinase gene, ppk, of P. gingivalis 381 is important in biofilm formation on polyvinylchloride and glass surfaces (Chen et al., 2002). Several laboratories are also currently utilizing transposon mutagenesis in P. gingivalis to identify additional genes which may be important in biofilm formation by these organisms (K. Honma, unpublished observations).
Using monospecific mutants of T. denticola, investigators have also been able to identify several mutants of these oral spirochetes which are attenuated in biofilm formation on fibronectin or P. gingivalis-coated surfaces (Vesey and Kuramitsu, unpublished observations). It is anticipated that similar genetic approaches will prove useful with other periodontal pathogens for the genetic characterization of biofilm formation by these organisms.
(c ) Interbacterial signaling
It is now evident that interbacterial interactions are an important factor in dental plaque formation (Kolenbrander, 1988). Such interactions can also affect the expression of potential virulence factors in these organisms (Xie et al., 2000). Likewise, recent results have suggested that some non-mutans streptococci can influence the com-dependent properties of S. mutans (Wang and Kuramitsu, unpublished observations). It is very likely that these results represent only a glimpse of the variety of bacterial interactions which occur in dental plaque. However, the mechanisms involved in such regulation still remain to be elucidated. Whether direct contact among organisms is involved and secretion of signaling molecules occurs are important questions which will need to be addressed. It will also be of interest to develop techniques for utilizing microarrays for individual organisms so that we can access the full complement of gene changes which occur when these organisms are in proximity to heterologous organisms in biofilms.
Another area of research which is rapidly evolving is the interaction of oral bacteria, particularly periodontal pathogens, with host cells. When molecular genetic approaches are combined with recently developed microscopic techniques (Rudney et al., 2001), it is now possible for bacterial attachment and invasion to be investigated in greater detail. As mentioned earlier, host cell invasion by periodontal bacteria such as A. actinomycetemcomitans as well as P. gingivalis in vivo has been somewhat controversial but has now been demonstrated in healthy buccal epithelial cells (Rudney et al., 2001). Genetic approaches have been used to define the attachment/invasion mechanisms utilized by P. gingivalis upon interaction with host cells (Lamont et al., 1995; Njoroge et al., 1997; Chen et al., 2001). These studies have demonstrated key roles for the major fimbrial protein, FimA, and cysteine proteinases in attachment. However, the genes specifically involved in invasion of these cells still remain to be identified. Although genes involved in cellular invasion by A. actinomycetemcomitans have not yet been characterized, the fibrils of these organisms appear to be involved in non-specific interactions with surfaces (Kachlany et al., 2001). In addition, microarrays are now being utilized to investigate the regulation of genes in both oral bacteria (see above) and host cells following interactions with oral bacteria.
(d ) Gene transfer among oral plaque bacteria
Earlier studies have documented the potential for transfer of antibiotic-resistant markers in dental plaque (LeBlanc et al., 1978). This suggested that these organisms may have the ability to transfer DNA, and this was subsequently confirmed (Kuramitsu and Trapa, 1984). In addition, it might also be possible that antibiotic-resistant plaque organisms could serve as a reservoir for subsequent horizontal transfer of the resistance genes to other non-oral organisms in transit through the oral cavity. More recently, the examination of the nucleotide sequences of several genes of P. gingivalis (Curtis et al., 1999) has suggested the likelihood of horizontal gene transfer occurring in these organisms. Furthermore, the presence of large numbers of insertion sequence (IS) elements in the genomes of P. gingivalis strains also suggests the possibility of transmission of these elements among individual members of this species in dental plaque (Califano et al., 2000). All of these observations are compatible with the ability of plaque micro-organisms to exchange genetic information with each other.
