- Email: [email protected]
Transcellular invasive mechanisms of Porphyromonas gingivalis in host–parasite interactions
Journal of Oral Biosciences 56 (2014) 58–62
Contents lists available at ScienceDirect
Journal of Oral Biosciences journal homepage: www.elsevier.com/locate/job
Review
Transcellular invasive mechanisms of Porphyromonas gingivalis in host–parasite interactions Atsuo Amano n, Masae Kuboniwa, Hiroki Takeuchi Department of Preventive Dentistry, Osaka University Graduate School of Dentistry, 1-8 Yamadaoka, Suita, Osaka 565-0871, Japan
art ic l e i nf o
a b s t r a c t
Article history: Received 31 January 2014 Received in revised form 19 February 2014 Accepted 19 February 2014 Available online 1 April 2014
Porphyromonas gingivalis, a well-known periodontal pathogen, expresses a number of virulence factors, including fimbriae and gingipains. In addition, the pathogen utilizes two unique transcellular invasive mechanisms that cause cellular impairment in periodontal tissues; these mechanisms include bacterial entry into periodontal cells and bacterial shooting of outer membrane vesicles (OMVs) into those cells. Gingival epithelial cells function as innate host defense barriers to prevent intrusion by periodontal bacteria. Nevertheless, P. gingivalis can enter these cells and pass through the epithelial barrier into deeper tissues. Fimbriae of P. gingivalis specifically interact with α5β1-integrin of epithelial cells, which induces cellular invagination to internalize the pathogen. Following their entry, intracellular P. gingivalis impairs fundamental cellular functions, while some intracellular bacteria are finally sorted to lytic compartments, including autolysosomes and late endosomes/lysosomes. Furthermore, a considerable number of organisms are sorted into recycling endosomes, leading to bacterial exit from infected cells to neighboring cells, a mechanism of cell-to-cell spread within periodontal tissues. Most gram-negative bacteria, including P. gingivalis, produce OMVs, which serve as bacterial “bullets” for directed intercellular transport of bacterial virulence factors into host cells and tissues. Following entry into the cells, OMVs can use bacterial gingipains to degrade cellular functional components. Although OMVs do not escape from lytic compartments, they survive within lysosomes for more than 24 h, resulting in significant formation of acidified compartments. This review addresses the remarkable transcellular strategies that are used by P. gingivalis to destroy periodontal tissues. & 2014 Japanese Association for Oral Biology. Published by Elsevier B.V. All rights reserved.
Keywords: Membrane trafficking Porphyromonas gingivalis Outer membrane vesicles Periodontitis
Contents 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Intracellular infection by P. gingivalis . . . . . . . . . . . . . . . . . 3. Intracellular trafficking routes of P. gingivalis . . . . . . . . . . . 4. P. gingivalis exit from periodontal cells. . . . . . . . . . . . . . . . 5. Cellular impairment by intracellular P. gingivalis . . . . . . . . 6. Entry mechanism of P. gingivalis OMVs . . . . . . . . . . . . . . . 7. Cellular impairment by intracellular OMVs of P. gingivalis 8. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conflicts of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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1. Introduction
n
Corresponding author. Tel.: þ 81 6 6879 2921; fax: þ 81 6 6879 2925. E-mail address: [email protected] (A. Amano).
Gingival epithelial cells function as an innate host defense system to prevent intrusion by periodontal bacteria [1]. However, persistent contact of subgingival bacterial biofilm with gingival crevices induces bacterial penetration into periodontal tissues.
http://dx.doi.org/10.1016/j.job.2014.02.001 1349-0079/& 2014 Japanese Association for Oral Biology. Published by Elsevier B.V. All rights reserved.
