Autophagy - Taylor & Francis Online

[Autophagy 2:3, 165-e6, July/August/September 2006]; ©2006 Landes Bioscience

Autophagy

Review: Spotlight on Bacterial Pathogenesis

A Highway for Porphyromonas gingivalis in Endothelial Cells ABSTRACT

Received 3/31/06; Accepted 4/6/06

Previously published online as an Autophagy E-publication: http://www.landesbioscience.com/journals/autophagy/abstract.php?id=2782

KEY WORDS autophagy, Porphyromonas gingivalis, lysosome, autophagosome, replicating vacuole

UT E

RIB

IST

Porphyromonas gingivalis, a Gram-negative anaerobic rod, is one of a group of periodontopathogens that are associated with adult periodontitis.1 Clinical and experimental evidence also suggests that periodontal disease and specifically P. gingivalis may be contributing factors in atherosclerosis and cardiovascular diseases (CVD).2-7 Atherosclerosis is a progressive disease that results from an excessive, inflammatory-fibroproliferative response to various forms of insult to the endothelium and smooth muscle of the artery wall.8 Like periodontal disease, atherosclerosis is mediated by an inflammatory process. Pro-inflammatory cytokines, such as C-reactive proteins, IL-1 and TNF-α, which are involved in CVD, may be stimulated by intracellular pathogens.9 P. gingivalis has been shown to stimulate the expression of such pro-inflammatory cytokines in various cell lines including endothelial cells.10-12 Therefore, invasion of coronary artery cells by P. gingivalis may aggravate an inflammatory response associated with atherosclerosis. It is well established that periodontal organisms such as P. gingivalis have a direct route to the circulatory system by the way of transient bacteremias (the presence of bacteria in the blood) and that these events occur commonly and frequently.13-16 Thus, the interactions of P. gingivalis and human coronary artery endothelial cells (HCAEC) may have a significant effect on the progression of atherosclerosis.

IEN

CE

ABBREVIATIONS

INTRODUCTION

OT D

*Correspondence to: Ann Progulske-Fox; Department of Oral Biology; University of Florida; P.O. Box 100424; Gainesville, Florida 32610-0424 USA; Tel.: 352.846.0770; Fax: 352.392.2361; Email: [email protected]

ON

†These authors contributed equally to this work.

.D

of Oral Biology, College of Dentistry; 2Department of Anatomy and Cell Biology, College of Medicine; Center for Molecular Microbiology; University of Florida; Gainesville, Florida USA 1Department

BIO

SC

cardiovascular diseases gingival epithelial cells glycosylphosphatidylinositol hemagglutinin A hemagglutinin B human coronary artery endothelial cells lysosomal associated protein-1 rough endoplasmic reticulum acid phosphatase

ND

ACKNOWLEDGEMENTS

ES

CVD GEC GPI HagA HagB HCAEC LAMP-1 RER AP

P. gingivalis, an important periodontal pathogen associated with adult periodontitis and a likely contributing factor to atherosclerosis and cardiovascular disease, traffics in endothelial cells via the autophagic pathway. Initially, P. gingivalis rapidly adheres to the host cell surface followed by internalization via lipid rafts and incorporation of the bacterium into early phagosomes. P. gingivalis activates cellular autophagy to provide a replicative niche while suppressing apoptosis. The replicating vacuole contains host proteins delivered by autophagy that are used by this asaccharolytic pathogen to survive and replicate within the host cell. When autophagy is suppressed by 3-methyladenine or wortmannin, internalized P. gingivalis transits to the phagolysosome where it is destroyed and degraded. Therefore, the survival of P. gingivalis depends upon the activation of autophagy and survival of the endothelial host cell, but the mechanism by which P. gingivalis accomplishes this remains unclear.

.

Myriam Bélanger1,† Paulo H. Rodrigues1,† William A. Dunn, Jr.2 Ann Progulske-Fox1,*

©

20

06

LA

This work was supported by grant NIH DE013545.

www.landesbioscience.com

CELLULAR INVASION

P. gingivalis can invade many cell types including HCAEC and coronary artery smooth muscle cells.17 The initial adherence of P. gingivalis to host cells is mediated by multiple adhesins including FimA and HagB.18-22 However, our knowledge of the initial events of P. gingivalis internalization is limited, especially with regard to internalization into HCAEC. There is some evidence that interaction with lipid rafts may serve as a portal of entry for P. gingivalis into host eukaryotic cells. Lipid rafts occur in both plasma and endosomal membranes of cells; they are rich in cholesterol, glycosphingolipids and glycosylphosphatidylinositol (GPI)-linked proteins that are anchored in the membrane. Although they have many functions such as polarized secretion, membrane transport, signal transduction, and transcytosis across epithelial monolayers, their roles in bacterial invasion are not as well understood.23 Many bacterial species, including Legionella pneumophila, and Brucella abortus, two bacterial intracellular pathogens that subvert the autophagic pathway, have been shown to use lipid rafts as a gateway to enter host cells.24-26 Recently, ICAM-1 and Autophagy

