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Analysis of a Band 7/MEC-2 Family Gene of Porphyromonas gingivalis
S. Walters1, ,
P. Rodrigues1,
M. Bélanger1,
J. Whitlock1 and
A. Progulske-Fox1,*
1 Department of Oral Biology, College of Dentistry and Center for Molecular Microbiology, University of Florida, Gainesville, FL 32610-0424, USA
Correspondence: * corresponding author, apfox{at}dental.ufl.edu
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ABSTRACT
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In vivo-induced antigen technology has previously been used to identify 115 genes induced in Porphyromonas gingivalis W83 during human infection. The aim of this study was to determine if one of these genes, PG1334, was important for the virulence of P. gingivalis. Analysis of plaque samples from persons with periodontitis revealed that PG1334 was expressed in 88.0% of diseased sites, compared with 42.1% of healthy sites, even though P. gingivalis was detected in equal numbers from both sites. A mutant of PG1334 was found to adhere to and to invade better than the parent strain, but did not persist as well in human coronary artery endothelial cells. Additionally, the mutant did not persist as well in a mouse abscess model. This gene appears to be important for the virulence of P. gingivalis, both in vivo and in vitro.
Key Words: Porphyromonas gingivalis • IVIAT • stomatin • periodontitis • cardiovascular disease
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INTRODUCTION
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Periodontal diseases affect at least 35% of adults over 30 years of age (Albandar et al., 1999). Periodontal disease is an inflammation of the periodontium that results in microbial plaque formation, periodontal inflammation, loss of attachment and alveolar bone, and consequent periodontal pocket formation (Newman, 2002). Porphyromonas gingivalis is recognized as an important species in the pathogenesis of periodontitis (Loesche et al., 1985; Slots et al., 1986; Haffajee and Socransky, 1994). More recently, P. gingivalis has been acknowledged as having an important role in systemic disease, such as cardiovascular diseases (Desvarieux et al., 2005; Kozarov et al., 2005) and atherosclerosis (Gibson et al., 2006). Study of the interaction between P. gingivalis and cells from the vasculature, such as human coronary artery endothelial cells (HCAEC), should provide insights into the pathogenic mechanisms of this important bacterium.
During an infection, bacterial gene expression constantly changes to adapt to the host environment (Chiang et al., 1999; Handfield et al., 2000, 2008). Many genes have stringent regulatory systems and are expressed only at the site of infection. Gene expression can also change with disease progression. Previously, in vivo-induced antigen technology (IVIAT) was used to identify 115 in vivo-induced genes of P. gingivalis strain W83 in human periodontitis (Song et al., 2002). One of these genes, PG1334 (http://cmr.jcvi.org/tigr-scripts/CMR/shared/GenePage.cgi?locus=PG_1334), shares homology with the band 7 family of proteins, including human erythrocyte protein band 7.2 (EPB72, stomatin) and stomatin-like protein 2 (SLP-2) (Tavernarakis et al., 1999; Wang and Morrow, 2000). Although orthologues appear in many other bacterial species, the functions of these genes in prokaryotes remain unknown; however, the band 7 gene frequently appears in operons with a membrane protein, often a protease (Green et al., 2004). We used reverse-transcriptase polymerase chain-reaction (RT-PCR) and real-time PCR to confirm that PG1334 is contained within an operon, and to confirm in vivo expression of PG1334 within patient plaque samples. Additionally, a mutation was constructed in PG1334, and both the mutant and the parent strain were tested for their ability to adhere, invade, and persist within HCAEC. We also compared the mutant and the parent strain in a mouse abscess model to determine if the mutation affected survival and phagocytosis by mouse neutrophils.
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MATERIALS & METHODS
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Bacterial and Cell Culture Conditions
P. gingivalis W83 and W83 1334 were grown in tryptic soy broth supplemented with hemin, vitamin K1, yeast extract, and L-cysteine hydrochloride as described previously (Bélanger et al., 2007). HCAEC (Lonza, Walkersville, MD, USA) were in endothelial cell basal medium-2 maintained at 37°C with 5% CO2 (EBM-2) supplemented with EGM-2-MV ‘singlequots’ (Lonza), according to the manufacturer’s protocols. HCAEC were seeded at 1 x 105 cells/well into 24-well tissue culture plates and incubated overnight in antibiotic-free medium.