A. Amano et al. / Journal of Oral Biosciences 56 (2014) 58–62
Immunofluorescence and immunohistochemical techniques have revealed the existence of Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, Prevotella intermedia, and Actinomyces naeslundii in gingival tissues [2]. In addition, intracellular localization of several periodontal bacteria, including P. gingivalis, A. actinomycetemcomitans, Tannerella forsythia, and Treponema denticola has been demonstrated using confocal scanning laser microscopy and in situ hybridization with 16 rRNA probes within epithelial cells obtained from periodontal pockets, gingival crevices, and buccal mucosa samples collected from subjects with or without chronic marginal periodontitis [2,3]. Periodontal bacteria appear to have co-evolved along with their host to maintain an ecologically balanced association with minimal harm inflicted on or by either party [4]. Disease only ensues when this interaction becomes unbalanced, an event termed as opportunistic infection. Bacterial entry into host cells allows pathogens to occupy various niches within the human body, which is required for successful establishment of a bacterial infection [5]. An intracellular location is considered advantageous for bacteria to escape from immune surveillance by the host and antibiotic pressure, leading to intracellular persistence, multiplication, and dissemination to adjacent tissues. Most periodontal pathogens likely have an ability to enter periodontal cells in the manner mentioned above and maintain a harmonious coexistence with the host. Nevertheless, once that balance is lost, these intracellular bacteria begin to attack the infected host cells [1,2,6]. However, it remains largely unknown how these microbes impair cellular processes, or if such intracellular bacteria can survive and multiply, or are killed by the host. P. gingivalis, a primary etiological agent of several forms of severe periodontal diseases, is most virulent under an ecologically imbalanced condition [4]. The pathogen possesses numerous potent virulence factors, including fimbriae, lipopolysaccharide, and proteases termed gingipains, which are aimed at neutralizing local host defenses and destroying periodontal tissues. In addition to these virulence factors, most gram-negative bacteria, including P. gingivalis, produce outer membrane vesicles (OMVs), which serve as bacterial “bullets” for directed intercellular transport of bacterial virulence factors into host cells and tissues. The pathogen can utilize two unique transcellular invasive mechanisms that result in cellular impairment and thus lead to periodontal destruction. One is direct bacterial entry into periodontal cells [6] and the other is bacterial shooting of OMVs into the cells [7]. This review addresses the remarkable strategies used by intracellular P. gingivalis and its OMVs.
2. Intracellular infection by P. gingivalis The intracellular infection process for most invasive microorganisms can be divided into four phases—adhesion, entry, survival, and exit [8]. Exit from infected cells enables further penetration into host tissues and provides the pathogenic bacteria a means to replicate further within infected tissues, thus promoting persistent infection. Invasive bacteria are able to induce their own uptake by non-phagocytic host cells and primarily use two mechanical methods of invasion, a trigger mechanism and a zipper mechanism [4,9], both of which mediate swift entry of bacteria into host cells. P. gingivalis exploits the cellular endocytosis pathway to enter cells in a trigger mechanism-like fashion [10,11]. Pathogen adherence to and entry into host epithelial and endothelial cells are mediated by interactions between bacterial fimbriae and cellular α5β1 integrin [11]. Subsequently, P. gingivalis is engulfed via actin filament assembly and finally internalized by encapsulation within an early endosome.
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3. Intracellular trafficking routes of P. gingivalis Intracellular P. gingivalis reportedly localize to various cellular compartments such as the cytoplasm [12–15], endosomes [13–17], and autophagosomes being distinct double-membrane vacuoles part of the autophagy pathway [17–19]. Bacterial localization within autophagosomes has been reported in endothelial and smooth muscle cells [18], but not in gingival epithelial cells [12], whereas there are no reports of bacterial presence in the cytoplasmic spaces of endothelial cells. In a previous study, we found that P. gingivalis organisms were internalized into early endosomes and about half of those intracellular bacteria were then sorted to lytic compartments, including autolysosomes and late endosomes/ lysosomes [20]. This finding suggests that intracellular trafficking of P. gingivalis depends on the phenotypic and genotypic heterogeneity of host cells. The intracellular fate (survival or death) of P. gingivalis has been examined by various research groups. The pathogen seems to be able to replicate inside cells during the first several hours after entry [12,18,21], after which bacterial load decreases and disappears by 48 h after infection [21,22]. Of note is that P. gingivalis can survive within lysosomes for more than 24 h, which is significantly longer than the period required for protein degradation (2–3 h) [23]. In addition, wortmannin (an autophagy inhibitor) can reduce the number of intracellular bacteria, suggesting that bacterial survival is dependent on autophagy [20]. However, the role of autophagy in survival of intracellular P. gingivalis is not well defined and is currently under investigation in our laboratory.
4. P. gingivalis exit from periodontal cells The cellular recycling pathway functions to recycle most endocytosed proteins and lipids back to the plasma membrane [24]. This pathway is exploited by intracellular P. gingivalis to allow the bacteria to exit from infected cells and move to neighboring cells by a mechanism of cell-to-cell spread [20]. Using such a mechanism, P. gingivalis can expand its population within infected tissues and allow for persistent infection. The pathogen becomes internalized within early endosomes positive for the FYVE zinc finger domain of early endosome antigen 1 (an early endosome marker) and transferrin receptor (TfR). While about half of the intracellular bacteria can be found within lytic compartments, a considerable number of the remaining organisms are sorted to a recycling pathway [20]. Bacterial exit is dependent on actin polymerization, lipid rafts, and microtubule assembly. Dominant negative forms as well as RNAi-knockdown of Ras-related proteins Rab11 and RalA, and exocyst complex subunits significantly disrupt the exit of P. gingivalis. It should be noted that intracellular bacteria were sorted to three different destinations within the same cells. These results imply the existence of unknown mechanism(s) that distribute bacteria among multiple sorting pathways destined for autophagy, lysosomes, or recycling.