Recently, it was demonstrated that bacteria bathed in saliva could be transformed with exogenous DNA (Mercer et al., 2001). Likewise, S. mutans grown as artificial biofilms could be shown to be transformable at even higher frequencies than in broth cultures (Li et al., 2001a). In addition, gene transfer from T. denticola to S. gordonii was demonstrated recently both in broth cultures and in biofilms in vitro (B-Y Wang et al., 2002). Therefore, it is likely that genetic exchange can occur in dental plaque among selected organisms. In this regard, many, but not all, plaque streptococci are naturally transformable (Lundsford, 1998). In addition, P. gingivalis can undergo conjugation-mediated genetic exchange with E. coli (Dyer et al., 1992). Likewise, two distinct species of oral spirochetes, T. denticola and T. socranskii, apparently naturally harbor the same plasmid, pTS1 (Chan et al., 1996). This later observation suggests possible gene transfer between these two organisms, but this has not yet been demonstrated.
It has not yet been directly demonstrated that genetic exchange among oral micro-organisms has directly influenced the virulence of these organisms. However, this has been suggested by the observation that P. gingivalis genes coding for the rag genes, putative virulence factors of these organisms (Curtis et al., 1999), contain distinct G+C ratios relative to the rest of the strain W50 genome. This suggested the possibility of horizontal genetic exchange of these genes from another organism, likely one in dental plaque. Such transfer of “pathogenicity islands” appears to be involved in the virulence of several micro-organisms (Ingersoll et al., 2002).
(I) Genetic Approaches for Examining the Relationship between Oral and Systemic Diseases
As mentioned previously, molecular genetic analysis of oral streptococci implicated in endocarditis is currently in progress. More recently, based initially on epidemiological studies, it has been suggested that periodontitis may be a risk factor for some systemic diseases, including atherosclerosis, diabetes, and preterm births (Williams and Offenbacher, 2000). Although the relationship between periodontitis and atherosclerosis is still somewhat controversial (Hujoel et al., 2000), mutants of P. gingivalis have been utilized to investigate the molecular mechanisms involved in the possible relationship between these two diseases (Kang and Kuramitsu, 2002; Nassar et al., 2002). Other results have also suggested that the potent gingipain cysteine proteinases of these organisms could be important factors in platelet aggregation (Lourbakos et al., 2001) and low-density lipoprotein aggregation (Miyakawa et al., unpublished observations), two properties associated with atherosclerosis. In addition, since the stimulation of immunological mediators of inflammation may be a factor in these diseases, the effects of P. gingivalis on endothelial cells may also be relevant (Kang and Kuramitsu, 2002; Nassar et al., 2002). So far, comparable approaches relating the properties of periodontal pathogens to preterm birth or diabetes have not yet been reported.
(J) Novel Therapies Based upon Genetic Approaches
(a ) Identification of antibacterial targets
One approach to reducing the incidence of dental caries and periodontitis would be to eliminate or retard the growth of the bacteria responsible for these diseases. The application of broad-spectrum antibacterial agents such as chlorhexidine, alcohols, etc., has demonstrated only limited utility in this regard. Ideally, inhibitors of greater specificity might prove useful, since they would minimize potential effects of drastically altering the oral ecology of the mouth. This approach is dependent upon the identification of essential genes of the etiological agents for development of specific growth inhibitors. The sequencing of many bacterial genomes has revealed that a significant fraction of each genome consists of genes of unknown function, some of which appear to be unique to each organism. A portion of these may prove to be essential for the organism in question and may therefore encode potential targets for species-specific inhibitors. Until very recently, identification of such genes required labor-intensive whole-genome mutagenesis and analysis of the resulting mutants. However, a recently developed antisense RNA strategy has been utilized to simplify the identification of such genes in Staphylococcus aureus (Ji et al., 2001). A similar strategy is currently being examined to identify specific essential genes of S. mutans (Wang and Kuramitsu, unpublished observations). However, it is unlikely that a similar strategy could be used for most periodontal pathogens, since this strategy depends upon relatively high-frequency gene transfer in the respective organisms.
