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Dual lifestyle of Porphyromonas gingivalis in biofilm and gingival cells
Microbial Pathogenesis xxx (2015) 1e6
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Review
Dual lifestyle of Porphyromonas gingivalis in biofilm and gingival cells Akito Sakanaka, Hiroki Takeuchi, Masae Kuboniwa, Atsuo Amano* Department of Preventive Dentistry, Osaka University Graduate School of Dentistry, 1-8 Yamadaoka, Suita, Osaka 565-0871, Japan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 26 September 2015 Received in revised form 1 October 2015 Accepted 3 October 2015 Available online xxx
Porphyromonas gingivalis is deeply involved in the pathogenesis of marginal periodontitis, and recent findings have consolidated its role as an important and unique pathogen. This bacterium has a unique dual lifestyle in periodontal sites including subgingival dental plaque (biofilm) and gingival cells, as it has been clearly shown that P. gingivalis is able to exert virulence using completely different tactics in each environment. Inter-bacterial cross-feeding enhances the virulence of periodontal microflora, and such metabolic and adhesive interplay creates a supportive environment for P. gingivalis and other species. Human oral epithelial cells harbor a large intracellular bacterial load, resembling the polymicrobial nature of periodontal biofilm. P. gingivalis can enter gingival epithelial cells and pass through the epithelial barrier into deeper tissues. Subsequently, from its intracellular position, the pathogen exploits cellular recycling pathways to exit invaded cells, by which it is able to control its population in infected tissues, allowing for persistent infection in gingival tissues. Here, we outline the dual lifestyle of P. gingivalis in subgingival areas and its effects on the pathogenesis of periodontitis. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Biofilm Inter-bacterial cross-feeding Membrane trafficking Porphyromonas gingivalis Periodontitis
Contents 1. 2. 3. 4. 5. 6. 7. 8.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Lifestyle of P. gingivalis in biofilm . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . P. gingivalis and accessory pathogens in biofilm . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Inter-bacterial cross-feeding in biofilm . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Metabolic and adhesive interplay among S. gordonii, F. nucleatum, and P. gingivalis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Lifestyle of P. gingivalis in gingival cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Exit of P. gingivalis from periodontal cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Introduction Marginal periodontitis is a chronic inflammatory disease that leads to destruction of tooth supporting tissues. Although not fatal, it can cause tooth loss and marked reduction in quality of life. It is well-accepted that periodontitis is initiated by microbes embedded in subgingival dental plaque (biofilm) and related to complex interactions of bacteria with the host [1,2]. The Gram-negative asaccharolytic bacterium Porphyromonas gingivalis has long been
* Corresponding author. E-mail address: [email protected] (A. Amano).
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associated with periodontitis. Recent sequencing analysis of ancient dental calculus specimens revealed that P. gingivalis was already present in the human oral cavity at least 7000 years ago [3], suggesting that the pathogen has evolved along with humans over an extended period to acquire a unique lifestyle pattern. It is now clear that P. gingivalis has dual lifestyles based on its site of periodontal habitation; subgingival dental biofilm and gingival cells. More importantly, the dual lifestyle of P. gingivalis has detrimental effects on the host by subverting periodontal host responses and transforming the biofilm community structure, though many of those are just beginning to be explored in detail. The goal of this paper is to provide a brief overview of the lifestyle of
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Please cite this article in press as: A. Sakanaka, et al., Dual lifestyle of Porphyromonas gingivalis in biofilm and gingival cells, Microbial Pathogenesis (2015), http://dx.doi.org/10.1016/j.micpath.2015.10.003
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P. gingivalis in biofilm and gingival cells so as to provide a more detailed understanding of the contributions of this organism to the etiology of periodontitis. 2. Lifestyle of P. gingivalis in biofilm P. gingivalis has been shown to be a late colonizer of periodontal biofilm in a model of periodontopathic biofilm development, which is thought to occur in three successive stages [4]. According to this model, early colonizers, including Streptococci and Actinomyces, adhere to salivary pellicles on teeth by utilizing adhesins such as fimbriae and polysaccharide. Fusobacterium nucleatum, which possesses multiple adhesins, is an intermediate colonizer that serves as a central bridging bacterium by selective co-aggregation with a variety of microbes, thus attracting late colonizers. Previously, the pathogenicity of periodontitis was thought to be associated with an increase in abundance of late colonizers such as P. gingivalis. However, recent next-generation sequencing studies of periodontal microbiota have provided novel insights into the unique tactics employed by P. gingivalis in biofilm communities, as reviewed elsewhere [5,6]. For example, it has been shown that P. gingivalis, even in low volume, can elevate the virulence of periodontal biofilm by subverting host responses, altering biofilm community structures, and facilitating an increase in overall bacterial load, leading to its designation as a keystone pathogen. Interestingly, a previous study found that the relative abundance of P. gingivalis indicated a possible negative correlation with total bacterial load in patients with periodontitis, suggesting that the properties of the overall biofilm community contribute more directly to periodontal pathogenesis than abundance of the pathogen [7]. It was also shown that P. gingivalis exerts virulence in the context of the polymicrobial community and did not cause disease by itself in germ-free mice [5]. These findings imply that its incorporation into communities with a web of interconnected relationships with other microbes elicits the full range of pathogenicity of P. gingivalis. Taking into account the presence of a dynamic network of interactions among community members in periodontal biofilm [8], inter-bacterial interactions between P. gingivalis and other community members are considered to play a critical role in promoting the pathogenicity of this bacterium in periodontal biofilm.