5. Cellular impairment by intracellular P. gingivalis Cellular integrins provide a physical link between the extracellular environment and the intracellular cytoskeleton via focal adhesions [25], which are intimately involved in cellular anchoring and directed migration. They are also involved in signal transduction pathways that control wound healing and regeneration, as well as tissue integrity [26]. During these events, paxillin and focal adhesion kinase (FAK) play important roles, and FAK phosphorylation is a central regulator of cell migration during integrin-mediated control of cell behavior. Paxillin has been shown to be localized in
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cultured cells, primarily to sites of cell adhesion to the extracellular matrix (i.e., focal adhesions), and its activation following integrin activation is necessary for actin-cytoskeleton formation, as well as the recruitment of FAK to robust focal adhesions. Intracellular P. gingivalis can degrade paxillin and FAK, resulting in impaired cellular function during wound healing and periodontal tissue regeneration [27,28]. However, despite the burden of intracellular bacteria, infected gingival epithelial cells do not undergo apoptotic or necrotic death [29], a distinctive characteristic of opportunistic bacteria.
6. Entry mechanism of P. gingivalis OMVs P. gingivalis secretes OMVs that contain major outer membrane components, including lipopolysaccharide, muramic acid, capsule, fimbriae, and gingipains (Arg-gingipain [Rgp] and Lys-gingipain [Kgp]) [30]. Fimbriae mediate bacterial adherence to and entry into host cells [31], whereas gingipains contribute to the destruction of periodontal tissues [32]. These factors provide adhesive and proteolytic abilities, respectively, to OMVs, which, together with their small size, enable them to penetrate intact mucosa and enter underlying host tissues [7]. OMVs adhere to the surface of gingival
P. gingivalis OMVs
Encapsulation by early endosome
Degradation of TfR, paxillin and FAK Endosome
Excess acidification Lysosome Cellular impairment actin FAK
Degradation virulence factors
lipid raft
TfR
integrin
paxillin
Fig. 1. Intracellular trafficking of OMVs secreted from P. gingivalis (modified from Amano et al. [7]). OMVs are internalized by gingival epithelial cells, a process that is dependent on lipid rafts, Rac1, actin, and the activity of PI3 kinase. They are then transported to endosomes and eventually sequestered by lysosomes. Internalized OMVs degrade cellular TfR and focal adhesion complex proteins such as paxillin and FAK, after which the lytic compartment is acidified, causing lysosome-specific initiation of cellular impairment. P. gingivalis OMVs are finally degraded in lysosomal compartments.
epithelial cells in a fimbria-dependent manner via lipid rafts and actin filament assembly, following which they are internalized with early endosomes in the same manner as whole P. gingivalis organisms [33]. The entry is dependent on phosphatidylinositol 3-kinase and Rac1, whereas clathrin- and dynamin-dependent pathways are not involved. Subsequently, OMVs are routed to early endosomes and sorted to lysosomes within 90 min (Fig. 1).
7. Cellular impairment by intracellular OMVs of P. gingivalis OMVs attach to the surface of plasma membranes of gingival epithelial cells immediately before being engulfed by endocytic compartments, where they efficiently degrade focal adhesion complex proteins on the membrane, including paxillin and FAK [33]. Furthermore, cellular surface components such as TfR also reside in early endosomes and are sorted back to the plasma membrane via the recycling pathway. Although they are taken up by the cellular digestive machinery, OMVs can use gingipains to degrade TfR and focal adhesion complex proteins, resulting in depletion of intracellular transferrin and inhibition of cellular migration [34] (Fig. 2). TfR, a carrier protein for transferrin, is indispensable for iron metabolism; thus, OMVs disrupt fundamental cellular operations that depend on iron, including DNA synthesis and ATP generation [35]. In addition, Rgp is necessary for efficient entry by OMVs, as it may expose cellular cryptic ligands in a proteolytic manner and promote OMV entry into host cells [33]. It has also been shown that Kgp is responsible for degradation of integrin-related molecules, while Rgp is associated with TfR degradation. Although P. gingivalis OMVs cannot escape from endocytic compartments [33], they have been shown to survive within lysosomes for more than 24 h, during which there is significant formation of acidified compartment. Such cellular stress has been suggested to cause lysosome-specific initiation of cellular impairment [36]. On the basis of pathological features of chronic periodontitis caused by P. gingivalis, daily and continuous attacks by OMVs from oral microbial biofilm on gingival margins seem able to destroy periodontal tissue. Thus, P. gingivalis OMVs are suggested to promote chronic virulence mechanisms in the longstanding battle between bacterial biofilm and host.