165

Autophagy and P. gingivalis

caveolae were shown to be required for the invasion of P. gingivalis into KB cells.27 Furthermore, Tsuda et al. proposed that P. gingivalis utilizes membrane lipid rafts for its internalization into HeLa cells.28 Our data indicate P. gingivalis is also internalized into HCAEC via lipid rafts (unpublished observations).

INTRACELLULAR TRAFFICKING

Intracellular pathogens have evolved multiple pathways and mechanisms to traffic and survive once they are within the host cell.29,30 Upon internalization, bacteria may be delivered to vacuoles. To avoid lysosomal killing, some bacterial species, such as Listeria monocytogenes, Actinobacillus actinomycetemcomitans, Shigella flexneri, and Rickettsia conorii escape into the cytoplasm.31,32 Mycobacterium is part of a second group of intracellular pathogens, which remain inside phagosomes. These pathogens disturb the maturation of phagosomes by blocking fusion with lysosomes, therefore controlling the biogenesis of their compartments.30 A third strategy is to traffic quickly from early phagosomal vesicles into the autophagic pathway and create a nutrient-rich environment that enables the bacterium to survive and replicate. Since autophagy is a highly regulated pathway that is responsible for maintaining cellular amino acid pools by the sequestration of cytosolic substances and organelles (e.g., peroxisomes and mitochondria) within the autophagosomes, the autophagic vesicles are a nutrient-rich environment for proteolytic, asaccharolytic pathogens such as P. gingivalis. We have demonstrated that the nascent or early autophagosome originates from an invagination of the rough endoplasmic reticulum;33 however, other labs have shown that a preautophagosomal structure and a lipid-rich phagophore or isolation membrane likely contributes to the formation of this unique vacuole.34 The early autophagosome then matures into an acidic late autophagosome, which then fuses with a lysosome, thereby becoming an autolysosome in which degradation of the contents of the vacuole occurs.35 A number of genes required for autophagy have been identified and characterized.36 A few bacterial species such as B. abortus and L. pneumophila have been shown to subvert this pathway for their intracellular survival.37-39 Likewise, we have reported that P. gingivalis traffics from phagosomes to autophagosomes in HCAEC to eventually replicate in vacuoles resembling late autophagosomes.17,21,40 At 90 min of infection, internalized P. gingivalis were found within structures resembling early (Fig. 1A) and late (Fig. 1B) autophagosomes. The presence of cytoplasmic organelles and ground substance suggests that these vacuoles arose from autophagic events. At 24 h, the bacteria were found in large vacuoles that contain numerous tubular vesicles (Fig. 1C). Profiles of rough endoplasmic reticulum (RER) were routinely observed in close association with these vacuoles. These morphological data are consistent with the internalized P. gingivalis residing within autophagosome and autophagosome-like vacuoles that have been derived from the RER. Since our ultrastructural data (above) suggested that internalized P. gingivalis enter the autophagic pathway, we utilized protein markers of autophagy to investigate the intracellular trafficking of P. gingivalis in HCAEC. The intracellular distribution of P. gingivalis, visualized using mouse monoclonal antibodies specific for hemagglutinin A (HagA), was compared to autophagy marker proteins. HCAEC were grown on glass coverslips and infected with P. gingivalis for different times. Each time point was analyzed by examining at least 100 internalized bacteria. Upon internalization, P. gingivalis was shown to transiently localize to the early phagosomes containing Rab5 (Fig. 2A). 166

Figure 1. Internalized P. gingivalis in HCAEC. HCAEC were infected with P. gingivalis. After 90 min of infection (A and B), P. gingivalis could be observed in vacuoles that were morphologically similar to (A) early and (B) late autophagosomes. Early autophagosomes are bound by two membranes (arrows) and contain undegraded vesicles and cytoplasmic ground substance. Late autophagosomes are bound by one (arrowhead) or two (arrows) membranes and contain undegraded vesicles and a lumen that appears more dense that the surrounding cytoplasm. After 24 h of infection (C), P. gingivalis was found in large vacuoles (“replicating vacuole”) bound by one (arrowheads) or two (arrow) membranes and containing many vesicular structures. Pg, P. gingivalis; RER, rough endoplasmic reticulum; M, mitochondria.

