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Journal of Bacteriology, February 2008, p. 1436-1446, Vol. 190, No. 4
0021-9193/08/$08.00+0     doi:10.1128/JB.01632-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Role of the Clp System in Stress Tolerance, Biofilm Formation, and Intracellular Invasion in Porphyromonas gingivalis

Cindy A. Capestany,¹ Gena D. Tribble,^1, Kazuhiko Maeda,¹ Donald R. Demuth,² and Richard J. Lamont^1*

Department of Oral Biology and Center for Molecular Microbiology, College of Dentistry, University of Florida, Gainesville, Florida 32610,¹ Department of Periodontics, Endodontics and Dental Hygiene, University of Louisville School of Dentistry, Louisville, Kentucky 40292²

Received 8 October 2007/ Accepted 23 November 2007

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Clp proteases and chaperones are ubiquitous among prokaryotes and eukaryotes, and in many pathogenic bacteria the Clp stress response system is also involved in regulation of virulence properties. In this study, the roles of ClpB, ClpC, and ClpXP in stress resistance, homotypic and heterotypic biofilm formation, and intracellular invasion in the oral opportunistic pathogen Porphyromonas gingivalis were investigated. Absence of ClpC and ClpXP, but not ClpB, resulted in diminished tolerance to high temperatures. Response to oxidative stress was not affected by the loss of any of the Clp proteins. The clpC and clpXP mutants demonstrated elevated monospecies biofilm formation, and the absence of ClpXP also enhanced heterotypic P. gingivalis-Streptococcus gordonii biofilm formation. All clp mutants adhered to gingival epithelial cells to the same level as the wild type; however, ClpC and ClpXP were found to be necessary for entry into host epithelial cells. ClpB did not play a role in entry but was required for intracellular replication and survival. ClpXP negatively regulated the surface exposure of the minor fimbrial (Mfa) protein subunit of P. gingivalis, which stimulates biofilm formation but interferes with epithelial cell entry. Collectively, these results show that the Clp protease complex and chaperones control several processes that are important for the colonization and survival of P. gingivalis in the oral cavity.

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Porphyromonas gingivalis, a gram-negative anaerobe, is a causative agent of severe and chronic manifestations of periodontal disease, one of the most common microbial infections of humans (24, 61). The primary ecological niche of P. gingivalis is the gingival sulcus, and within this compartment, P. gingivalis colonizes different microenvironments. On tooth surfaces, P. gingivalis is a constituent of the complex multispecies biofilm known as dental plaque. Colonization of dental plaque is favored by the attachment of P. gingivalis cells to substrata of antecedent colonizers. In particular, P. gingivalis can adhere to cells of Streptococcus gordonii, an early colonizer of plaque, and subsequently accumulate into heterotypic biofilm microcolonies (11, 34, 37). P. gingivalis also colonizes the tissues and cells of the gingival epithelium (1, 36). The organism is intensely invasive, and high numbers of P. gingivalis cells rapidly accumulate in the perinuclear area of gingival epithelial cells (GECs) (2). Both host and internalized bacterial cells remain viable, and indeed, P. gingivalis can suppresses host cell apoptosis through modulation of intrinsic apoptotic pathways (44). In addition to the oral habitat, P. gingivalis can also spread systemically and locate in heart and gestational tissues (32, 41), bone (67), and cerebrospinal fluid (25).