8. Conclusions P. gingivalis can enter and exit periodontal cells by using a sophisticated strategy, which promotes the spread of periodontal infection. Such efficient bacterial entry likely contributes to periodontal destruction, which may be further enhanced by mutual communication within a complex polymicrobial etiology [4]. However, OMVs cannot escape from endocytic compartments, and the endosome-incorporated materials are thought to reside therein [5,7]. Thus, it is unclear how viable P. gingivalis promotes its own escape from the cells. Furthermore, the intracellular dynamics is likely dependent on bacterial virulence factors such as fimbriae and gingipains [6]. The protein expression profiles of viable bacteria can vary, and this may regulate their intracellular fates according to the circumstances. Further studies are necessary to understand the underlying mechanism of action. Although analyses of the bacterial strategies used by P. gingivalis to determine intercellular persistence, dissemination, and fate have provided important information, these have unveiled only part of the picture. Nevertheless, recent studies have shown that human oral epithelial cells harbor a large intracellular bacterial load, resembling the polymicrobial nature of tooth-surface biofilm [3]. In addition, no significant induction of apoptosis or necrosis has been observed in epithelial cells persistently infected by bacteria
A. Amano et al. / Journal of Oral Biosciences 56 (2014) 58–62
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Fig. 2. Degradation of transferrin receptor (TfR) and depletion of intracellular transferrin by internalized OMVs (modified from Furuta et al. [33]). (A) Epithelial cells were incubated with P. gingivalis OMVs (green) at 37 1C for 15 min, and the distribution of TfR (white) was analyzed with confocal microscopy. (B) Following incubation with OMVs for 15 min, cells were incubated with Alexa Fluor 594-conjugated transferrin (white) at 37 1C for 45 min. Cells were processed by staining with 40 ,6-diamidino2-phenylindole (blue) and observed by fluorescence microscopy. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
[37]. These new findings suggest that intracellular periodontal bacteria reside in a largely dormant fashion which allows them to persist, but not destroy the host.
Conflicts of interest There are no conflicts of interest to report. References [1] Amano A. Disruption of epithelial barrier and impairment of cellular function by Porphyromonas gingivalis. Front Biosci 2007;12:3965–74. [2] Lamont RJ, Yilmaz O. In or out: the invasiveness of oral bacteria. Periodontol 2000 2002;30:61–9. [3] Colombo AV, da Silva CM, Haffajee A, Colombo AP. Identification of intracellular oral species within human crevicular epithelial cells from subjects with chronic periodontitis by fluorescence in situ hybridization. J Periodontal Res 2007;42:236–43. [4] Hajishengallis G, Lamont RJ. Beyond the red complex and into more complexity: the polymicrobial synergy and dysbiosis model of periodontal disease etiology. Mol Oral Microbiol 2012;27:409–19. [5] Cossart P, Sansonetti PJ. Bacterial invasion: the paradigms of enteroinvasive pathogens. Science 2004;304:242–8. [6] Amano A, Furuta N, Tsuda K. Host membrane trafficking for conveyance of intracellular oral pathogens. Periodontol 2000 2010;52:84–93. [7] Amano A, Takeuchi H, Furuta N. Outer membrane vesicles function as offensive weapons in host–parasite interactions. Microbe Infect 2010;12: 791–8. [8] Casadevall A. Evolution of intracellular pathogens. Annu Rev Microbiol 2008;62:19–33. [9] Conner SD, Schmid SL. Regulated portals of entry into the cell. Nature 2003;422:37–44. [10] Tsuda K, Amano A, Umebayashi K, Inaba H, Nakagawa I, Nakanishi Y, Yoshimori T. Molecular dissection of internalization of Porphyromonas gingivalis by cells using fluorescent beads coated with bacterial membrane vesicle. Cell Struct Funct 2005;30:81–91. [11] Tsuda K, Furuta N, Inaba H, Kawai S, Hanada K, Yoshimori T, Amano A. Functional analysis of α5β1 integrin and lipid rafts in invasion of epithelial cells by Porphyromonas gingivlis using fluorescent beads coated with bacterial membrane vesicle. Cell Struct Funct 2008;33:123–32. [12] Lamont RJ, Chan A, Belton CM, Izutsu KT, Vasel D, Weinberg A. Porphyromonas gingivalis invasion of gingival epithelial cells. Infect Immun 1995;63:3878–85. [13] Sandros J, Papapanou P, Dahlen G. Porphyromonas gingivalis invades oral epithelial cells in vitro. J Periodontal Res 1993;28:219–26.