RNA Sample Collection
P. gingivalis W83 cultures were grown in supplemented brain heart infusion (BHI) broth and subcultured. After Institutional Review Board (IRB) approval and informed participant consent were obtained, subgingival plaque samples were collected from two healthy and two diseased sites of 20 persons according to criteria described previously (Shelburne et al., 2002). Plaque was removed from the curette by means of a sterile paper point and placed in 1 mL of Trizol (Invitrogen, Carlsbad, CA, USA). Samples were vortexed and stored at –80°C. RNA from P. gingivalis W83 and from the plaque samples was extracted as described below.
RNA Isolation and Reverse Transcription (RT)
RNA isolation was performed with Trizol and the RNeasy kit (Qiagen, Valencia, CA, USA) with DNase treatment as described previously (Yuan et al., 2007). RT was performed with Superscript III reverse transcriptase (Invitrogen) and random hexamer primers (Qiagen), following the manufacturers’ protocols, with reactions incubated at 48°C for 16 hrs. cDNA samples were purified by means of the PCR purification kit (Qiagen), following the manufacturer’s protocol.
PG1334 Operon Determination
We analyzed the putative PG1334 operon transcript to detect the co-expression of genes PG1333, PG1334, and PG1335. Flanking genes PG1332 and PG1337 were also analyzed. Total RNA was isolated and RT was performed as described above. PCR was performed, and the resulting products were visualized by electrophoresis in 1% agarose gel. To assay for any DNA contamination, we performed RT in the absence of reverse transcriptase. Primers are described in the Table .
Real-time PCR
Primers (Table ) were designed with Beacon Designer software (PREMIER Biosoft International, Palo Alto, CA, USA). P. gingivalis-specific 16S rRNA primers used were those previously reported (Shelburne et al., 2002). Real-time PCR was performed in an iCycler Thermal Cycler and with iQ SYBR green supermix, according to the manufacturer’s instructions (Bio-Rad Laboratories, Hercules, CA, USA). DNA fragments of each gene were used as internal controls and standard curves. All real-time PCR reactions were run at an annealing temperature of 55°C. Data normalization and analysis were performed with the iCycler and Microsoft Excel software. Gene expression was normalized to the P. gingivalis 16S rRNA expression.
Mutant Construction
W831334 was constructed by allelic replacement as described previously (Bélanger et al., 2007). Briefly, upstream and downstream regions of PG1334 were amplified by PCR with gene-specific primers (Table) and cloned into the suicide vector pPR-UF1 (Appendix Fig. 1). The mutation was confirmed by PCR (data not shown).
Adhesion Assays
Adhesion assays were performed as described previously (Tribble et al., 2006), with minor modifications. Briefly, P. gingivalis in EBM-2 was added to a multiplicity of infection of 100 and incubated with HCAEC for 30 min at 4°C. Cells were washed, and adherent bacteria were fixed with 5% formalin for 15 min at 37°C. Cells were washed and then blocked (5% BSA, 1% goat serum, and 0.1% Tween 20 in PBS) for 1 hr at room temperature. Adherent bacteria were detected with polyclonal rabbit anti-P. gingivalis serum, followed by a horseradish peroxidase-labeled mouse anti-rabbit antibody. The 3,3',5,5'-tetramethylbenzidine (Sigma, St. Louis, MO, USA) colorimetric substrate was added and the supernatant quantitated. An ELISA had previously been performed for confirmation that the polyclonal antibody bound similarly to both W83 and W831334.
Invasion and Persistence Assays
In total, 105 HCAEC/well were seeded into a 24-well tissue culture plate and incubated at 37°C overnight. P. gingivalis W83 or W831334 was added at a concentration of 107 bacterial cells/mL, and bacteria were allowed to invade the cells for 1.5 hrs. After cells were washed, 300 µg/mL gentamycin and 200 µg/mL metronidazole were added to kill extracellular bacteria. After an additional one-hour incubation, the cells were washed and either subjected to lysis with 1 mL of water for 20 min and plated for enumeration on blood agar plates, or antibiotic-free medium was added to the cells before re-incubation. In the latter case, at 6, 12, and 24 hrs post-inoculation, the supernatant was collected and centrifuged at 600 x g for 5 min. The pelleted bacteria were re-suspended in PBS and plated on blood agar for enumeration of the extracellular bacteria. Subsequently, HCAEC underwent lysis with water, and intracellular bacteria were enumerated by being plated on blood agar. All individual cell culture experiments were performed in triplicate wells, and each experiment was performed 3 times.