Transitioning among the various oral and nonoral and intra- and extracellular locations will subject P. gingivalis to environmental stresses, including temperature and redox potential. P. gingivalis is well equipped to respond to these stressors through the production of a series of stress-related proteins. These include heat shock proteins (HSPs), such as GroES/GroEL (Hsp60), DnaK/DnaJ/GrpE (Hsp70/Hsp40/Hsp70 cofactor), and HtpG (Hsp90) (21, 59), along with superoxide dismutase, alkyl hydroperoxide reductase, rubrerythrin, and the transcriptional activator OxyR, which are involved in aerotolerance (15, 28, 46, 49, 50). P. gingivalis also possesses ClpB, ClpC, ClpP, and ClpX, which are members of the Clp (caseinolytic protease, Hsp100) family. The Clp protease proteolytic subunit, ClpP, is a cytoplasmic, barrel-shaped serine protease composed of two heptameric rings (5, 17). Narrow axial pores prohibit access of globular proteins to the internal catalytic chamber, and in order to gain proteolytic activity, ClpP must associate with a Clp ATPase partner that possesses nucleotide binding domains characteristic of the AAA+ superfamily of ATPases (5). The Clp ATPase regulatory proteins ClpA, ClpC, and ClpX form hexameric rings and possess a ClpP recognition tripeptide that allows association with the ClpP peptidase. The resulting Clp proteolytic complex specifically targets damaged or misfolded proteins for translocation and degradation (31, 33, 69). The ATPases determine the protease substrate specificity, in some cases by recognition of an 11-amino-acid degradation tag known as SsrA (30, 69). The ClpB and ClpL ATPases do not appear to associate with ClpP but rather function solely as chaperones with protein reactivation and remodeling activities (5, 20, 45, 76).

The Clp system is involved in general stress responses as well as in the maintenance of cell morphology and virulence. For example, in Staphylococcus aureus, ClpP and ClpC are required for heat stress, while ClpXP is important for survival under osmotic stress, oxidative stress, and cold (16). Clp mutants of Salmonella, Streptococcus pneumoniae, and S. aureus all demonstrate attenuated virulence (5, 22). ClpC is required for optimal adhesion and invasion of Listeria monocytogenes and promotes early escape of the organism from the phagosomal compartment of macrophages (55). ClpP is also involved in the adaptive response of Listeria in macrophages, and both ClpP and ClpC affect the control of PrfA-regulated genes (inlA, inlB, actA, and hly) (18, 47). Differential biofilm formation also has been observed in Clp mutants, and either enhancement or diminution of biofilm formation can be observed, depending on the organisms and the nature of the Clp protein deficiency (16, 40). Hence, while the overall phenotypic properties controlled by the Clp system are similar among organisms, different species can adapt the Clp components to perform distinct roles.

The roles of the Clp proteins in P. gingivalis have not been investigated in a systematic manner. Our initial proteomic studies found that ClpP, ClpC, and ClpX were upregulated in response to an epithelial cell environment and that an insertional mutation in clpXP reduced the ability of P. gingivalis to invade GECs (75). Similarly, insertional inactivation of clpB was found to reduce invasion of P. gingivalis in epithelial and endothelial cells (74). In this study, we utilized deletion mutants of clpB, clpC, and clpXP, along with their genetic complements, to assess the functions of the corresponding proteins in P. gingivalis stress responses; single- and mixed-species biofilm formation; and adhesion, entry, and survival in epithelial cells. In addition, the impacts of the clp mutations on the expression of well-characterized virulence factors of P. gingivalis were evaluated.

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Bacteria and culture conditions. P. gingivalis strain ATCC 33277 and derivatives were grown on sheep blood agar or in trypticase soy broth supplemented with yeast extract (1 µg ml^–1), menadione (1 µg ml^–1), and hemin (5 µg ml^–1) at 37°C under anaerobic conditions of 85% N₂, 10% H₂, and 5% CO₂. When necessary, erythromycin at 10 µg ml^–1, tetracycline at 1 µg ml^–1, or gentamicin at 100 µg ml^–1 was added to the medium. S. gordonii DL1 was grown in chemically defined medium (3), with sucrose (0.8%) as the carbon source. Escherichia coli TOP10, J53, and DH5 were grown in Luria-Bertani (LB) broth containing, when necessary, ampicillin at 100 µg ml^–1. Solid medium was prepared by addition of 1.5% agar.