[14] Papapanou PN, Sandros J, Lindberg K, Duncan MJ, Niederman R, Nannmark U. Porphyromonas gingivalis may multiply and advance within stratified human junctional epithelium in vitro. J Periodontal Res 1994;29:374–5. [15] Sandros J, Papapanou PN, Nannmark U, Dahlen G. Porphyromonas gingivalis invades human pocket epithelium in vitro. J Periodontal Res 1994;29:62–9. [16] Duncan MJ, Nakao S, Skobe Z, Xie H. Interactions of Porphyromonas gingivalis with epithelial cells. Infect Immun 1993;61:2260–5. [17] Yamatake K, Maeda M, Kadowaki T, Takii R, Tsukuba T, Ueno T, Kominami E, Yokota S, Yamamoto K. Role for gingipains in Porphyromonas gingivalis traffic to phagolysosomes and survival in human aortic endothelial cells. Infect Immun 2007;75:2090–100. [18] Dorn BR, Dunn Jr WA, Progulske-Fox A. Porphyromonas gingivalis traffics to autophagosomes in human coronary artery endothelial cells. Infect Immun 2001;69:5698–708. [19] Belanger M, Rodrigues PH, Dunn Jr WA, Progulske-Fox A. Autophagy: a highway for Porphyromonas gingivalis in endothelial cells. Autophagy 2006;2: 165–70. [20] Takeuchi H, Furuta N, Morisaki I, Amano A. Exit of intracellular Porphyromonas gingivalis from gingival epithelial cells is mediated by endocytic recycling pathway. Cell Microbiol 2011;13:677–91. [21] Eick S, Reissmann A, Rodel J, Schmidt KH, Pfister W. Porphyromonas gingivalis survives within KB cells and modulates inflammatory response. Oral Microbiol Immunol 2006;21:231–7. [22] Li L, Michel R, Cohen J, Decarlo A, Kozarov E. Intracellular survival and vascular cell-to-cell transmission of Porphyromonas gingivalis. BMC Microbiol 2008;8: 26. [23] Sevlever D, Jiang P, Yen SH. Cathepsin D is the main lysosomal enzyme involved in the degradation of alpha-synuclein and generation of its carboxyterminally truncated species. Biochemistry 2008;47:9678–87. [24] He B, Guo W. The exocyst complex in polarized exocytosis. Curr Opin Cell Bio 2009;21:537–42. [25] Clark EA, King WG, Brugge JS, Symons M, Hynes RO. Integrin-mediated signals regulated by members of the Rho family of GTPases. J Cell Biol 1998;142: 573–86. [26] Hakkinen L, Uitto VJ, Larjava H. Cell biology of gingival wound healing. Periodontol 2000 2000;24:127–52. [27] Hintermann E, Haake SK, Christen U, Sharabi A, Quaranta V. Discrete proteolysis of focal contact and adherent junction components in Porphyromonas gingivalis-infected oral keratinocytes: a strategy for cell adhesion and migration disabling. Infect Immun 2002;70:5846–56. [28] Kato T, Kawai S, Nakano K, Inaba H, Kuboniwa M, Nakagawa I, Tsuda K, Omori H, Ooshima T, Yoshimori T, Amano A. Virulence of Porphyromonas gingivalis is altered by substitution of fimbria gene with different genotype. Cell Microbiol 2007;9:753–65. [29] Yilmaz O, Jungas T, Verbeke P, Ojcius DM. Activation of the phosphatidylinositol 3-kinase/Akt pathway contributes to survival of primary epithelial cells infected with the periodontal pathogen Porphyromonas gingivalis. Infect Immun 2004;72:3743–51.
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Contents lists available at ScienceDirect
Journal of Oral Biosciences journal homepage: www.elsevier.com/locate/job
Review
Transcellular invasive mechanisms of Porphyromonas gingivalis in host–parasite interactions Atsuo Amano n, Masae Kuboniwa, Hiroki Takeuchi Department of Preventive Dentistry, Osaka University Graduate School of Dentistry, 1-8 Yamadaoka, Suita, Osaka 565-0871, Japan
art ic l e i nf o
a b s t r a c t
Article history: Received 31 January 2014 Received in revised form 19 February 2014 Accepted 19 February 2014 Available online 1 April 2014
Porphyromonas gingivalis, a well-known periodontal pathogen, expresses a number of virulence factors, including fimbriae and gingipains. In addition, the pathogen utilizes two unique transcellular invasive mechanisms that cause cellular impairment in periodontal tissues; these mechanisms include bacterial entry into periodontal cells and bacterial shooting of outer membrane vesicles (OMVs) into those cells. Gingival epithelial cells function as innate host defense barriers to prevent intrusion by periodontal bacteria. Nevertheless, P. gingivalis can enter these cells and pass through the epithelial barrier into deeper tissues. Fimbriae of P. gingivalis specifically interact with α5β1-integrin of epithelial cells, which induces cellular invagination to internalize the pathogen. Following their entry, intracellular P. gingivalis impairs fundamental cellular functions, while some intracellular bacteria are finally sorted to lytic compartments, including autolysosomes and late endosomes/lysosomes. Furthermore, a considerable number of organisms are sorted into recycling endosomes, leading to bacterial exit from infected cells to neighboring cells, a mechanism of cell-to-cell spread within periodontal tissues. Most gram-negative bacteria, including P. gingivalis, produce OMVs, which serve as bacterial “bullets” for directed intercellular transport of bacterial virulence factors into host cells and tissues. Following entry into the cells, OMVs can use bacterial gingipains to degrade cellular functional components. Although OMVs do not escape from lytic compartments, they survive within lysosomes for more than 24 h, resulting in significant formation of acidified compartments. This review addresses the remarkable transcellular strategies that are used by P. gingivalis to destroy periodontal tissues. & 2014 Japanese Association for Oral Biology. Published by Elsevier B.V. All rights reserved.