At 25–35 min, over 70% of the bacteria were found in these vacuoles. At 60 min post-infection, only 20% of the bacteria colocalized with Rab5. Within 15 min of infection, 42% of the internalized bacteria colocalized with BiP, an RER lumenal protein and marker for the early stages of autophagy (Fig. 2B). At 30 to 90 min, 68 to 80% of the P. gingivalis were found in BiP-positive compartments. Even at 120 min, 52% of the bacteria were localized to the BiP compartment. We also examined the distribution of Atg7, another early stage marker of autophagosomes (Fig. 2B). Within 15 min, 75% of the internalized P. gingivalis cells were localized to vacuoles containing Atg7, and this value decreased over time to 30% at 120 min. These data suggest that soon after internalization, P. gingivalis transits to the early phagosome and subsequently becomes associated with an early autophagosome containing BiP and Atg7. We also demonstrated that P. gingivalis trafficks to the late autophagosomes (Lamp1) and eventually to the autolysosome (containing cathepsin L) for degradation. Within 60 min of invasion, 82% of the P. gingivalis were in Lamp1-positive vacuoles (Fig. 2B). At the same point, only 6% of the bacteria were in an autolysosome or phagolysosome as determined by the presence of cathepsin L (Fig. 2C). In addition, we evaluated whether the cellular distribution of P. gingivalis in HCAEC would be altered if autophagy were inhibited by wortmannin. Under these conditions, we have shown that P. gingivalis was not observed in BiP-positive vacuoles and less than 9% of the internalized P. gingivalis localized to the Atg7-positive vacuoles. In contrast, 78% of the internalized P. gingivalis cells were localized to vacuoles that contained cathepsin L (Fig. 2C). Furthermore, we have shown that when autophagy is suppressed by 3-methyladenine or wortmannin, P. gingivalis instead traffics to phagolysosomes where it is destroyed.41 These data indicate that P. gingivalis traffics to a late autophagosome that fails to mature to an autolysosome, but the bacterium is transported to phagolysosomes when autophagy is inhibited by wortmannin. The localization of P. gingivalis in vacuoles

Autophagy

2006; Vol. 2 Issue 3


A

engulfed by these vacuoles, we suggest that the phagosome fuses with the outer membrane of the autophagosome as proposed for the entry of endocytosed proteins into autophagosomes.42 Upon entry of the bacterium into the space between the two limiting membranes of the autophagosome, the inner membrane would be broken possibly by the bacterial proteinases (e.g., gingipains) allowing the bacterium to enter the lumen of the autophagosome. When the formation of the early autophagosome is suppressed by 3-methyladenine or wortmannin, internalized P. gingivalis transits to a single-membrane bound late phagosome containing Rab1 and mannose 6-phosphate receptor from which the bacterium then enters the phagolysosome where it is degraded. Therefore, our results demonstrate that survival of P. gingivalis depends upon the activation of autophagy, but it is uncertain how autophagy is activated by P. gingivalis. Furthermore, it remains unclear whether P. gingivalis must be internalized for autophagy to be activated. Once internalized, this bacterium is sorted to a vacuole morphologically similar to late autophagosomes containing BiP, a rough endoplasmic reticulum protein, and Lamp1, a lysosomal membrane protein but devoid of cathepsin L, a lysosomal proteinase. The fact that this vacuole does not attain cathepsin L indicates that P. gingivalis has evolved a mechanism to prevent maturation of the autophagosome into an autolysosome. Eventually, P. gingivalis resides in a “replicating vacuole” that has characteristics of the late autophagosome lacking digestive enzymes and containing host cell proteins sequestered by autophagy. P. gingivalis is an asaccharolytic bacterium, which can utilize peptides for its carbon and energy source. Therefore, autophagic vesicles would be an ideal niche (or microenvironment) for P. gingivalis where the host cell delivers proteins that can be used by the bacterium for growth and division. Upon replication, the bacterium then exits the replicating vacuole and the host cell by yet unknown events that do not appear to be harmful to the host cell.