General molecular techniques. Recombinant-DNA techniques were performed as described previously (57). Restriction enzymes and DNA-modifying enzymes were purchased from Promega. P. gingivalis cells were lysed with Trizol (Invitrogen). Chromosomal DNA was isolated using a Wizard genomic DNA purification kit (Promega). RNA was extracted with phenol-chloroform and precipitated with isopropanol. RNA preparations were washed with 70% ethanol, dissolved in RNase-free H₂O, and treated with RNase-free DNase I (Ambion). The RNA was then purified on RNeasy columns (Qiagen). Plasmid DNA was isolated from E. coli by using a QiaMiniprep kit (Qiagen). Standard PCR conditions were 95°C for 6 min and 30 cycles of 94°C for 30 s, 57°C for 30 s, and 72°C for 2 min, followed by a final extension at 72°C for 7 min. For Southern blots, DNA probes were labeled and hybridization was detected using the Gene Images AlkPhos direct labeling and detection system (Amersham). P. gingivalis competent cells were prepared by suspending early-log-phase cells in electroporation buffer (10% glycerol, 1.0 mM MgCl₂). For electroporation, cells were incubated with 2 µg of a PCR product in Tris-EDTA buffer and pulsed with a Bio-Rad gene pulser at 2.5 kV.

Construction of mutant and complemented strains. Mutants of the clpB and clpC genes were created by allelic replacement. To create the clpB mutant, a 4.4-kb region encompassing the entire clpB gene was PCR amplified with primers ClpB-upper (5'-GGCTGAGGTCTAGAGACTGCCTGCGTTGTGGTTG-3') and ClpB-lower (5'-TGCGTATTTGGTGGTTATTG-3'). The resulting PCR product was digested with restriction enzymes XbaI and HindIII, and the fragment was cloned into the same restriction sites of pUC19. The resulting plasmid was then digested with PstI to remove a 2-kb internal fragment from clpB. This region was replaced with a 1.1-kb insert containing the ermF allele from pVA3000 (39). For clpC, PCR primers ClpC-upper (5'-GGGAAGGCGAATTCGGCGGAGGCGATACTACCAC-3') and ClpC-lower (5'-GGGCTTGCATGGCACCCATG-3') were used to amplify a 2.2-kb region containing the 3' region of the clpC gene. The resulting PCR product was digested with EcoRI and HindIII and cloned into the complementary sites of pUC18. The resulting plasmid was digested with SphI to remove a 303-bp internal fragment of clpC. This fragment was replaced by ligation to the ermF gene, contained on a 1.1-kb SphI fragment. For both the clpB and clpC mutants, the final plasmid constructs were linearized by digestion with ScaI and transformed into P. gingivalis strain 33277 by electroporation, and allelic replacement mutants were selected with erythromycin. Insertion of the replacement allele was confirmed by PCR and Southern hybridization, and loss of mRNA was established by reverse transcription-PCR (RT-PCR).

The clpXP mutant was constructed by gene replacement using a PCR fusion technique (60). A DNA sequence containing 542 bp upstream of the clpP initiation codon was amplified using the primers CLPP-upF (5'-TCAGTGGCAGCGGAGATG-3') and CLPP-upR (5'-AACGGGCAATTTCTTTTTTGTCATTGTCTTTACTGTGATCATATTTGTTTT-3'). A 799-bp region downstream of the clpX stop codon was amplified using CLPX-downF (5'-GTCCCTGAAAAATTTCATCCTTCGTAGTAGAAGTACCCCCAAGAGAATC-3') and CLPX-downR (5'-CCGGCTGGGATAGGATGTA-3'). To replace the clpXP genes, an ermF cassette was amplified from pVA3000 by using primers ERMF-F (5'-ATGACAAAAAAGAAATTGCCCGTT-3') and ERMF-R (5'-CTACGAAGGATGAAATTTTTCAGGGAC-3'), which contain 5' homology to CLPP-upR and CLPX-downF, and a fusion PCR product was produced (35). To confirm product size and purity, the fusion PCR product was size fractionated by agarose gel electrophoresis. The final construct was purified with the Wizard PCR cleanup system (Promega) and introduced into P. gingivalis by electroporation. A double-crossover recombination event was selected by plating with erythromycin selection. Insertion of the replacement allele was confirmed by PCR and Southern hybridization, and loss of mRNA was established by RT-PCR.