Keywords: Membrane trafficking Porphyromonas gingivalis Outer membrane vesicles Periodontitis
Contents 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Intracellular infection by P. gingivalis . . . . . . . . . . . . . . . . . 3. Intracellular trafficking routes of P. gingivalis . . . . . . . . . . . 4. P. gingivalis exit from periodontal cells. . . . . . . . . . . . . . . . 5. Cellular impairment by intracellular P. gingivalis . . . . . . . . 6. Entry mechanism of P. gingivalis OMVs . . . . . . . . . . . . . . . 7. Cellular impairment by intracellular OMVs of P. gingivalis 8. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conflicts of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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1. Introduction
n
Corresponding author. Tel.: þ 81 6 6879 2921; fax: þ 81 6 6879 2925. E-mail address: [email protected] (A. Amano).
Gingival epithelial cells function as an innate host defense system to prevent intrusion by periodontal bacteria [1]. However, persistent contact of subgingival bacterial biofilm with gingival crevices induces bacterial penetration into periodontal tissues.
http://dx.doi.org/10.1016/j.job.2014.02.001 1349-0079/& 2014 Japanese Association for Oral Biology. Published by Elsevier B.V. All rights reserved.
A. Amano et al. / Journal of Oral Biosciences 56 (2014) 58–62
Immunofluorescence and immunohistochemical techniques have revealed the existence of Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, Prevotella intermedia, and Actinomyces naeslundii in gingival tissues [2]. In addition, intracellular localization of several periodontal bacteria, including P. gingivalis, A. actinomycetemcomitans, Tannerella forsythia, and Treponema denticola has been demonstrated using confocal scanning laser microscopy and in situ hybridization with 16 rRNA probes within epithelial cells obtained from periodontal pockets, gingival crevices, and buccal mucosa samples collected from subjects with or without chronic marginal periodontitis [2,3]. Periodontal bacteria appear to have co-evolved along with their host to maintain an ecologically balanced association with minimal harm inflicted on or by either party [4]. Disease only ensues when this interaction becomes unbalanced, an event termed as opportunistic infection. Bacterial entry into host cells allows pathogens to occupy various niches within the human body, which is required for successful establishment of a bacterial infection [5]. An intracellular location is considered advantageous for bacteria to escape from immune surveillance by the host and antibiotic pressure, leading to intracellular persistence, multiplication, and dissemination to adjacent tissues. Most periodontal pathogens likely have an ability to enter periodontal cells in the manner mentioned above and maintain a harmonious coexistence with the host. Nevertheless, once that balance is lost, these intracellular bacteria begin to attack the infected host cells [1,2,6]. However, it remains largely unknown how these microbes impair cellular processes, or if such intracellular bacteria can survive and multiply, or are killed by the host. P. gingivalis, a primary etiological agent of several forms of severe periodontal diseases, is most virulent under an ecologically imbalanced condition [4]. The pathogen possesses numerous potent virulence factors, including fimbriae, lipopolysaccharide, and proteases termed gingipains, which are aimed at neutralizing local host defenses and destroying periodontal tissues. In addition to these virulence factors, most gram-negative bacteria, including P. gingivalis, produce outer membrane vesicles (OMVs), which serve as bacterial “bullets” for directed intercellular transport of bacterial virulence factors into host cells and tissues. The pathogen can utilize two unique transcellular invasive mechanisms that result in cellular impairment and thus lead to periodontal destruction. One is direct bacterial entry into periodontal cells [6] and the other is bacterial shooting of OMVs into the cells [7]. This review addresses the remarkable strategies used by intracellular P. gingivalis and its OMVs.
2. Intracellular infection by P. gingivalis The intracellular infection process for most invasive microorganisms can be divided into four phases—adhesion, entry, survival, and exit [8]. Exit from infected cells enables further penetration into host tissues and provides the pathogenic bacteria a means to replicate further within infected tissues, thus promoting persistent infection. Invasive bacteria are able to induce their own uptake by non-phagocytic host cells and primarily use two mechanical methods of invasion, a trigger mechanism and a zipper mechanism [4,9], both of which mediate swift entry of bacteria into host cells. P. gingivalis exploits the cellular endocytosis pathway to enter cells in a trigger mechanism-like fashion [10,11]. Pathogen adherence to and entry into host epithelial and endothelial cells are mediated by interactions between bacterial fimbriae and cellular α5β1 integrin [11]. Subsequently, P. gingivalis is engulfed via actin filament assembly and finally internalized by encapsulation within an early endosome.