B

C

Figure 2. Colocalization of P. gingivalis with protein markers of the autophagic pathway. HCAEC were incubated with P. gingivalis for 15 to 120 min. The cells were fixed and the localization of P. gingivalis (mouse anti-HagA) compared to (A) Rab5 and (B) BiP, ATG7 and Lamp1 was visualized by fluorescence microscopy. The data were expressed as the percentage (mean + SE) of P. gingivalis vacuoles that contained the marker protein. In (C), HCAEC were incubated with P. gingivalis in the absence (solid bars) and presence (open bars) of 10 nM wortmannin. The cells were fixed and immunofluorescence localization of P. gingivalis (mouse anti-HagA) was compared to lysosomal cathepsin L.

containing cathepsin L is consistent with the profiles of degraded bacteria observed in these vacuoles as well as the decrease in bacterial persistence within these cells (unpublished observations). Based on our immunofluorescence and ultrastructural data, we present a model of P. gingivalis invasion of and trafficking within endothelial cells (Fig. 3). We propose that within 0–15 min after coculture, the bacterium adheres to the cell surface of HCAEC via multiple adhesins including FimA and HagB. This is followed by internalization (15–60 min) via lipid rafts and incorporation of the bacterium into early Rab5-positive phagosomes. The next series of events involve the trafficking and survival of P. gingivalis within autophagosomes (60–240 min). That is, the early phagosome either “fuses” with or is sequestered within a double-membrane bound early autophagosome and rapidly acquires Atg7 and BiP, which likely represents the first step in the autophagic sequestration of P. gingivalis. Since the bacteria within the autophagosomes do not appear to be membrane bound as would be the case if the phagosome was www.landesbioscience.com

BACTERIAL PERSISTENCE AND HOST CELL SURVIVAL

Upon invasion, P. gingivalis has been shown to persist and replicate in a number of epithelial and endothelial cell lines.18,21,43-45 In gingival epithelial cells (GEC), P. gingivalis accumulates in the perinuclear area and neither apoptosis nor necrotic cell death is observed.43,46,47 However, in KB cells the bacteria are found free in the cytosol or within single-membrane bound vacuoles.19,48-50 P. gingivalis has been shown to promote cell death in KB cells as well as in gingival fibroblasts.51,52 Our data suggest that P. gingivalis does not promote cell death in HCAEC (unpublished observations). However, purified gingipains, major extracellular and cell-associated proteinases, from P. gingivalis have been shown to induce apoptosis of endothelial cells.53 Increasing evidence indicates that by utilizing various strategies, several bacterial species can modulate the apoptotic signaling cascade of host cells, thereby playing a role in pathogenesis. For example, intracellular bacterial pathogens, such as Shigella, Salmonella and Yersinia, can induce apoptosis to destroy macrophages.54,55 Apoptosis of immune cells enables bacteria to evade and/or decrease the effectiveness of the host immune responses.56 The type III secretion system and the secreted protein, SipB, which activates caspase-1 have been shown to be essential for Salmonellainduced macrophage cell death.57 Alternatively, intracellular bacteria can inhibit apoptosis to survive and replicate within the host cells, extending the life of the host cell, while sheltering the bacterium from the immunologic system and gaining access to a nutritionally rich environment. It appears that P. gingivalis can either induce

Autophagy

167


apoptosis or perhaps shut it down, depending on the host cell. In some endothelial cells, P. gingivalis stimulates autophagy, which has been implicated in both cell death and survival;58 however, the relationship between autophagy and the survival of bacteria-infected cells has not been investigated. Nevertheless, autophagy has been shown to defend against invading Mycobacterium and Streptococcus.59,60 Further experiments will be required to better define the control and effect P. gingivalis exerts on autophagy and apoptosis in HCAEC and to understand the possible role of autophagy in cell survival.