P. gingivalis exert reciprocal influence on the phenotype of each, and optimizes their fitness advantages by fostering metabolic dependency on another organism. Therefore, in order to understand the lifestyle of P. gingivalis in periodontal biofilm, it is important to untangle these complex interactions, notably that related to metabolism, mediated by accessory pathogens.
4. Inter-bacterial cross-feeding in biofilm The metabolic interactions of accessory pathogens are increasingly being shown to be important determinants of community structure and pathogenesis in periodontal biofilm [9,17]. A previous study found that Veillonella atypica, an anaerobic early colonizer, consumes lactate produced by S. gordonii as an end-product of glycolysis, leading to enhanced growth of both species in vitro [18]. This type of metabolic interaction is referred to as cross-feeding, in which metabolic by-products of one microbe are utilized by others as an energy or nutrient source, and, in many cases, support the growth of both organisms [19,20]. Indeed, a recent microbiome analysis study confirmed that the amounts of Streptococcus and Veillonella in subgingival biofilm samples obtained from periodontal patients were positively correlated, suggesting a mutually beneficial relationship between these species in subgingival areas [7]. Furthermore, cross-feeding on lactate has been shown to occur between S. gordonii and Aggregatibacter actinomycetemcomitans [21e23].
3. P. gingivalis and accessory pathogens in biofilm Of particular interest are interactions between P. gingivalis and a subset of oral streptococci that have traditionally been considered to be commensal organisms. These streptococcal species, now referred to as accessory pathogens, enhance the virulence of P. gingivalis in the heterotypic community via various microbial interactions, including quorum sensing, cell surface adhesionmediated co-aggregation, and metabolic cooperation [9]. For example, P. gingivalis binds to the accessory pathogen Streptococcus gordonii by a coordinated adhesion system, in which FimA and Mfa1 fimbriae of P. gingivalis bind to GAPDH and SspA/B surface proteins of S. gordonii, respectively [10e13]. In addition to surface molecules, several gene functions in S. gordonii have been shown to be required for development of mutualistic biofilm communities with P. gingivalis [14]. The putative functions of the encoded proteins include maintenance of cell wall integrity (murE), intercellular signaling (cbe), and regulation of redox state (spxB and msrA). Recent proteomic analyses have also revealed that S. gordonii and P. gingivalis display different proteome profiles in co-cultures with each other, and that a substantial portion of differentially expressed proteins participate in metabolism regulation [15,16]. Collectively, these results indicate that interactions between S. gordonii and
Fig. 1. Inter-bacterial cross-feeding in biofilm. (a) Overview of metabolic interactions fostered by a variety of periodontal bacteria. All metabolic interactions depicted here can support growth of the recipients, except that between P. gingivalis and S. intermedius/S. cristatus, in which biofilm formation by P. gingivalis is diminished by extracellular conversion of arginine into citrulline by these streptococci. (b) Metabolic and adhesive interplay among S. gordonii, F. nucleatum, and P. gingivalis. This triad state seems to create a supportive environment for each member, thus maximizing their fitness advantages. Abbreviations: ADS, arginine deaminase system; CPS, capsular polysaccharide; LPS, lipopolysaccharide; ODC, ornithine decarboxylase.
Please cite this article in press as: A. Sakanaka, et al., Dual lifestyle of Porphyromonas gingivalis in biofilm and gingival cells, Microbial Pathogenesis (2015), http://dx.doi.org/10.1016/j.micpath.2015.10.003
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Another important metabolite that likely contributes to the metabolic interactions of accessory pathogens is arginine, as a number of studies have indicated its involvement in inter-bacterial interactions between oral streptococci and other bacterial species. In particular, this metabolite seems to have a profound influence on the lifestyle of P. gingivalis. Arginine was reported to be utilized by P. gingivalis to promote its biofilm formation [24], while others presented interesting findings showing that the FimA expression of P. gingivalis is inhibited by arginine deaminase (ArcA) secreted by Streptococcus intermedius and Streptococcus cristatus, leading to diminished biofilm formation [25,26]. In turn, this down-regulation was shown to be a result of the enzymatic activity of extracellular ArcA, which catalyzes the conversion of arginine to citrulline [24]. Together, these findings suggest negative effects of extracellular arginine depletion and/or citrulline accumulation on FimA expression and biofilm formation by P. gingivalis (Fig. 1a). In contrast to S. gordonii, these streptococci are not considered to be accessory pathogens. The distribution of S. cristatus in subgingival plaque is negatively correlated with that of P. gingivalis [27] and S. cristatus mitigates P. gingivalis-induced bone loss in mice [28]. What causes the difference between those streptococcal species is currently unknown, though one possibility is extracellular localization of ArcA. Extracellular ArcA of S. gordonii has not been reported, whereas S. gordonii ArcA has been well characterized in the context of the arginine deaminase system (ADS), which catalyzes the intracellular conversion of arginine to ammonia and CO2, along with concomitant production of ATP [29e31]. Although it is unclear whether S. gordonii secretes ArcA, it seems likely that distinctive responses to P. gingivalis within oral streptococci are due to the ability to transform