Mouse Abscess Model
We obtained IRB approval prior to performing this experiment. P. gingivalis W83 or W831334 was injected intradermally (6 mice/group) at an inoculum of 5 x 109 bacteria in 100 µL PBS, following the mouse abscess model as described previously (Wu et al., 2002), except that liver samples were collected, homogenized, and plated on blood agar for enumeration of bacteria. Additional mice were infected as described above, except that an approximately 1 cm3 skin sample was excised for histological sectioning and examination (see Appendix).
Neutrophil Killing
The susceptibility of P. gingivalis to neutrophil killing was measured as described previously, with minor modifications (Cutler et al., 1991). Briefly, overnight cultures of Escherichia coli S17-1 (control) and P. gingivalis W83 and W831334 were pelleted and re-suspended to 1 x 108 CFU/mL in PBS. Each bacterial suspension (70 µL) was opsonized with 50 µL of polyclonal rabbit anti-P. gingivalis serum. After opsonization, a 0.5-mL quantity of the neutrophil suspension (5 x 105 polymorphonuclear leukocytes) was added to the bacteria/serum mixture and incubated at 37°C at 300 rpm in a thermomixer. After 4 hrs, a 30-µL sample was collected and plated. Experiments were performed in triplicate.
Statistical Analysis
Comparisons between groups were done by the Student t test. Data that were not normally distributed were compared by the Mann-Whitney Rank sum test. Differences in proportions were compared by Fisher’s exact test plus Yates’ correction. A value of P < 0.05 was considered statistically significant.
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RESULTS
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Analysis of the Putative PG1334 Operon
The existence of a PG1334 operon in P. gingivalis was examined. The P. gingivalis database at The Institute for Genomic Research (TIGR) describes PG1334 between 2 genes: PG1333 (hypothetical protein) and PG1335 (putative membrane protein) (Fig. 1A). Thus, primers for RT-PCR were designed to amplify the regions between PG1333 and PG1334 as well as between PG1334 and PG1335. As controls, primers were also designed to amplify flanking regions from PG1332 to PG1333 and PG1335 to PG1337 (Table). Following RT-PCR, single bands of the expected sizes were obtained for the sequences overlapping genes PG1333, PG1334, and PG1335 (Fig. 1B). No products were obtained for similar sequences overlapping PG1332 and PG1337 (Fig. 1B).
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Figure 1. Characterization of the P. gingivalis W83 PG1333-1334-1335 operon and the flanking genes. (A) Arrows show the direction of transcription. Additional information can be found at http://cmr.jcvi.org/cgi-bin/CMR/shared/RegionView.cgi?not_first_time=1&locus=PG_1334&feat_type=ORF. (B) Confirmation of operon PG1333-1334-1335. Specific primers overlapping genes PG1332, PG1333, PG1334, PG1335, and PG1336 (Table) were used to amplify the putative mRNAs from total RNA isolated from P. gingivalis. (Lanes 1, 4, 7, 10) DNA isolated from P. gingivalis were used as positive control. (Lanes 2, 5, 8, 11) No reverse transcriptase added to the reaction (negative control). (Lanes 3, 6, 9, 12) Template RNA.
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Confirmation of IVIAT Gene Expression in Human Plaque Samples
We used real-time PCR to assay in vivo expression of gene PG1334 in plaque samples from persons with periodontitis. No statistically significant differences were observed in P. gingivalis detection (P > 0.05) between the plaque sites; 73.5% of samples taken from the diseased sites were positive for P. gingivalis, as measured by P. gingivalis-specific 16S primers. while 54.3% of healthy plaque samples tested positive for P. gingivalis. However, when the expression patterns were compared in diseased vs. healthy plaque sites, PG1334 was expressed significantly more in diseased sites (88.0%) than in healthy sites (42.1%, P < 0.01).