MOLECULAR EVENTS OF BACTERIAL INVASION

We propose that either cell surface or secreted proteins of P. gingivalis are responsible for the activation of cellular autophagy and the intracellular trafficking of the bacterium to the autophagosome. Therefore, we examined by microarrays the expression of P. gingivalis proteins that may be involved in these events.61 A bacterial suspension was added to confluent HCAEC monolayers and incubated at 37˚C for 5 min, 0.5 h, 1.0 h and 2.5 h (unpublished data). Total RNA was collected, cleaned and cDNAs were obtained by reverse transcription. The cDNA samples were labeled and then interrogated using microarray slides that were prepared based on the known genomic sequence of P. gingivalis (www.tigr.org). Our data revealed that a total of 265 genes were found to be differentially regulated during invasion. Of these, 86 genes were upregulated and Figure 3. Model of P. gingivalis invasion of endothelial cells. AP: acid proteinases. 179 genes were downregulated. Based on expression profiles, we have classified the upregulated genes into three groups. Those genes that upregulate hemolysins in Escherichia coli63-65 as well as in the secretion of toxins early but transiently would likely be involved in adhesion, internal- in other bacterial species.66,67 Several invasive bacterial species use ization, and/or activation of autophagy. Genes whose expression is hemolytic factors to invade human cells by modulating microfilaments elevated at 1 h but decreased at 2.5 h may be required for trafficking and microtubules while some species use them to escape from vacuoles of the bacterium to the “replicating vacuole.” Finally, genes that are and to spread to adjacent cells.68 PG0280 encodes a putative ABC upregulated late, at 2.5 h or later, would likely be involved in bacterial transporter permease protein that is organized as a channeling pore persistence and host cell survival. complex through the membrane.62 The ABC transporter superfamily Among the upregulated genes are several that may be involved in is responsible for the translocation of a wide variety of substances intracellular trafficking and/or interactions with autophagosomal into or out of cells; however, the substrate of this particular ABC vesicles or other virulence functions. For instance, PG0092 encodes transporter has not yet been described. Genes PG1682 (glycosyl a putative transporter of unknown substrate. It appears to be in an transferase) and PG1683 (conserved hypothetical protein which has operon with two other genes; PG0093, a HlyD family secretion homology to α-amylases) have been suggested to be involved in the protein, and PG0094, a putative outer membrane efflux protein. It attachment of P. gingivalis to epithelial cells and coaggregation of is proposed that these proteins are organized as a channel complex P. gingivalis with other oral bacterial species.69,70 Genes PG1682 and through the inner and outer membranes.62 The HlyD family of PG1683 might also be involved in the coaggregation of P. gingivalis secretion proteins is involved in the activation and release of with cell membranes (autophagosomes). Perhaps related, PG1286 168

Autophagy



(ftn) encodes a ferritin. Ferritin is one of the intracellular iron-storage proteins and may also contribute to the protection of organisms against oxidative stresses.71 This gene has also been shown to be upregulated when P. gingivalis is invading epithelial cells.72 PG0686, a conserved hypothetical protein, is another gene also shown to be upregulated when P. gingivalis is invading epithelial72 and endothelial cells. PG1172 encodes a putative iron-sulfur cluster binding protein, a prosthetic group present in a diverse set of proteins involved in environmental sensing, gene regulation, and substrate activation. Out of the 179 downregulated genes, 26 genes have products that are involved in the cell envelope composition, which suggests that P. gingivalis modifies its membrane composition upon entry of HCAEC, perhaps adapting to the intracellular environment. The characterization of the function of these proteins will help us better define the molecular events of bacterial invasion and replication within host cells.