arginine into citrulline in an extracellular manner, leading to reduced availability of arginine caused by P. gingivalis and/or a negative influence of citrulline on that bacterium. Arginine and its derivatives also contribute to interactions between oral streptococci and F. nucleatum. This fusiform-shaped anaerobic bacterium is referred to as a “bridging organism” that links early and late colonizers of oral microbial communities, and contributes to the pathogenicity of periodontitis [32e34]. F. nucleatum harbors an adhesin that is inhibited by arginine (RadD), which is responsible for its adherence to oral streptococci, and high concentrations of arginine inhibit cell-to-cell contact (i.e., coaggregation) between these species [35]. More recently, we reported a novel cross-feeding interaction between S. gordonii and F. nucleatum [36]. This interaction involves ornithine, which is exported by the arginine-ornithine antiporter ArcD in the ADS of S. gordonii. Dual-species biofilm experimental results demonstrated that deletion of arcD attenuates accumulation of F. nucleatum in S. gordonii biofilm, while ornithine supplementation restored the biovolume of F. nucleatum in mono-species biofilms as well as dual-species biofilms with the S. gordonii DarcD mutant, suggesting that ArcD-exported ornithine supports the growth of F. nucleatum and bolsters the development of its biofilm. In addition, a previous study found that F. nucleatum exhibited a significant increase in the level of protein expression of ornithine decarboxylase (ODC), an enzyme responsible for conversion of ornithine/arginine to putrescine, in community biofilms formed with S. gordonii [37]. Therefore, this pathogen seems to utilize ornithine released by S. gordonii ArcD as a substrate of ODC. Collectively, ArcD of S. gordonii helps to enhance community development by these species by mediating crossfeeding of ornithine, as well as reinforcing coaggregation between S. gordonii and F. nucleatum. These findings suggest that sustained delivery of ornithine from accessory pathogens underlies the conversion of periodontal microbiota from a symbiotic to dysbiotic state, highlighting the role of accessory pathogens in
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periodontal disease initiation. 5. Metabolic and adhesive interplay among S. gordonii, F. nucleatum, and P. gingivalis An interesting finding showed that loss of ArcD does not affect the interaction between S. gordonii and P. gingivalis [36]. Furthermore, S. gordonii was reported to exhibit distinct patterns of protein abundance in communities including F. nucleatum or P. gingivalis, especially in relation to the ADS component enzymes ArcA, ArcB (catabolic ornithine carbamoyltransferase), ArcC (carbamate kinase), and ArcD [16]. Also, the levels of ArcA, ArcB, and ArcC of S. gordonii were reduced in community biofilms formed with P. gingivalis as compared to mono-species biofilms, while the level of ArcD protein was below the limit of detection. On the other hand, community biofilms formed with F. nucleatum showed markedly increased levels of ArcA, ArcB, and ArcC despite a significantly reduced level of ArcD in S. gordonii. A recent study demonstrated that arcABC of S. gordonii is upregulated in the presence of a high concentration of arginine, whereas arcD is repressed [38], suggesting that arginine is donated by F. nucleatum to S. gordonii and competed for by P. gingivalis. Taking into account the avid adhesive interactions among these 3 species [8,39,40], the state of the triad could reach an interbacterial stability point and maximize their fitness advantages (Fig. 1b). Although further studies are needed to examine the arginine-related metabolic interactions among periodontal bacteria, these examples are indicative of metabolic selectivity in periodontal microflora. Metabolic cooperation between traditional periodontal pathogens has also been reported, such as P. gingivalis and Treponema denticola, which are known to co-localize in subgingival biofilms in vivo and display mutualistic biofilm growth in vitro, as well as synergistic virulence upon coinfection in animal models of alveolar bone loss [41e45]. In co-cultures, P. gingivalis produces isobutyric acid and T. denticola produces succinic acid, both of which can be used as nutrient substrates by the other [46,47]. A recent study also demonstrated that T. denticola can stimulate peptide hydrolysis by P. gingivalis to release free glycine, which T. denticola uses as a major carbon source, leading to up-regulation of T. denticola genes encoding virulence factors and glycine catabolic pathways [48]. Coculture of these species also up-regulates RgpA, Kgp, gingipains, and HagA in P. gingivalis, which in turn enhance its binding to substrates including epithelial cells and S. gordonii [49]. Given that S. gordonii provides metabolic support for P. gingivalis, these findings suggest that metabolic interactions among periodontal microbes are likely far more diverse and interconnected than currently understood (Fig. 1a). 6. Lifestyle of P. gingivalis in gingival cells Gingival epithelial cells function as an innate physical barrier to prevent invasion by periodontal bacteria [50]. Nevertheless, several strains of periodontal bacteria, including P. gingivalis, Tannerella forsythia, and T. denticola, have been detected among epithelial cells obtained from periodontal pockets, gingival crevices, and buccal mucosa specimens collected from both periodontitis patients and subjects with healthy gingivae [51,52]. P. gingivalis is able to enter those cells and pass through the epithelial barrier into deeper tissues, after which intracellular P. gingivalis impairs fundamental cellular functions such as cellular migration and proliferation [53]. The pathogen can also enter gingival fibroblasts [54] and osteoblasts [55]. An intracellular location is considered advantageous for escape from immune surveillance by the host and antibiotic pressure, leading to intracellular persistence, multiplication, and