Adherence, Invasion, and Persistence
Mutant W831334 demonstrated significantly enhanced adherence to HCAEC at 30 min when compared with W83 (2.7-fold, P < 0.001, Fig. 2A), and the ability to invade HCAEC was also increased by 2.5-fold for W831334 at 2.5 hrs (P < 0.001, Fig. 2B). Thus, when invasion efficiency was normalized to the adherence data, W831334 showed no increase in invasion. The persistence of W83 and W831334 within HCAEC was also examined. The results are expressed as the percentage of the wild-type W83 recovered at 2.5 hrs post-inoculation. When compared with W83, a lower percentage of W831334 was recovered from the medium at both 12 and 24 hrs (2.4-fold, P < 0.001; 6.0-fold, P < 0.001, Fig. 3), but no difference was detected at 6 hrs. After 6 and 12 hrs, a similar percentage of W831334 was recovered from within the cells when compared with W83 (Fig. 3). However, at 24 hrs, fewer W831334 (3.5-fold, P < 0.01) were recovered compared with W83 (Fig. 3).
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Figure 2. Infection of HCAEC by P. gingivalis wild-type (black bar) and mutant strain (white bar). (A) Adherence of P. gingivalis to HCAEC at 4°C after 30 min and measured by colorimetric assay. (B) Invasion of HCAEC (2.5 hrs) by P. gingivalis, expressed as the percentage of inoculum. Error bars indicate the standard deviation obtained from triplicate (N) analyses. *P < 0.001.
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Figure 3. Persistence of P. gingivalis in HCAEC. P. gingivalis W83 (black bar) and mutant W831334 (white bar) detected extracellularly (A) or intracellularly (B) at 6, 12, and 24 hrs after inoculation of HCAEC, expressed as the percentage of that strain recovered at 2.5 hrs post-inoculation. Error bars indicate the standard deviation obtained from triplicate (N) analyses. *P < 0.001.
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Mouse Abscess Model
P. gingivalis was detected in the livers of 5 out of 6 mice infected with wild-type W83, while bacteria could be detected in the liver of only 1 of the 6 mice infected with the mutant. However, these results did not reach a level of significance, due to the small sample size (P = 0.08). When the numbers of bacteria recovered from mice were calculated and compared, there was a statistically significant difference observed between the groups (P = 0.026), since more P. gingivalis were recovered from the livers of mice infected with W83 (mean, 9.9 x 108 CFU/gram) than from the liver of the mouse infected with W831334 (mean, 1.1 x 105 CFU/gram). Additionally, histological examinations revealed significantly less inflammation in mice infected with W831334 than in those infected with W83 (Appendix Table, Appendix Fig. 2).
Neutrophil Killing
There was no statistical difference in the susceptibility to killing by neutrophils when P. gingivalis W831334 was compared with W83 (Appendix Fig. 3).
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DISCUSSION
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P. gingivalis gene PG1334 is a band 7/MEC-2 homologue identified by IVIAT as in vivo-induced. Analysis of the data reported here indicates that the expression of this gene is highly associated with periodontal disease sites, with 88% of plaque samples from diseased sites testing positive for expression of PG1334 compared with 42% in healthy sites, even though P. gingivalis was detected equally from both sites. Other studies have also demonstrated that P. gingivalis could be detected in high frequency from periodontally healthy and diseased sites (Gornitsky et al., 1991; Wilson et al., 1993).
The band 7/MEC-2 family includes stomatin, prohibitin, flotillin, and the HflC/K (SPFH) domain. In P. gingivalis W83, PG1334 is contiguous with and in the same orientation as PG1333 and PG1335. Analysis of our data indicated that PG1334 is co-transcribed with genes PG1333 and PG1335, and thus these 3 genes constitute an operon. Prokaryotic stomatin homologues are frequently found in operons with a membrane protein, often a protease (Green et al., 2004). The putative membrane protein (PG1335) in P. gingivalis does have domain homology to membrane proteins involved in the regulation of membrane protease activity. It has been suggested that the likely role for these prokaryotic stomatins is that of a partner protein for the membrane-bound protease, either as a chaperone, a substrate, or a regulator (Green et al., 2004). The interaction of these genes was studied in Pyrococcus horikoshii, where the membrane protease is assumed to be a serine protease that is capable of cleaving the C-terminal region of the stomatin homologue (Yokoyama and Matsui, 2005). It is proposed that, in P. horikoshii, the PG1334 and PG1335 homologues regulate an ion channel (Yokoyama and Matsui, 2005). Based on the P. horikoshii data, it is probable that PG1334 and PG1335 interact, although further studies need to be completed to discern the nature of this interaction and the function of these 2 proteins in P. gingivalis.