For complementation of the clp mutations, a region containing the appropriate clp open reading frame, plus no fewer than an additional 100 bp upstream and 30 bp downstream, was PCR amplified using ExTaq (Takara). The primers were engineered to contain the following restriction sites: SalI-HindIII for clpB, NheI-HindIII for clpC, and NheI-BamHI for clpXP. The shuttle vector plasmid pT-COW (19) was digested with the appropriate restriction enzymes to allow cloning of the PCR product into the tetC region. The resulting plasmids were transformed into E. coli TOP10 and selected on ampicillin plates. Colonies were screened by colony direct PCR and restriction digestion. The plasmids with the correct restriction profile were designated pT-ClpB, pT-ClpC, and pT-ClpXP, and these plasmids were introduced into the corresponding clp strain by conjugation. The conjugation reaction mixture also contained helper E. coli J53 containing R751, an IncP plasmid used to mobilize vectors from E. coli to a Bacteroides recipient. The conjugation mixture had a donor-to-recipient ratio of 0.2. The mating was performed in a candle jar on prereduced blood agar plates for 16 h, and transconjugants were selected with gentamicin and tetracycline. The presence of pT-COW-derived plasmid, and of the ermF gene on the chromosome in the transconjugants, was confirmed by PCR. The resulting strains were designated CclpB, CclpC, and CclpXP.

P. gingivalis biofilm formation. Homotypic biofilm formation by P. gingivalis was quantified by a microtiter plate assay (51), as adapted for P. gingivalis (6). Parental and mutant strains in early log phase (2 x 10⁸ cells) were incubated at 37°C anaerobically for 24 h. The resulting biofilms were washed, stained with 1% crystal violet, and destained with 95% ethanol. Absorbance at 595 nm was determined with a Benchmark microplate reader. Biofilm assays were repeated independently three times with each strain in triplicate.

Monospecies P. gingivalis biofilms and heterotypic P. gingivalis-S. gordonii biofilms on glass plates were generated and analyzed by confocal microscopy as described previously (6, 34). For homotypic biofilms, P. gingivalis strains in early log phase were labeled with 5 (and 6)-carboxyfluorescein succinimidyl ester (4 µg ml^–1; Molecular Probes). Labeled bacterial cells (5 x 10⁷) were added to CultureWell chambered cover glass 16-well slides (Grace Bio Labs) and incubated anaerobically for 24 h at 37°C with rocking. For heterotypic P. gingivalis-S. gordonii biofilms, S. gordonii cells were labeled with hexidium iodide (15 µg ml^–1; Molecular Probes) and then cultured anaerobically at 37°C for 16 h with rocking in individual chambers of the CultureWell cover glass system. Fluorescein-labeled P. gingivalis cells (2 x 10⁶ in prereduced phosphate-buffered saline [PBS]) were reacted with the S. gordonii biofilm for 24 h anaerobically at 37°C with rocking. The resultant single- or mixed-species biofilms were examined with a Bio-Rad MRC600 confocal scanning laser microscope (Kr/Ar) system with an MS plan 40x 1.4-numerical-aperture objective. Images were digitally reconstructed (x-z section and z projection of x-y sections) with Image J V1.33u (NIH). Quantitation of P. gingivalis-specific fluorescence, along with the heights of the biofilms, was determined using DAIME software (13). Quantitation of S. gordonii-specific fluorescence ensured that equivalent levels of the streptococcal substratum were present in each experiment. Biofilm assays were repeated independently three times with each strain in triplicate.

GEC culture and P. gingivalis invasion. Primary cultures of GECs were generated as described previously (36, 48). Briefly, healthy gingival tissue was collected from patients undergoing surgery for removal of impacted third molars, following Institutional Review Board guidelines. Basal epithelial cells were separated and cultured in keratinocyte growth medium (Cambrix) at 37°C in 5% CO₂. GECs were used at passages 3 to 5 and at 80% confluence. Invasion assays were repeated three times in triplicate with GECs from different donors.