Please cite this article in press as: A. Sakanaka, et al., Dual lifestyle of Porphyromonas gingivalis in biofilm and gingival cells, Microbial Pathogenesis (2015), http://dx.doi.org/10.1016/j.micpath.2015.10.003
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Fig. 2. Sorting routes of intracellular P. gingivalis. Following cellular entry, P. gingivalis organisms are initially localized within endocytic vacuoles (early endosomes). Thereafter, some are routed to late endosomes, then subsequently sorted to lysosomes for degradation. Other bacteria likely promote their own entry into the autophagic pathway by bacterial escape from endosomes and sorted to autolysosomes formed by the fusion of autophagosomes with lysosomes for degradation. A large number of intracellular P. gingivalis organisms escape from these lytic compartments via endocytosis and autophagy pathways by hijacking a fast recycling pathway to exit from primarily infected host cells into intercellular space and then enter new host cells, thus enabling further penetration of host tissues in a trans-cellular manner. EEA1: early endosome antigen 1; RUFY1: RUN and FYVE domain containing 1; BMX: bone marrow tyrosine kinase gene in chromosome X protein; SNARE: soluble N-ethylmaleimide-sensitive factor attachment protein receptor; VAMP2: vesicle-associated membrane protein 2; EXOC2: exocyst complex component 2; EXOC3: exocyst complex component 3; RalA: v-Ral simian leukemia viral oncogene homolog A; CDC42: cell division cycle 42; LC3: microtubule-associated protein light chain 3.
dissemination to adjacent tissues [50,52,56]. However, it remains largely unknown how intracellular P. gingivalis can survive and spread its infection into deeper tissues. The intracellular infection process is divided into four phases; adhesion, entry, intracellular trafficking, and exit [57]. Exit from invaded cells promotes further infection of neighboring gingival cells, which leads to persistent infection in tissues. P. gingivalis exploits the cellular endocytosis pathway to enter cells [58,59]. First, the pathogen adheres to the cell surface by interactions between bacterial FimA fimbriae and cellular a5b1 integrin, then is subsequently engulfed by actin filament assembly and internalized by encapsulation within an early endosome. 7. Exit of P. gingivalis from periodontal cells Following encapsulation, some intracellular microorganisms are sorted to lytic compartments such as autolysosomes and late endosomes/lysosomes [60] (Fig. 2). Although autophagy has been suggested to mediate bacterial survival, its role in the fate of intracellular P. gingivalis is still unclear. A considerable number of the remaining organisms are sorted to a recycling pathway [60], which recycles functional proteins and lipids after undergoing endocytosis back to the plasma membrane [61]. Dominant negative forms as well as RNAi-knockdown of recycling pathway-related molecules, such as Rab11 and RalA, and exocyst complex subunits have been shown to significantly disrupt the exit of P. gingivalis [60]. Intracellular P. gingivalis organisms likely hijack this pathway to exit from cells and invade neighboring cells by a cell-to-cell spreading mechanism, which allows expansion of the bacterial population
within infected tissues for persistent infection [60]. 8. Conclusion Detailed exploration of metabolic cross-feeding in periodontal microflora has only just begun and a vast constellation of microbial metabolites employed by that process is awaiting discovery. Recent metagenomics studies have found that genes encoding enzymes associated with the metabolism of amino acids including arginine had a higher abundance in subgingival biofilm specimens obtained from periodontal patients [62,63]. Furthermore, a recent study that utilized transcriptomics showed that disease-associated periodontal communities in periodontitis patients display highly conserved metabolic profiles, despite a diverse microbial composition [64]. These findings suggest that similar alterations of metabolic interactions in periodontal microflora underlie the transition from healthy to periodontopathic biofilm, and that control of these metabolic shifts, rather than elimination of P. gingivalis, may be a fruitful approach for development of novel therapeutic strategies. However, the overall profiles of metabolic interactions in periodontal microflora remain largely unknown, though taxonomic and genetic profiles are beginning to be revealed as nextgeneration sequencing technologies progress. Thus, deciphering the metabolic profiles of periodontal microflora is at the cutting edge of research in this field. It has been shown that human oral epithelial cells harbor a large intracellular bacterial load, resembling the polymicrobial nature of periodontal biofilm [51]. Furthermore, negligible levels of apoptosis and necrosis have been observed in epithelial cells infected by
Please cite this article in press as: A. Sakanaka, et al., Dual lifestyle of Porphyromonas gingivalis in biofilm and gingival cells, Microbial Pathogenesis (2015), http://dx.doi.org/10.1016/j.micpath.2015.10.003
A. Sakanaka et al. / Microbial Pathogenesis xxx (2015) 1e6
bacteria [65]. These findings indicate that intracellular periodontal bacteria reside in a largely dormant fashion, which allows them to persist but not destroy the host. Using a sophisticated strategy, P. gingivalis organisms enter and exit gingival cells, which promotes persistent infection. Such efficient bacterial movement contributes to periodontal destruction, which is further enhanced by mutual communication within the complex polymicrobial etiology [66].