Our hypothesis is that PG1334 is involved in the activity of PG1335; thus, the absence of PG1334 could lead to increased proteolytic activity. This enhanced proteolysis could create additional sites for bacterial adherence. P. gingivalis has been shown to enter HCAEC quickly (Dorn et al., 1999). Therefore, if adherence to HCAEC is increased, invasion is most likely increased as well. Most significantly, PG1334 appears to be important for survival within the invaded cells, since the mutation in this gene greatly reduced its persistence in HCAEC when compared with that of the parent strain. The mutation in PG1334 may allow greater numbers of bacteria to enter the cell due to enhanced adherence, but this mutation may also be detrimental to survival within the cells. In addition, PG1334 also appears to be responsible, at least in part, for in vivo persistence in the animal. However, loss of this protein does not render P. gingivalis more susceptible to killing by neutrophils. Additional experiments need to be performed to define the exact role of this protein in the disease process. Overall, defining the role of this virulence factor of P. gingivalis could lead to the development of a vaccine and/or a new antimicrobial agent.
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ACKNOWLEDGMENTS
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We thank Ikramuddin Aukhil and Luciana Shaddox for collection of plaque samples. This work was supported by the UFL Center for Molecular Microbiology and the NIDCR, NIH grant DE13957.
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FOOTNOTES
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 current address, Immunology Branch, US Army Dental and Trauma Research Detachment, Walter Reed Army Institute of Research, Great Lakes Naval Training Center, Great Lakes, IL 60088, USA 
A supplemental appendix to this article is published electronically only at http://jdr.sagepub.com/supplemental.
Received for publication April 21, 2008.
Revision received September 9, 2008.
Accepted for publication October 14, 2008.
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REFERENCES
|
|---|
- Albandar JM, Brunelle JA, Kingman A (1999). Destructive periodontal disease in adults 30 years of age and older in the United States, 1988–1994. J Periodontol 70:13–29; erratum in J Periodontol 70:351, 1999.[CrossRef][Medline]
[Order article via Infotrieve]
- Bélanger M, Rodrigues P, Progulske-Fox A (2007). Genetic manipulation of Porphyromonas gingivalis. Curr Protoc Microbiol. Chapter 13; Unit 13C.2 (online publication).
- Chiang SL, Mekalanos JJ, Holden DW (1999). In vivo genetic analysis of bacterial virulence. Annu Rev Microbiol 53:129–154.[CrossRef][Medline]
[Order article via Infotrieve]
- Cutler CW, Kalmar JR, Arnold RR (1991). Phagocytosis of virulent Porphyromonas gingivalis by human polymorphonuclear leukocytes requires specific immunoglobulin G. Infect Immun 59:2097–2104.[Abstract/Free Full Text]
- Desvarieux M, Demmer RT, Rundek T, Boden-Albala B, Jacobs DR Jr, Sacco RL, et al. (2005). Periodontal microbiota and carotid intima-media thickness: the Oral Infections and Vascular Disease Epidemiology Study (INVEST). Circulation 111:576–582.[Abstract/Free Full Text]
- Dorn BR, Dunn WA Jr, Progulske-Fox A (1999). Invasion of human coronary artery cells by periodontal pathogens. Infect Immun 67: 5792–5798.[Abstract/Free Full Text]
- Gibson FC 3rd, Yumoto H, Takahashi Y, Chou HH, Genco CA (2006). Innate immune signaling and Porphyromonas gingivalis-accelerated atherosclerosis. J Dent Res 85:106–121.[Abstract/Free Full Text]
- Gornitsky M, Clark DC, Siboo R, Amsel R, Iugovaz I, Wooley C, et al. (1991). Clinical documentation and occurrence of putative periodontopathic bacteria in human immunodeficiency virus-associated periodontal disease. J Periodontol 62:576–585.[Medline]
[Order article via Infotrieve]
- Green JB, Fricke B, Chetty MC, von During M, Preston GF, Stewart GW (2004). Eukaryotic and prokaryotic stomatins: the proteolytic link. Blood Cells Mol Dis 32:411–422.[CrossRef][Medline]
[Order article via Infotrieve]
- Haffajee AD, Socransky SS (1994). Microbial etiological agents of destructive periodontal diseases. Periodontol 2000 5:78–111.[CrossRef]
- Handfield M, Brady LJ, Progulske-Fox A, Hillman JD (2000). IVIAT: a novel method to identify microbial genes expressed specifically during human infections. Trends Microbiol 8:336–339.[CrossRef][Medline]
[Order article via Infotrieve]
- Handfield M, Baker HV, Lamont RJ (2008). Beyond good and evil in the oral cavity: insights into host-microbe relationships derived from transcriptional profiling of gingival cells. J Dent Res 87:203–223.[Abstract/Free Full Text]
- Kozarov EV, Dorn BR, Shelburne CE, Dunn WA Jr, Progulske-Fox A (2005). Human atherosclerotic plaque contains viable invasive Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis. Arterioscler Thromb Vasc Biol 25:e17–e18.[Free Full Text]
- Loesche WJ, Syed SA, Schmidt E, Morrison EC (1985). Bacterial profiles of subgingival plaques in periodontitis. J Periodontol 56:447–456.[Medline]
[Order article via Infotrieve]
- Newman C (2002). Carranza’s clinical periodontology. 9th ed. Philadelphia: W.B. Saunders Co.