Alternatively, it may be possible to identify genes of a pathogen which are important for survival in vivo. Such an approach is possible for some organisms based upon the in vivo expression technology (IVET) strategy (Slauch et al., 1994). This strategy has been adapted for the identification of such genes in P. gingivalis (Wu et al., 2002) and oral streptococci (Kili et al., 1999). Another novel approach for identifying genes which are specifically expressed in vivo has also been developed (Handfield et al., 2000). This in vivo induced antigen technology (IVIAT) strategy is designed to identify gene products of an organism which are expressed in vivo by using antiserum from infected animals or individuals to screen gene libraries relative to uninfected animal or healthy sera. It is likely that some of the genes detected may correspond to those which are essential for in vivo growth or virulence. This approach appears to be adaptable to a wide range of micro-organisms (bacteria, fungi, and viruses) and is currently being evaluated for a broad range of pathogens. Another advantage of this strategy is that it does not depend upon genetic manipulation of the target organism. Based upon information obtained from these novel screening strategies, it may be possible to design specific inhibitors of the gene products based upon the deduced properties of the protein or by combinatorial chemistry screening approaches (Nielsen, 2002).
Besides eliminating the etiological agents of oral diseases, another potential approach to controlling these infections would be to neutralize the virulence factors of the organisms involved. For example, the demonstrated important roles for the SpaP surface adhesin and Gtfs of S. mutans in producing caries in rodent models (Kuramitsu, 2000) have suggested that these protein molecules might serve as targets for developing inhibitors of cariogenesis. In this regard, sugar substitutes provide one practical means of reducing glucan formation by S. mutans when incorporated into some food products (Ooshima et al., 1990). More recently, a peptide inhibitor which mimics the active site of the SpaP adhesin has been synthesized and was shown to attenuate S. mutans tooth colonization in human volunteers (Kelly et al., 1999). Moreover, a gene corresponding to the peptide has been genetically engineered into corn plants which in turn produce active inhibitor (Kelly et al., 2001). This suggests the possibility that such inhibitors could be expressed in food products such as cereals or milk as a means of reducing caries in children. However, several concerns need to be addressed before such genetically engineered foods could be used as a prophylactic approach.
(b ) Vaccines against oral diseases
The development of an anti-caries as well as an anti-periodontitis vaccine is another strategy which could be used to neutralize oral pathogens. This review will not survey this area of research in detail, and the reader should refer to several recent comprehensive reviews of this subject for a broader perspective (Abiko, 2000; Michalek et al., 2001). Despite the fact that anti-caries vaccines have been demonstrated to be effective in animal models (Smith and Taubman, 1995), they have not yet been examined in human trials. Anti-caries vaccines based upon Gtf and SpaP antigens continue to be of interest in terms of optimizing the routes of immunization (Michalek et al., 2001). Additionally, the utilization of DNA vaccines against dental caries may represent a novel approach to control this disease (Fan et al., 2002).
Since P. gingivalis is an important periodontal pathogen, recent efforts have been made in developing a vaccine against this organism. It was demonstrated that the utilization of a FimA fimbrial subunit vaccine was effective in protecting rats against alveolar bone loss (Evans et al., 1992). In addition, the utilization of the gingipain proteinases of these organisms as antigens has been recently evaluated and shown to provide some protection against P. gingivalis-mediated virulence in animal models (Gibson and Genco, 2001; Rajapakse et al., 2002). More recently, a hemoglobin-binding domain vaccine has been successfully utilized to protect rats against P. gingivalis-induced bone loss (DeCarlo et al., 2003). It will be of interest to determine if a vaccine targeted to a specific periodontal pathogen will have a significant impact on a disease for which multiple organisms have been implicated as virulence factors.
In addition to active immunization, passive immunization is another approach which may prove useful in controlling oral diseases (Abiko, 2000). Recently, Koga and colleagues (Mitoma et al., 2002) have demonstrated passive protection of rats against dental caries using milk produced from cows vaccinated with a Pac-Gtf fusion protein antigen. This suggests the possibility of using foods containing antibodies as a means of passively protecting children against dental caries.
(c ) Other genetically based therapies
An interesting approach for potentially controlling dental caries has been proposed by Hillman and colleagues (Hillman, 2002). This replacement therapy strategy involves the colonization of the oral cavity with an S. mutans strain which has been genetically engineered to be defective in lactic acid production. Such strains, which also express elevated levels of an anti-S. mutans mutacin, could displace the normally cariogenic strains of these organisms in the oral cavity. It is anticipated that this strategy may be examined in human clinical trials in the near future.