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Contents lists available at ScienceDirect
Microbial Pathogenesis journal homepage: www.elsevier.com/locate/micpath
Review
Dual lifestyle of Porphyromonas gingivalis in biofilm and gingival cells Akito Sakanaka, Hiroki Takeuchi, Masae Kuboniwa, Atsuo Amano* Department of Preventive Dentistry, Osaka University Graduate School of Dentistry, 1-8 Yamadaoka, Suita, Osaka 565-0871, Japan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 26 September 2015 Received in revised form 1 October 2015 Accepted 3 October 2015 Available online xxx
Porphyromonas gingivalis is deeply involved in the pathogenesis of marginal periodontitis, and recent findings have consolidated its role as an important and unique pathogen. This bacterium has a unique dual lifestyle in periodontal sites including subgingival dental plaque (biofilm) and gingival cells, as it has been clearly shown that P. gingivalis is able to exert virulence using completely different tactics in each environment. Inter-bacterial cross-feeding enhances the virulence of periodontal microflora, and such metabolic and adhesive interplay creates a supportive environment for P. gingivalis and other species. Human oral epithelial cells harbor a large intracellular bacterial load, resembling the polymicrobial nature of periodontal biofilm. P. gingivalis can enter gingival epithelial cells and pass through the epithelial barrier into deeper tissues. Subsequently, from its intracellular position, the pathogen exploits cellular recycling pathways to exit invaded cells, by which it is able to control its population in infected tissues, allowing for persistent infection in gingival tissues. Here, we outline the dual lifestyle of P. gingivalis in subgingival areas and its effects on the pathogenesis of periodontitis. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Biofilm Inter-bacterial cross-feeding Membrane trafficking Porphyromonas gingivalis Periodontitis
Contents 1. 2. 3. 4. 5. 6. 7. 8.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Lifestyle of P. gingivalis in biofilm . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . P. gingivalis and accessory pathogens in biofilm . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Inter-bacterial cross-feeding in biofilm . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Metabolic and adhesive interplay among S. gordonii, F. nucleatum, and P. gingivalis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Lifestyle of P. gingivalis in gingival cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Exit of P. gingivalis from periodontal cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Introduction Marginal periodontitis is a chronic inflammatory disease that leads to destruction of tooth supporting tissues. Although not fatal, it can cause tooth loss and marked reduction in quality of life. It is well-accepted that periodontitis is initiated by microbes embedded in subgingival dental plaque (biofilm) and related to complex interactions of bacteria with the host [1,2]. The Gram-negative asaccharolytic bacterium Porphyromonas gingivalis has long been
* Corresponding author. E-mail address: [email protected] (A. Amano).
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associated with periodontitis. Recent sequencing analysis of ancient dental calculus specimens revealed that P. gingivalis was already present in the human oral cavity at least 7000 years ago [3], suggesting that the pathogen has evolved along with humans over an extended period to acquire a unique lifestyle pattern. It is now clear that P. gingivalis has dual lifestyles based on its site of periodontal habitation; subgingival dental biofilm and gingival cells. More importantly, the dual lifestyle of P. gingivalis has detrimental effects on the host by subverting periodontal host responses and transforming the biofilm community structure, though many of those are just beginning to be explored in detail. The goal of this paper is to provide a brief overview of the lifestyle of
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P. gingivalis in biofilm and gingival cells so as to provide a more detailed understanding of the contributions of this organism to the etiology of periodontitis. 2. Lifestyle of P. gingivalis in biofilm P. gingivalis has been shown to be a late colonizer of periodontal biofilm in a model of periodontopathic biofilm development, which is thought to occur in three successive stages [4]. According to this model, early colonizers, including Streptococci and Actinomyces, adhere to salivary pellicles on teeth by utilizing adhesins such as fimbriae and polysaccharide. Fusobacterium nucleatum, which possesses multiple adhesins, is an intermediate colonizer that serves as a central bridging bacterium by selective co-aggregation with a variety of microbes, thus attracting late colonizers. Previously, the pathogenicity of periodontitis was thought to be associated with an increase in abundance of late colonizers such as P. gingivalis. However, recent next-generation sequencing studies of periodontal microbiota have provided novel insights into the unique tactics employed by P. gingivalis in biofilm communities, as reviewed elsewhere [5,6]. For example, it has been shown that P. gingivalis, even in low volume, can elevate the virulence of periodontal biofilm by subverting host responses, altering biofilm community structures, and facilitating an increase in overall bacterial load, leading to its designation as a keystone pathogen. Interestingly, a previous study found that the relative abundance of P. gingivalis indicated a possible negative correlation with total bacterial load in patients with periodontitis, suggesting that the properties of the overall biofilm community contribute more directly to periodontal pathogenesis than abundance of the pathogen [7]. It was also shown that P. gingivalis exerts virulence in the context of the polymicrobial community and did not cause disease by itself in germ-free mice [5]. These findings imply that its incorporation into communities with a web of interconnected relationships with other microbes elicits the full range of pathogenicity of P. gingivalis. Taking into account the presence of a dynamic network of interactions among community members in periodontal biofilm [8], inter-bacterial interactions between P. gingivalis and other community members are considered to play a critical role in promoting the pathogenicity of this bacterium in periodontal biofilm.