SUMMARY

Intracellular pathogens have evolved ways to invade, survive and replicate within eukaryotic cells in order to evade host immunologic defenses. P. gingivalis can invade HCAEC, activate cellular autophagy, and suppress apoptosis. This bacterium appears to replicate within autophagosomes that contain host cell proteins that had been sequestered for lysosomal degradation. However, there remain a number of unanswered questions. For example: (1) How does P. gingivalis stimulate autophagy (i.e., from the outside or from within the host cell)? (2) How does P. gingivalis prevent the late autophagosome from acquiring lysosomal hydrolases and maturing into an autolysosome? (3) How does the bacterium escape the replicating vacuole and host cell while suppressing cell death? As trafficking of P. gingivalis into the autophagic pathway appears to be dependent upon the host cell, it is likely that P. gingivalis induces autophagy in some cell types and suppresses cell death while creating a microenvironment favorable to its replication. References 1. Socransky SS, Haffajee AD. The bacterial etiology of destructive periodontal disease: Current concepts. J Periodontol 1992; 63:322-31. 2. Zhang SH, Reddick RL, Piedrahita JA, Maeda N. Spontaneous hypercholesterolemia and arterial lesions in mice lacking apolipoprotein E. Science 1992; 258:468-71. 3. Gibson FCr, Hong C, Chou HH, Yumoto H, Chen J, Lien E, Wong J, Genco CA. Innate immune recognition of invasive bacteria accelerates atherosclerosis in apolipoprotein E-deficient mice. Circulation 2004; 109:2801-6. 4. Lalla E, Lamster IB, Hofmann MA, Bucciarelli L, Jerud AP, Tucker S, Lu Y, Papapanou PN, Schmidt AM. Oral infection with a periodontal pathogen accelerates early atherosclerosis in apolipoprotein E-null mice. Arterioscler Thromb Vasc Biol 2003; 23:1405-11. 5. Li L, Messas E, Batista EL, Jr., Levine RA, Amar S. Porphyromonas gingivalis infection accelerates the progression of atherosclerosis in a heterozygous apolipoprotein E-deficient murine model. Circulation 2002; 105:861-7. 6. Haraszthy VI, Zambon JJ, Trevisan M, Zeid M, Genco RJ. Identification of periodontal pathogens in atheromatous plaques. J Periodontol 2000; 71:1554-60. 7. Kozarov EV, Dorn BR, Shelburne CE, Dunn Jr WA, Progulske-Fox A. Human atherosclerotic plaque contains viable invasive Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis. Arterioscler Thromb Vasc Biol 2005; 25:e17-8. 8. Ross R. Atherosclerosis-an inflammatory disease. N Engl J Med 1999; 340:115-26. 9. Valtonen VV. Role of infections in atherosclerosis. Am Heart J 1999; 138:S431-3. 10. Bosshardt DD, Lang NP. The junctional epithelium: From health to disease. J Dent Res 2005; 84:9-20. 11. O’Brien-Simpson NM, Veith PD, Dashper SG, Reynolds EC. Porphyromonas gingivalis gingipains: The molecular teeth of a microbial vampire. Curr Protein Pept Sci 2003; 4:409-26. 12. Seymour GJ, Gemmell E. Cytokines in periodontal disease: Where to from here? Acta Odontol Scand 2001; 59:167-73. 13. Roberts GJ. Dentists are innocent! “Everyday” bacteremia is the real culprit: A review and assessment of the evidence that dental surgical procedures are a principal cause of bacterial endocarditis in children. Pediatr Cardiol 1999; 20:317-25.