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3. Intracellular trafficking routes of P. gingivalis Intracellular P. gingivalis reportedly localize to various cellular compartments such as the cytoplasm [12–15], endosomes [13–17], and autophagosomes being distinct double-membrane vacuoles part of the autophagy pathway [17–19]. Bacterial localization within autophagosomes has been reported in endothelial and smooth muscle cells [18], but not in gingival epithelial cells [12], whereas there are no reports of bacterial presence in the cytoplasmic spaces of endothelial cells. In a previous study, we found that P. gingivalis organisms were internalized into early endosomes and about half of those intracellular bacteria were then sorted to lytic compartments, including autolysosomes and late endosomes/ lysosomes [20]. This finding suggests that intracellular trafficking of P. gingivalis depends on the phenotypic and genotypic heterogeneity of host cells. The intracellular fate (survival or death) of P. gingivalis has been examined by various research groups. The pathogen seems to be able to replicate inside cells during the first several hours after entry [12,18,21], after which bacterial load decreases and disappears by 48 h after infection [21,22]. Of note is that P. gingivalis can survive within lysosomes for more than 24 h, which is significantly longer than the period required for protein degradation (2–3 h) [23]. In addition, wortmannin (an autophagy inhibitor) can reduce the number of intracellular bacteria, suggesting that bacterial survival is dependent on autophagy [20]. However, the role of autophagy in survival of intracellular P. gingivalis is not well defined and is currently under investigation in our laboratory.
4. P. gingivalis exit from periodontal cells The cellular recycling pathway functions to recycle most endocytosed proteins and lipids back to the plasma membrane [24]. This pathway is exploited by intracellular P. gingivalis to allow the bacteria to exit from infected cells and move to neighboring cells by a mechanism of cell-to-cell spread [20]. Using such a mechanism, P. gingivalis can expand its population within infected tissues and allow for persistent infection. The pathogen becomes internalized within early endosomes positive for the FYVE zinc finger domain of early endosome antigen 1 (an early endosome marker) and transferrin receptor (TfR). While about half of the intracellular bacteria can be found within lytic compartments, a considerable number of the remaining organisms are sorted to a recycling pathway [20]. Bacterial exit is dependent on actin polymerization, lipid rafts, and microtubule assembly. Dominant negative forms as well as RNAi-knockdown of Ras-related proteins Rab11 and RalA, and exocyst complex subunits significantly disrupt the exit of P. gingivalis. It should be noted that intracellular bacteria were sorted to three different destinations within the same cells. These results imply the existence of unknown mechanism(s) that distribute bacteria among multiple sorting pathways destined for autophagy, lysosomes, or recycling.
5. Cellular impairment by intracellular P. gingivalis Cellular integrins provide a physical link between the extracellular environment and the intracellular cytoskeleton via focal adhesions [25], which are intimately involved in cellular anchoring and directed migration. They are also involved in signal transduction pathways that control wound healing and regeneration, as well as tissue integrity [26]. During these events, paxillin and focal adhesion kinase (FAK) play important roles, and FAK phosphorylation is a central regulator of cell migration during integrin-mediated control of cell behavior. Paxillin has been shown to be localized in
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cultured cells, primarily to sites of cell adhesion to the extracellular matrix (i.e., focal adhesions), and its activation following integrin activation is necessary for actin-cytoskeleton formation, as well as the recruitment of FAK to robust focal adhesions. Intracellular P. gingivalis can degrade paxillin and FAK, resulting in impaired cellular function during wound healing and periodontal tissue regeneration [27,28]. However, despite the burden of intracellular bacteria, infected gingival epithelial cells do not undergo apoptotic or necrotic death [29], a distinctive characteristic of opportunistic bacteria.