P. gingivalis exert reciprocal influence on the phenotype of each, and optimizes their fitness advantages by fostering metabolic dependency on another organism. Therefore, in order to understand the lifestyle of P. gingivalis in periodontal biofilm, it is important to untangle these complex interactions, notably that related to metabolism, mediated by accessory pathogens.
4. Inter-bacterial cross-feeding in biofilm The metabolic interactions of accessory pathogens are increasingly being shown to be important determinants of community structure and pathogenesis in periodontal biofilm [9,17]. A previous study found that Veillonella atypica, an anaerobic early colonizer, consumes lactate produced by S. gordonii as an end-product of glycolysis, leading to enhanced growth of both species in vitro [18]. This type of metabolic interaction is referred to as cross-feeding, in which metabolic by-products of one microbe are utilized by others as an energy or nutrient source, and, in many cases, support the growth of both organisms [19,20]. Indeed, a recent microbiome analysis study confirmed that the amounts of Streptococcus and Veillonella in subgingival biofilm samples obtained from periodontal patients were positively correlated, suggesting a mutually beneficial relationship between these species in subgingival areas [7]. Furthermore, cross-feeding on lactate has been shown to occur between S. gordonii and Aggregatibacter actinomycetemcomitans [21e23].
3. P. gingivalis and accessory pathogens in biofilm Of particular interest are interactions between P. gingivalis and a subset of oral streptococci that have traditionally been considered to be commensal organisms. These streptococcal species, now referred to as accessory pathogens, enhance the virulence of P. gingivalis in the heterotypic community via various microbial interactions, including quorum sensing, cell surface adhesionmediated co-aggregation, and metabolic cooperation [9]. For example, P. gingivalis binds to the accessory pathogen Streptococcus gordonii by a coordinated adhesion system, in which FimA and Mfa1 fimbriae of P. gingivalis bind to GAPDH and SspA/B surface proteins of S. gordonii, respectively [10e13]. In addition to surface molecules, several gene functions in S. gordonii have been shown to be required for development of mutualistic biofilm communities with P. gingivalis [14]. The putative functions of the encoded proteins include maintenance of cell wall integrity (murE), intercellular signaling (cbe), and regulation of redox state (spxB and msrA). Recent proteomic analyses have also revealed that S. gordonii and P. gingivalis display different proteome profiles in co-cultures with each other, and that a substantial portion of differentially expressed proteins participate in metabolism regulation [15,16]. Collectively, these results indicate that interactions between S. gordonii and
Fig. 1. Inter-bacterial cross-feeding in biofilm. (a) Overview of metabolic interactions fostered by a variety of periodontal bacteria. All metabolic interactions depicted here can support growth of the recipients, except that between P. gingivalis and S. intermedius/S. cristatus, in which biofilm formation by P. gingivalis is diminished by extracellular conversion of arginine into citrulline by these streptococci. (b) Metabolic and adhesive interplay among S. gordonii, F. nucleatum, and P. gingivalis. This triad state seems to create a supportive environment for each member, thus maximizing their fitness advantages. Abbreviations: ADS, arginine deaminase system; CPS, capsular polysaccharide; LPS, lipopolysaccharide; ODC, ornithine decarboxylase.
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Another important metabolite that likely contributes to the metabolic interactions of accessory pathogens is arginine, as a number of studies have indicated its involvement in inter-bacterial interactions between oral streptococci and other bacterial species. In particular, this metabolite seems to have a profound influence on the lifestyle of P. gingivalis. Arginine was reported to be utilized by P. gingivalis to promote its biofilm formation [24], while others presented interesting findings showing that the FimA expression of P. gingivalis is inhibited by arginine deaminase (ArcA) secreted by Streptococcus intermedius and Streptococcus cristatus, leading to diminished biofilm formation [25,26]. In turn, this down-regulation was shown to be a result of the enzymatic activity of extracellular ArcA, which catalyzes the conversion of arginine to citrulline [24]. Together, these findings suggest negative effects of extracellular arginine depletion and/or citrulline accumulation on FimA expression and biofilm formation by P. gingivalis (Fig. 1a). In contrast to S. gordonii, these streptococci are not considered to be accessory pathogens. The distribution of S. cristatus in subgingival plaque is negatively correlated with that of P. gingivalis [27] and S. cristatus mitigates P. gingivalis-induced bone loss in mice [28]. What causes the difference between those streptococcal species is currently unknown, though one possibility is extracellular localization of ArcA. Extracellular ArcA of S. gordonii has not been reported, whereas S. gordonii ArcA has been well characterized in the context of the arginine deaminase system (ADS), which catalyzes the intracellular conversion of arginine to ammonia and CO2, along with concomitant production of ATP [29e31]. Although it is unclear whether S. gordonii secretes ArcA, it seems likely that distinctive responses to P. gingivalis within oral streptococci are due to the ability to transform arginine into citrulline in an extracellular manner, leading to reduced availability of arginine caused by P. gingivalis and/or a negative influence of citrulline on that bacterium. Arginine and its derivatives also contribute to interactions between oral streptococci and F. nucleatum. This fusiform-shaped anaerobic bacterium is referred to as a “bridging organism” that links early and late colonizers of oral microbial communities, and contributes to the pathogenicity of periodontitis [32e34]. F. nucleatum harbors an adhesin that is inhibited by arginine (RadD), which is responsible for its adherence to oral streptococci, and high concentrations of arginine inhibit cell-to-cell contact (i.e., coaggregation) between these species [35]. More recently, we reported a novel cross-feeding interaction between S. gordonii and F. nucleatum [36]. This interaction involves ornithine, which is exported by the arginine-ornithine antiporter ArcD in the ADS of