www.landesbioscience.com

14. Carroll GC, Sebor RJ. Dental flossing and its relationship to transient bacteremia. J Periodontol 1980; 51:691-2. 15. Sconyers JR, Crawford JJ, Moriarty JD. Relationship of bacteremia to toothbrushing in patients with periodontitis. J Am Dent Assoc 1973; 87:616-22. 16. Silver JG, Martin AW, McBride BC. Experimental transient bacteraemias in human subjects with varying degrees of plaque accumulation and gingival inflammation. J Clin Periodontol 1977; 4:92-9. 17. Dorn BR, Dunn Jr WA, Progulske-Fox A. Invasion of human coronary artery cells by periodontal pathogens. Infect Immun 1999; 67:5792-8. 18. Deshpande RG, Khan MB, Genco CA. Invasion of aortic and heart endothelial cells by Porphyromonas gingivalis. Infect Immun 1998; 66:5337-43. 19. Njoroge T, Genco RJ, Sojar HT, Hamada N, Genco CA. A role for fimbriae in Porphyromonas gingivalis invasion of oral epithelial cells. Infect Immun 1997; 65:1980-4. 20. Xie H, Cai S, Lamont RJ. Environmental regulation of fimbrial gene expression in Porphyromonas gingivalis. Infect Immun 1997; 65:2265-71. 21. Dorn BR, Burks JN, Seifert KN, Progulske-Fox A. Invasion of endothelial and epithelial cells by strains of Porphyromonas gingivalis. FEMS Microbiol Let 2000; 187:139-44. 22. Song H, Bélanger M, Whitlock J, Kozarov E, Progulske-Fox A. Hemagglutinin B is involved in the adherence of Porphyromonas gingivalis to human coronary artery endothelial cells. Infect Immun 2005; 73:7267-73. 23. Simons K, Ikonen E. Functional rafts in cell membranes. Nature 1997; 387:569-72. 24. Duncan MJ, Li G, Shin JS, Carson JL, Abraham SN. Bacterial penetration of bladder epithelium through lipid rafts. J Biol Chem 2004; 279:18944-51. 25. Watarai M. Interaction between Brucella abortus and cellular prion protein in lipid raft microdomains. Microbes Infect 2004; 6:93-100. 26. Duncan MJ, Shin JS, Abraham SN. Microbial entry through caveolae: Variations on a theme. Cell Microbiol 2002; 4:783-91. 27. Tamai R, Asai Y, Ogawa T. Requirement for intercellular adhesion molecule 1 and caveolae in invasion of human oral epithelial cells by Porphyromonas gingivalis. Infect Immun 2005; 73:6290-8. 28. 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. 29. Hackstadt T. Redirection of host vesicle trafficking pathways by intracellular parasites. Traffic 2000; 1:93-9. 30. Meresse S, Steele-Mortimer O, Moreno E, Desjardins M, Finlay B, Gorvel JP. Controlling the maturation of pathogen-containing vacuoles: A matter of life and death. Nat Cell Biol 1999; 1:E183-8. 31. Gouin E, Gantelet H, Egile C, Lasa I, Ohayon H, Villiers V, Gounon P, Sansonetti PJ, Cossart P. A comparative study of the actin-based motilities of the pathogenic bacteria Listeria monocytogenes, Shigella flexneri and Rickettsia conorii. J Cell Sci 1999; 112:1697-708. 32. Meyer DH, Rose JE, Lippmann JE, Fives-Taylor PM. Microtubules are associated with intracellular movement and spread of the periodontopathogen Actinobacillus actinomycetemcomitans. Infect Immun 1999; 67:6518-25. 33. Dunn Jr WA,. Studies on the mechanisms of autophagy: Formation of the autophagic vacuole. J Cell Biol 1990; 110:1923-33. 34. Shintani T, Klionsky DJ. Autophagy in health and disease: A double-edged sword. Science 2004; 306:990-5. 35. Dunn Jr WA. Studies on the mechanisms of autophagy: Maturation of the autophagic vacuole. J Cell Biol 1990; 110:1935-45. 36. Klionsky DJ, Cregg JM, Dunn Jr WA, Emr SD, Sakai Y, Sandoval IV, Sibirny A, Subramani S, Thumm M, Veenhuis M, Ohsumi Y. A unified nomenclature for yeast autophagy-related genes. Dev Cell 2003; 5:539-45. 37. Pizarro-Cerda J, Meresse S, Parton RG, van der Goot G, Sola-Landa A, Lopez-Goni I, Moreno E, Gorvel JP. Brucella abortus transits through the autophagic pathway and replicates in the endoplasmic reticulum of nonprofessional phagocytes. Infect Immun 1998; 66:5711-24. 38. Pizarro-Cerda J, Moreno E, Sanguedolce V, Mege JL, Gorvel JP. Virulent Brucella abortus prevents lysosome fusion and is distributed within autophagosome-like compartments. Infect Immun 1998; 66:2387-92. 39. Swanson MS, Isberg RR. Association of Legionella pneumophila with the macrophage endoplasmic reticulum. Infect Immun 1995; 63:3609-20. 40. Progulske-Fox A, Kozarov E, Dorn B, Dunn Jr W, Burks J, Wu Y. Porphyromonas gingivalis virulence factors and invasion of cells of the cardiovascular system. J Periodontal Res 1999; 34:393-9. 41. 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. 42. Berg TO, Fengsrud M, Stromhaug PE, Berg T, Seglen PO. Isolation and characterization of rat liver amphisomes. Evidence for fusion of autophagosomes with both early and late endosomes. J Biol Chem 1998; 273:21883-92. 43. Nakhjiri SF, Park Y, Yilmaz O, Chung WO, Watanabe K, El-Sabaeny A, Park K, Lamont RJ. Inhibition of epithelial cell apoptosis by Porphyromonas gingivalis. FEMS Microbiol Lett 2001; 200:145-9. 44. Deshpande RG, Khan M, Genco CA. Invasion strategies of the oral pathogen Porphyromonas gingivalis: Implications for cardiovascular disease. Invasion Metastasis 1998; 18:57-69. 45. Dorn BR, Dunn Jr WA, Progulske-Fox A. Bacterial interactions with the autophagic pathway. Cell Microbiol 2002; 4:1-10.