Overall invasion and survival of P. gingivalis in GECs were determined by an antibiotic protection assay (36). GECs were reacted with P. gingivalis strains at a multiplicity of infection (MOI) of 100 at 37°C in 5% CO₂ for 2 h. Infected GECs were incubated with metronidazole (200 µg/ml) and gentamicin (300 µg/ml) for 1 h to kill extracellular bacteria and lysed with sterile, distilled water for 15 min. The intracellular P. gingivalis bacteria released by cell lysis were enumerated by viable counting on blood agar plates.

Internalization of P. gingivalis strains was visualized and quantified by confocal microscopy. GECs were cultivated on glass coverslips (Lab-Tek) and reacted P. gingivalis strains at an MOI of 100 at 37°C in 5% CO₂ for 30 min. Cells were fixed with 4% paraformaldehyde for 20 min, washed with PBS, and blocked overnight at 4°C in 10% normal goat serum in PBS. The cells were permeabilized for 20 min at room temperature (RT) with 10% normal goat serum and 0.2% saponin in PBS and then incubated with rabbit polyclonal antibody to P. gingivalis 33277 (72) at 1:5,000 for 2 h at RT. Fluorescein isothiocyanate (FITC)-conjugated Affini-Pure F(ab')₂ fragment goat anti-rabbit immunoglobulin G (heavy plus light chains; Jackson ImmunoReasearch Laboratories) was added at 1:500 for 2 h at RT. Actin microfilaments were stained with tetramethyl rhodamine isocyanate-phalloidin (Sigma) at 1:200 for 15 min at RT. Images were acquired with a Bio-Rad MRC600 confocal scanning laser microscope (Kr/Ar) system with an MS plan 40x 1.4-numerical-aperture objective. A series of fluorescent optical x-y sections were collected to create digitally reconstructed images (x-z section and z projection of x-y sections) with Image J 1.35c and Adobe Photoshop 6.0 software. Total P. gingivalis fluorescent accumulations in the stack of z projections representing whole cells were quantified with Image J 1.35c using the area calculator plugin.

Attachment of P. gingivalis strains to GEC was determined by an enzyme-linked immunosorbent assay (ELISA) as described previously (53). GECs were cultured on 96-well plates, fixed with 5% buffered formalin, and reacted with P. gingivalis strains at an MOI of 100 for 30 min at 37°C. Cells were washed with PBS, and surface bacteria were immunolabeled with P. gingivalis whole-cell antibodies (1:1,000) and horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G (ICN Biochemicals). The adherence of each strain was determined by a colorimetric reaction using the 3,3',5,5'-tetramethylbenzidine (TMB) liquid substrate system (Sigma).

Detection of Mfa, FimA, and SerB. For Western blotting, P. gingivalis strains in mid-log phase were lysed with radioimmunoprecipitation assay buffer (Santa Cruz) and proteins (10 µg) separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane by electroblotting. Membranes were blocked and reacted with rabbit polyclonal antibodies against recombinant Mfa (rMfa), rFimA, and rSerB (1:5,000) followed by horseradish peroxidase-conjugated goat anti-rabbit secondary antibody. Bound antibody was detected with an enhanced chemiluminescence system (Amersham).

The levels of cell surface Mfa and FimA proteins were determined by an ELISA with formalin-fixed whole cells. Bacteria were reacted with rMfa or rFimA antibodies (1:10,000) followed by peroxidase-conjugated secondary antibody. Antigen-antibody binding was determined by a colorimetric reaction with TMB. Antiserum to P. gingivalis 33277 whole cells was used as a control to ensure that consistent numbers of cells were fixed in each well.