S. gordonii. Dual-species biofilm experimental results demonstrated that deletion of arcD attenuates accumulation of F. nucleatum in S. gordonii biofilm, while ornithine supplementation restored the biovolume of F. nucleatum in mono-species biofilms as well as dual-species biofilms with the S. gordonii DarcD mutant, suggesting that ArcD-exported ornithine supports the growth of F. nucleatum and bolsters the development of its biofilm. In addition, a previous study found that F. nucleatum exhibited a significant increase in the level of protein expression of ornithine decarboxylase (ODC), an enzyme responsible for conversion of ornithine/arginine to putrescine, in community biofilms formed with S. gordonii [37]. Therefore, this pathogen seems to utilize ornithine released by S. gordonii ArcD as a substrate of ODC. Collectively, ArcD of S. gordonii helps to enhance community development by these species by mediating crossfeeding of ornithine, as well as reinforcing coaggregation between S. gordonii and F. nucleatum. These findings suggest that sustained delivery of ornithine from accessory pathogens underlies the conversion of periodontal microbiota from a symbiotic to dysbiotic state, highlighting the role of accessory pathogens in
3
periodontal disease initiation. 5. Metabolic and adhesive interplay among S. gordonii, F. nucleatum, and P. gingivalis An interesting finding showed that loss of ArcD does not affect the interaction between S. gordonii and P. gingivalis [36]. Furthermore, S. gordonii was reported to exhibit distinct patterns of protein abundance in communities including F. nucleatum or P. gingivalis, especially in relation to the ADS component enzymes ArcA, ArcB (catabolic ornithine carbamoyltransferase), ArcC (carbamate kinase), and ArcD [16]. Also, the levels of ArcA, ArcB, and ArcC of S. gordonii were reduced in community biofilms formed with P. gingivalis as compared to mono-species biofilms, while the level of ArcD protein was below the limit of detection. On the other hand, community biofilms formed with F. nucleatum showed markedly increased levels of ArcA, ArcB, and ArcC despite a significantly reduced level of ArcD in S. gordonii. A recent study demonstrated that arcABC of S. gordonii is upregulated in the presence of a high concentration of arginine, whereas arcD is repressed [38], suggesting that arginine is donated by F. nucleatum to S. gordonii and competed for by P. gingivalis. Taking into account the avid adhesive interactions among these 3 species [8,39,40], the state of the triad could reach an interbacterial stability point and maximize their fitness advantages (Fig. 1b). Although further studies are needed to examine the arginine-related metabolic interactions among periodontal bacteria, these examples are indicative of metabolic selectivity in periodontal microflora. Metabolic cooperation between traditional periodontal pathogens has also been reported, such as P. gingivalis and Treponema denticola, which are known to co-localize in subgingival biofilms in vivo and display mutualistic biofilm growth in vitro, as well as synergistic virulence upon coinfection in animal models of alveolar bone loss [41e45]. In co-cultures, P. gingivalis produces isobutyric acid and T. denticola produces succinic acid, both of which can be used as nutrient substrates by the other [46,47]. A recent study also demonstrated that T. denticola can stimulate peptide hydrolysis by P. gingivalis to release free glycine, which T. denticola uses as a major carbon source, leading to up-regulation of T. denticola genes encoding virulence factors and glycine catabolic pathways [48]. Coculture of these species also up-regulates RgpA, Kgp, gingipains, and HagA in P. gingivalis, which in turn enhance its binding to substrates including epithelial cells and S. gordonii [49]. Given that S. gordonii provides metabolic support for P. gingivalis, these findings suggest that metabolic interactions among periodontal microbes are likely far more diverse and interconnected than currently understood (Fig. 1a). 6. Lifestyle of P. gingivalis in gingival cells Gingival epithelial cells function as an innate physical barrier to prevent invasion by periodontal bacteria [50]. Nevertheless, several strains of periodontal bacteria, including P. gingivalis, Tannerella forsythia, and T. denticola, have been detected among epithelial cells obtained from periodontal pockets, gingival crevices, and buccal mucosa specimens collected from both periodontitis patients and subjects with healthy gingivae [51,52]. P. gingivalis is able to enter those cells and pass through the epithelial barrier into deeper tissues, after which intracellular P. gingivalis impairs fundamental cellular functions such as cellular migration and proliferation [53]. The pathogen can also enter gingival fibroblasts [54] and osteoblasts [55]. An intracellular location is considered advantageous for escape from immune surveillance by the host and antibiotic pressure, leading to intracellular persistence, multiplication, and
Please cite this article in press as: A. Sakanaka, et al., Dual lifestyle of Porphyromonas gingivalis in biofilm and gingival cells, Microbial Pathogenesis (2015), http://dx.doi.org/10.1016/j.micpath.2015.10.003
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Fig. 2. Sorting routes of intracellular P. gingivalis. Following cellular entry, P. gingivalis organisms are initially localized within endocytic vacuoles (early endosomes). Thereafter, some are routed to late endosomes, then subsequently sorted to lysosomes for degradation. Other bacteria likely promote their own entry into the autophagic pathway by bacterial escape from endosomes and sorted to autolysosomes formed by the fusion of autophagosomes with lysosomes for degradation. A large number of intracellular P. gingivalis organisms escape from these lytic compartments via endocytosis and autophagy pathways by hijacking a fast recycling pathway to exit from primarily infected host cells into intercellular space and then enter new host cells, thus enabling further penetration of host tissues in a trans-cellular manner. EEA1: early endosome antigen 1; RUFY1: RUN and FYVE domain containing 1; BMX: bone marrow tyrosine kinase gene in chromosome X protein; SNARE: soluble N-ethylmaleimide-sensitive factor attachment protein receptor; VAMP2: vesicle-associated membrane protein 2; EXOC2: exocyst complex component 2; EXOC3: exocyst complex component 3; RalA: v-Ral simian leukemia viral oncogene homolog A; CDC42: cell division cycle 42; LC3: microtubule-associated protein light chain 3.