Autophagy

169


46. Belton CM, Izutsu KT, Goodwin PC, Park Y, Lamont RJ. Fluorescence image analysis of the association between Porphyromonas gingivalis and gingival epithelial cells. Cell Microbiol 1999; 1:215-23. 47. 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. 48. Duncan MJ, Nakao S, Skobe Z, Xie H. Interactions of Porphyromonas gingivalis with epithelial cells. Infect Immun 1993; 61:2260-5. 49. Houalet-Jeanne S, Pellen-Mussi P, Tricot-Doleux S, Apiou J, Bonnaure-Mallet M. Assessment of internalization and viability of Porphyromonas gingivalis in KB epithelial cells by confocal microscopy. Infect Immun 2001; 69:7146-51. 50. Sandros J, Madianos PN, Papapanou PN. Cellular events concurrent with Porphyromonas gingivalis invasion of oral epithelium in vitro. Eur J Oral Sci 1996; 104:363-71. 51. Chen Z, Casiano CA, Fletcher HM. Protease-active extracellular protein preparations from Porphyromonas gingivalis W83 induce N-cadherin proteolysis, loss of cell adhesion, and apoptosis in human epithelial cells. J Periodontol 2001; 72:641-50. 52. Wang PL, Shirasu S, Shinohara M, Daito M, Oido M, Kowashi Y, Ohura K. Induction of apoptosis in human gingival fibroblasts by a Porphyromonas gingivalis protease preparation. Arch Oral Biol 1999; 44:337-42. 53. Sheets SM, Potempa J, Travis J, Casiano CA, Fletcher HM. Gingipains from Porphyromonas gingivalis W83 induce cell adhesion molecule cleavage and apoptosis in endothelial cells. Infect Immun 2005; 73:1543-52. 54. Hueffer K, Galan JE. Salmonella-induced macrophage death: Multiple mechanisms, different outcomes. Cell Microbiol 2004; 6:1019-25. 55. Monack D, Falkow S. Apoptosis as a common bacterial virulence strategy. Int J Med Microbiol 2000; 290:7-13. 56. Navarre WW, Zychlinsky A. Pathogen-induced apoptosis of macrophages: A common end for different pathogenic strategies. Cell Microbiol 2000; 2:265-73. 57. Hernandez LD, Pypaert M, Flavell RA, Galan JE. A Salmonella protein causes macrophage cell death by inducing autophagy. J Cell Biol 2003; 163:1123-31. 58. Codogno P, Meijer AJ. Autophagy and signaling: Their role in cell survival and cell death. Cell Death Differ 2005; 12(Suppl 2):1509-18. 59. Gutierrez MG, Master SS, Singh SB, Taylor GA, Colombo MI, Deretic V. Autophagy is a defense mechanism inhibiting BCG and Mycobacterium tuberculosis survival in infected macrophages. Cell 2004; 119:753-66. 60. Nakagawa I, Amano A, Mizushima N, Yamamoto A, Yamaguchi H, Kamimoto T, Nara A, Funao J, Nakata M, Tsuda K, Hamada S, Yoshimori T. Autophagy defends cells against invading group A Streptococcus. Science 2004; 306:1037-40. 61. Rodrigues PH, Progulske-Fox A. Gene expression profile analysis of Porphyromonas gingivalis during invasion of human coronary artery endothelial cells. Infect Immun 2005; 73:6169-73. 62. Nelson KE, Fleischmann RD, DeBoy RT, Paulsen IT, Fouts DE, Eisen JA, Daugherty SC, Dodson RJ, Durkin AS, Gwinn M, Haft DH, Kolonay JF, Nelson WC, Mason T, Tallon L, Gray J, Granger D, Tettelin H, Dong H, Galvin JL, Duncan MJ, Dewhirst FE, Fraser CM. Complete genome sequence of the oral pathogenic bacterium Porphyromonas gingivalis strain W83. J Bacteriol 2003; 185:5591-601. 63. Trent MS, Worsham LM, Ernst-Fonberg ML. The biochemistry of hemolysin toxin activation: characterization of HlyC, an internal protein acyltransferase. Biochemistry 1998; 37:4644-52. 64. Wang RC, Seror SJ, Blight M, Pratt JM, Broome-Smith JK, Holland IB. Analysis of the membrane organization of an Escherichia coli protein translocator, HlyB, a member of a large family of prokaryote and eukaryote surface transport proteins. J Mol Biol 1991; 217:441-54. 65. Holland IB, Kenny B, Blight M. Haemolysin secretion from Escherichia coli. Biochimie 1990; 72:131-41. 66. Highlander SK, Engler MJ, Weinstock GM. Secretion and expression of the Pasteurella haemolytica leukotoxin. J Bacteriol 1990; 172:2343-50. 67. Jansen R, Briaire J, van Geel AB, Kamp EM, Gielkens AL, Smits MA. Genetic map of the Actinobacillus pleuropneumoniae RTX-toxin (Apx) operons: Characterization of the ApxIII operons. Infect Immun 1994; 62:4411-8. 68. Meyer DH, Mintz KP, Fives-Taylor PM. Models of invasion of enteric and periodontal pathogens into epithelial cells: A comparative analysis. Crit Rev Oral Biol Med 1997; 8:389-409. 69. Kamaguchi A, Baba H, Hoshi M, Inomata K. Coaggregation between Porphyromonas gingivalis and mutans streptococci. Microbiol Immunol 1994; 38:457-60. 70. Ellen RP, Lepine G, Nghiem PM. In vitro models that support adhesion specificity in biofilms of oral bacteria. Adv Dent Res 1997; 11:33-42. 71. Andrews SC. Iron storage in bacteria. Adv Microb Physiol 1998; 40:281-351. 72. Hosogi Y, Duncan MJ. Gene expression in Porphyromonas gingivalis after contact with human epithelial cells. Infect Immun 2005; 73:2327-35.

170

Autophagy