Quantitative RT-PCR. Real-time RT-PCR was used to quantify mRNA levels for mfa and fimA by using the following primers, designed with Beacon Designer 2.0 software (Palo Alto, CA): mfa, 5'-TGCGGCGAAGTCGTATATG-3' and 5'-ATCTTCAGCACTCTCCACAAG-3'; and fimA, 5'-TTGTTGGGACTTGCTGCTCTTG-3' and 5'-TTCGGCTGATTTGATGGCTTCC-3'. Total RNA was reverse transcribed to cDNA by using Superscript III. Specific DNA standards for mfa and fimA were synthesized from chromosomal DNA by using standard PCR. Real-time RT-PCR was performed with a Bio-Rad iCycler using SYBR green Supermix (Bio-Rad). Results were analyzed with iCycler iQ Optical System software version 3.0a. The melt curve profiles were examined to verify a single peak for each sample, indicating primer specificity, and transcript copy number was calculated (73). RNA extracts were prepared in duplicate from independent experiments, and cDNA samples were loaded in triplicate.

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The clp genes in P. gingivalis: construction of mutants and complemented mutant strains. The genome of the sequenced strain of P. gingivalis, W83, possesses clpB (PG1118), clpC (PG0010), clpP (PG0418), and clpX (PG0417), with the latter two adjacent to each other on the chromosome. P. gingivalis lacks clpA, which is usually present in gram-negative but absent in gram-positive bacteria (17), although P. gingivalis does have a relatively low GC content (48%). Proteins corresponding to each of the P. gingivalis clp gene products have been detected in strain 33277 (75), which was adopted in this study as it is more virulent than W83 in rodent models of periodontitis (29). RT-PCR demonstrated that clpP and clpX are cotranscribed in 33277 (not shown). Allelic replacement mutations were created in the clpB and clpC genes. The cotranscribed genes clpP and clpX were deleted together to produce a double knockout. RT-PCR showed that transcription of the upstream and downstream genes was unaffected by the mutations (not shown). Furthermore, all mutations were complemented with the wild-type allele(s) in trans using the E. coli-P. gingivalis shuttle vector pT-COW (19). As a control for the complemented strains, pT-COW without an insert was introduced into 33277.

Inactivation of clpC and clpXP impairs growth at high temperatures. The survival and growth of wild-type, mutant, and complemented mutant strains under conditions of elevated temperature or oxidative stress were compared to assess the contribution of the Clps to environmental stress resistance. For heat stress, P. gingivalis cells were cultured to mid-exponential phase and dilutions spotted in duplicate on blood agar plates which were incubated anaerobically for 5 days at either 37°C or 42°C. At the control temperature (37°C), the wild type and clp mutants maintained similar viabilities (Fig. 1A). At the higher temperature (42°C), the clpC and clpXP mutants showed 3-log decreases in viable counts compared to wild-type levels. In contrast, the clpB mutant formed colonies with sizes and numbers similar to those of the parental strain. These results differ from those of a study with P. gingivalis strain W83 that showed increased susceptibility to death at 50°C for a clpB mutant, although the difference between parent and mutant levels was less than 1 log (74). We did not observe a change in resistance to thermal stress in the 33277 clpB mutant at this higher temperature, and thus there may be some strain-specific effects in P. gingivalis related to the functionality of ClpB. Sensitivity to higher temperatures in the clpC and clpXP mutants was lost when the strains were genetically complemented (Fig. 1B). Exposure to 30 mM hydrogen peroxide or atmospheric air for 5 h did not result in any significant differences in viability between parental and mutant strains (not shown). The Clp system in strain 33277, therefore, does not appear to be involved in protection against oxidative stress; however, ClpC and ClpXP are required for optimal resistance to thermal stress.

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FIG. 1. ClpC and ClpXP are required for optimal growth at high temperatures. P. gingivalis cultures at mid-exponential phase were serially diluted and spotted onto blood agar plates. The plates were incubated anaerobically at the indicated temperatures and colonies (CFU) counted. (A) Parental strain 33277 and clp mutants clpB, clpC, and clpXP. (B) 33277 with pT-COW (CWT) and complemented strains CclpC and CclpXP. Experiments were repeated three times in duplicate with independent cultures. * denotes P values of <0.0001 (t test) for comparison to 33277.