Another approach to the control of dental caries would be genetic engineering of oral commensal organisms to antagonize the cariogenicity of S. mutans strains. In this regard, previous results have indicated that S. gordonii strains expressing a heterologous glucanase gene inhibited the sucrose-dependent colonization of smooth surfaces by S. sobrinus in vitro (Kubo et al., 1993). However, it has not yet been demonstrated that such a strategy is effective in vivo. Likewise, increasing the alkalinity of dental plaque by genetic engineering of commensal organisms could be envisioned as a novel means of controlling dental caries (Burne and Marquis, 2001). The introduction of the genes for urease or arginine deiminase into commensal oral streptococci may serve to increase the alkalinity of dental plaque and thereby retard caries development. Again, these novel strategies involving the utilization of genetically engineered organisms will likely face both scientific and ethical scrutiny in terms of future application.
(K) Future Outlook
It is quite clear that the application of molecular genetic approaches for the examination of the virulence of oral pathogens in the past two decades has greatly increased our knowledge of the etiology of the two major oral diseases, dental caries and periodontitis. It is also difficult to argue that such progress could have occurred without the introduction of these revolutionary techniques. Nevertheless, it is also true that much of this information has not yet been successfully transferred from the laboratory bench into dental practice in terms of having a significant impact on these diseases. However, this author is confident that we now stand at the threshold of a period when practical applications will be forthcoming at frequencies which have not occurred in the past. This will result not only from an increased understanding of the etiology of these diseases but also from new fiscal pressures placed upon both industrial as well as academic laboratories to stimulate translational research.
Although rapid progess is being made in determining the molecular basis for the pathogenicity of many oral bacteria, several significant issues still remain to be addressed. For example, because of the vast complexity of the microbial oral flora, it is likely that we have not yet identified all of the organisms involved in the development of dental caries and periodontitis. This recognition, together with the application of newer genetic approaches (Kroes et al., 1999), will lead to “filling in the gaps” in this regard. Such cataloging of organisms will then necessitate the application of more traditional methods for determining their respective roles in pathogenicity involving both clinical and basic science approaches. It can be predicted that it will be necessary not only to identify potential pathogens but also to understand how these organisms interact with other plaque bacteria to understand disease etiology. This ecological approach will usher in an era where the emphasis will be placed on the combined activities of groups of micro-organisms rather than on those of a single organism. This will be especially relevant to oral microbiology, which may serve as a convenient model system for the exploration of such interactions. Thus, it might be predicted that oral microbiology may play an especially important role in this emerging field.
The increasing emphasis on “holistic” approaches to human well-being underlines the interactions between and among different organ systems. Logically, then, it is not surprising that infectious diseases can have an impact on those diseases which have been traditionally classified as “systemic”. It is likely that the recent interest in the role of periodontitis in diseases such as atherosclerosis, preterm birth abnormalities, and diabetes will be expanded to include other diseases, such as osteoporosis, arthritis, and perhaps even neurological diseases. This should also increase the emphasis on periodontal microbiology and immunology. Therefore, it is reasonable to predict that it will be increasingly important for investigators to understand the molecular basis for the potential virulence of oral micro-organisms.
Looking back at the near-exponential growth in our understanding of the pathogenicity of micro-organisms over the past two decades, it cannot be readily predicted what new technologies will soon emerge to continue this progress. At present, we have only a glimpse of how future single-cell analytical techniques, proteomics, bioinformatics, new animal models, etc., will affect this scenario. Whether a new technology with the impact of the genomics revolution is “just around the corner” is something to be keenly anticipated.
Footnotes
Acknowledgements
This review is dedicated to the memory of Maria K. Yeung, who uniquely represented the pioneering spirit involved in developing molecular genetic approaches for examining the etiology of oral microbial diseases. The author gratefully acknowledges the sharing of unpublished results by numerous colleagues. Because of the scope of the material covered, adequate acknowledgment of all significant contributions to this area of research was not possible, and such oversights solely reflect author bias. Work cited from the author’s laboratory was supported by NIH grants DE03258, DE08293, and DE09821.