6. Entry mechanism of P. gingivalis OMVs P. gingivalis secretes OMVs that contain major outer membrane components, including lipopolysaccharide, muramic acid, capsule, fimbriae, and gingipains (Arg-gingipain [Rgp] and Lys-gingipain [Kgp]) [30]. Fimbriae mediate bacterial adherence to and entry into host cells [31], whereas gingipains contribute to the destruction of periodontal tissues [32]. These factors provide adhesive and proteolytic abilities, respectively, to OMVs, which, together with their small size, enable them to penetrate intact mucosa and enter underlying host tissues [7]. OMVs adhere to the surface of gingival
P. gingivalis OMVs
Encapsulation by early endosome
Degradation of TfR, paxillin and FAK Endosome
Excess acidification Lysosome Cellular impairment actin FAK
Degradation virulence factors
lipid raft
TfR
integrin
paxillin
Fig. 1. Intracellular trafficking of OMVs secreted from P. gingivalis (modified from Amano et al. [7]). OMVs are internalized by gingival epithelial cells, a process that is dependent on lipid rafts, Rac1, actin, and the activity of PI3 kinase. They are then transported to endosomes and eventually sequestered by lysosomes. Internalized OMVs degrade cellular TfR and focal adhesion complex proteins such as paxillin and FAK, after which the lytic compartment is acidified, causing lysosome-specific initiation of cellular impairment. P. gingivalis OMVs are finally degraded in lysosomal compartments.
epithelial cells in a fimbria-dependent manner via lipid rafts and actin filament assembly, following which they are internalized with early endosomes in the same manner as whole P. gingivalis organisms [33]. The entry is dependent on phosphatidylinositol 3-kinase and Rac1, whereas clathrin- and dynamin-dependent pathways are not involved. Subsequently, OMVs are routed to early endosomes and sorted to lysosomes within 90 min (Fig. 1).
7. Cellular impairment by intracellular OMVs of P. gingivalis OMVs attach to the surface of plasma membranes of gingival epithelial cells immediately before being engulfed by endocytic compartments, where they efficiently degrade focal adhesion complex proteins on the membrane, including paxillin and FAK [33]. Furthermore, cellular surface components such as TfR also reside in early endosomes and are sorted back to the plasma membrane via the recycling pathway. Although they are taken up by the cellular digestive machinery, OMVs can use gingipains to degrade TfR and focal adhesion complex proteins, resulting in depletion of intracellular transferrin and inhibition of cellular migration [34] (Fig. 2). TfR, a carrier protein for transferrin, is indispensable for iron metabolism; thus, OMVs disrupt fundamental cellular operations that depend on iron, including DNA synthesis and ATP generation [35]. In addition, Rgp is necessary for efficient entry by OMVs, as it may expose cellular cryptic ligands in a proteolytic manner and promote OMV entry into host cells [33]. It has also been shown that Kgp is responsible for degradation of integrin-related molecules, while Rgp is associated with TfR degradation. Although P. gingivalis OMVs cannot escape from endocytic compartments [33], they have been shown to survive within lysosomes for more than 24 h, during which there is significant formation of acidified compartment. Such cellular stress has been suggested to cause lysosome-specific initiation of cellular impairment [36]. On the basis of pathological features of chronic periodontitis caused by P. gingivalis, daily and continuous attacks by OMVs from oral microbial biofilm on gingival margins seem able to destroy periodontal tissue. Thus, P. gingivalis OMVs are suggested to promote chronic virulence mechanisms in the longstanding battle between bacterial biofilm and host.
8. Conclusions P. gingivalis can enter and exit periodontal cells by using a sophisticated strategy, which promotes the spread of periodontal infection. Such efficient bacterial entry likely contributes to periodontal destruction, which may be further enhanced by mutual communication within a complex polymicrobial etiology [4]. However, OMVs cannot escape from endocytic compartments, and the endosome-incorporated materials are thought to reside therein [5,7]. Thus, it is unclear how viable P. gingivalis promotes its own escape from the cells. Furthermore, the intracellular dynamics is likely dependent on bacterial virulence factors such as fimbriae and gingipains [6]. The protein expression profiles of viable bacteria can vary, and this may regulate their intracellular fates according to the circumstances. Further studies are necessary to understand the underlying mechanism of action. Although analyses of the bacterial strategies used by P. gingivalis to determine intercellular persistence, dissemination, and fate have provided important information, these have unveiled only part of the picture. Nevertheless, recent studies have shown that human oral epithelial cells harbor a large intracellular bacterial load, resembling the polymicrobial nature of tooth-surface biofilm [3]. In addition, no significant induction of apoptosis or necrosis has been observed in epithelial cells persistently infected by bacteria
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Fig. 2. Degradation of transferrin receptor (TfR) and depletion of intracellular transferrin by internalized OMVs (modified from Furuta et al. [33]). (A) Epithelial cells were incubated with P. gingivalis OMVs (green) at 37 1C for 15 min, and the distribution of TfR (white) was analyzed with confocal microscopy. (B) Following incubation with OMVs for 15 min, cells were incubated with Alexa Fluor 594-conjugated transferrin (white) at 37 1C for 45 min. Cells were processed by staining with 40 ,6-diamidino2-phenylindole (blue) and observed by fluorescence microscopy. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
[37]. These new findings suggest that intracellular periodontal bacteria reside in a largely dormant fashion which allows them to persist, but not destroy the host.
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