- Shelburne CE, Gleason RM, Germaine GR, Wolff LF, Mullally BH, Coulter WA, et al. (2002). Quantitative reverse transcription polymerase chain reaction analysis of Porphyromonas gingivalis gene expression in vivo. J Microbiol Methods 49:147–156.[CrossRef][Medline]
[Order article via Infotrieve]
- Slots J, Bragd L, Wikström M, Dahlén G (1986). The occurrence of Actinobacillus actinomycetemcomitans, Bacteroides gingivalis and Bacteroides intermedius in destructive periodontal disease in adults. J Clin Periodontol 13:570–577.[CrossRef][Medline]
[Order article via Infotrieve]
- Song YH, Kozarov EV, Walters SM, Cao SL, Handfield M, Hillman JD, et al. (2002). Genes of periodontopathogens expressed during human disease. Ann Periodontol 7:38–42.[CrossRef][Medline]
[Order article via Infotrieve]
- Tavernarakis N, Driscoll M, Kyrpides NC (1999). The SPFH domain: implicated in regulating targeted protein turnover in stomatins and other membrane-associated proteins. Trends Biochem Sci 24:425–427.[CrossRef][Medline]
[Order article via Infotrieve]
- Tribble GD, Mao S, James CE, Lamont RJ (2006). A Porphyromonas gingivalis haloacid dehalogenase family phosphatase interacts with human phosphoproteins and is important for invasion. Proc Natl Acad Sci USA 103:11027–11032.[Abstract/Free Full Text]
- Wang Y, Morrow JS (2000). Identification and characterization of human SLP-2, a novel homologue of stomatin (band 7.2b) present in erythrocytes and other tissues. J Biol Chem 275:8062–8071.[Abstract/Free Full Text]
- Wilson M, Lopatin D, Osborne G, Kieser JB (1993). Prevalence of Treponema denticola and Porphyromonas gingivalis in plaque from periodontally-healthy and periodontally-diseased sites. J Med Microbiol 38:406–410.[Abstract/Free Full Text]
- Wu Y, Lee SW, Hillman JD, Progulske-Fox A (2002). Identification and testing of Porphyromonas gingivalis virulence genes with a pPGIVET system. Infect Immun 70:928–937.[Abstract/Free Full Text]
- Yokoyama H, Matsui I (2005). A novel thermostable membrane protease forming an operon with a stomatin homolog from the hyper-thermophilic archaebacterium Pyrococcus horikoshii. J Biol Chem 280: 6588–6594.[Abstract/Free Full Text]
- Yuan L, Rodrigues PH, Bélanger M, Dunn W Jr, Progulske-Fox A (2007). The Porphyromonas gingivalis clpB gene is involved in cellular invasion in vitro and virulence in vivo. FEMS Immunol Med Microbiol 51: 388–398.[CrossRef][Medline]
[Order article via Infotrieve]
Journal of Dental Research, Vol. 88, No. 1,
34-38 (2009)
DOI: 10.1177/0022034508328381
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ABSTRACT
INTRODUCTION