dissemination to adjacent tissues [50,52,56]. However, it remains largely unknown how intracellular P. gingivalis can survive and spread its infection into deeper tissues. The intracellular infection process is divided into four phases; adhesion, entry, intracellular trafficking, and exit [57]. Exit from invaded cells promotes further infection of neighboring gingival cells, which leads to persistent infection in tissues. P. gingivalis exploits the cellular endocytosis pathway to enter cells [58,59]. First, the pathogen adheres to the cell surface by interactions between bacterial FimA fimbriae and cellular a5b1 integrin, then is subsequently engulfed by actin filament assembly and internalized by encapsulation within an early endosome. 7. Exit of P. gingivalis from periodontal cells Following encapsulation, some intracellular microorganisms are sorted to lytic compartments such as autolysosomes and late endosomes/lysosomes [60] (Fig. 2). Although autophagy has been suggested to mediate bacterial survival, its role in the fate of intracellular P. gingivalis is still unclear. A considerable number of the remaining organisms are sorted to a recycling pathway [60], which recycles functional proteins and lipids after undergoing endocytosis back to the plasma membrane [61]. Dominant negative forms as well as RNAi-knockdown of recycling pathway-related molecules, such as Rab11 and RalA, and exocyst complex subunits have been shown to significantly disrupt the exit of P. gingivalis [60]. Intracellular P. gingivalis organisms likely hijack this pathway to exit from cells and invade neighboring cells by a cell-to-cell spreading mechanism, which allows expansion of the bacterial population
within infected tissues for persistent infection [60]. 8. Conclusion Detailed exploration of metabolic cross-feeding in periodontal microflora has only just begun and a vast constellation of microbial metabolites employed by that process is awaiting discovery. Recent metagenomics studies have found that genes encoding enzymes associated with the metabolism of amino acids including arginine had a higher abundance in subgingival biofilm specimens obtained from periodontal patients [62,63]. Furthermore, a recent study that utilized transcriptomics showed that disease-associated periodontal communities in periodontitis patients display highly conserved metabolic profiles, despite a diverse microbial composition [64]. These findings suggest that similar alterations of metabolic interactions in periodontal microflora underlie the transition from healthy to periodontopathic biofilm, and that control of these metabolic shifts, rather than elimination of P. gingivalis, may be a fruitful approach for development of novel therapeutic strategies. However, the overall profiles of metabolic interactions in periodontal microflora remain largely unknown, though taxonomic and genetic profiles are beginning to be revealed as nextgeneration sequencing technologies progress. Thus, deciphering the metabolic profiles of periodontal microflora is at the cutting edge of research in this field. It has been shown that human oral epithelial cells harbor a large intracellular bacterial load, resembling the polymicrobial nature of periodontal biofilm [51]. Furthermore, negligible levels of apoptosis and necrosis have been observed in epithelial cells infected by
Please cite this article in press as: A. Sakanaka, et al., Dual lifestyle of Porphyromonas gingivalis in biofilm and gingival cells, Microbial Pathogenesis (2015), http://dx.doi.org/10.1016/j.micpath.2015.10.003
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bacteria [65]. These findings indicate that intracellular periodontal bacteria reside in a largely dormant fashion, which allows them to persist but not destroy the host. Using a sophisticated strategy, P. gingivalis organisms enter and exit gingival cells, which promotes persistent infection. Such efficient bacterial movement contributes to periodontal destruction, which is further enhanced by mutual communication within the complex polymicrobial etiology [66].
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Please cite this article in press as: A. Sakanaka, et al., Dual lifestyle of Porphyromonas gingivalis in biofilm and gingival cells, Microbial Pathogenesis (2015), http://dx.doi.org/10.1016/j.micpath.2015.10.003