Homotypic biofilm formation by P. gingivalis is enhanced in the absence of ClpC and ClpXP. The ability of P. gingivalis to form biofilms on oral surfaces contributes to the persistence of the organism and increases the potential for periodontal tissue destruction. Stress responses, including those mediated by the Clp system, are known to be important for biofilm formation by other pathogenic organisms, such as S. aureus, Streptococcus mutans, and Pseudomonas fluorescens (16, 40, 51). Therefore, we examined the relative biofilm forming capabilities of the clp mutant strains of P. gingivalis. Monospecies biofilm formation was first tested in the microtiter plate assay with crystal violet staining (51). As shown in Fig. 2A, the clpC and clpXP mutants showed greater biofilm accumulation than the parental strain, and biofilm formation by the complemented ClpC and ClpXP strains was restored to wild-type levels (Fig. 2B). To observe and quantify biofilm architectural features, biofilms of fluorescein-labeled P. gingivalis cells were generated on glass surfaces and then examined by confocal microscopy with digitally reconstructed image analysis. Biofilms formed by the ClpC- and ClpXP-deficient cells were visibly denser (Fig. 3A) than those of the parent, and total accumulation was significantly increased (Fig. 3B). Furthermore, the average heights of the clpC and clpXP mutant biofilms were significantly elevated compared to that of the parent (Fig. 3C). The biofilm accumulations of the complemented clpC and clpXP mutant strains were indistinguishable from those of the parental control (Fig. 3D and E). Hence, ClpC and ClpXP are required to constrain P. gingivalis homotypic biofilm development, and in the absence of ClpC and ClpXP, more luxuriant biofilms are formed by P. gingivalis.

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FIG. 2. ClpC and ClpXP control homotypic P. gingivalis biofilm growth. Microtiter plate biofilms after 24 h were stained with crystal violet and washed, and then the crystal violet was released with 95% ethanol. Biofilm accumulation was measured by absorbance at 595 nm. (A) Parental strain 33277 and clp mutants clpB, clpC, and clpXP. (B) 33277 with pT-COW (CWT) and complemented strains CclpC and CclpXP. Results for the complemented strains are expressed as percentages of CWT biofilm growth. * denotes P values of <0.05 (t test) for comparison to 33277.

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FIG. 3. ClpC and ClpXP regulate P. gingivalis monospecies biofilm architecture. Biofilm accumulations of fluorescein-labeled P. gingivalis strains on glass plates were viewed by confocal microscopy, and fluorescent optical x-y sections were reconstructed with Image J and analyzed with DAIME software. (A) Confocal laser scanning microscopy images of x-y and x-z projections of parental strain 33277 and clp mutants clpB, clpC, and clpXP. Magnification, x40. Total biofilm accumulation was measured by grain area analysis of 268.6- by 268.6-µm x-y sections for 33277, clpB, clpC, and clpXP strains (B) or 33277 with pT-COW (CWT), CclpC, and CclpXP (D). Average biofilm height of P. gingivalis accumulation was measured across three random x-z sections for 33277, clpB, clpC, and clpXP strains (C) or CWT, CclpC, and CclpXP (E). Results for the complemented strains are expressed as percentages of CWT levels. * denotes P values of <0.05 (t test) for comparison to 33277.

ClpXP deficiency enhances accumulation of heterotypic P. gingivalis-S. gordonii biofilms. The biofilms that develop in vivo on oral surfaces are complex multispecies communities (54). Successful bacterial colonizers of oral biofilms, therefore, are frequently capable of interacting synergistically with other biofilm inhabitants. Of particular relevance to this study, the presence of a substratum of the antecedent colonizer S. gordonii stimulates attachment and biofilm formation by P. gingivalis (34, 37). Hence, the role of the Clp proteins in the formation of mixed P. gingivalis-S. gordonii biofilms was determined. A biofilm of hexidium iodide-labeled S. gordonii cells was first generated on glass coverslips. Fluorescein-labeled P. gingivalis cells were reacted anaerobically with the S. gordonii biofilm, and accumulations of heterotypic biofilms were observed by confocal microscopy as described above. As the P. gingivalis cells were suspended in buffer, any increase in number is unlikely to be due to cell division. Therefore, this assay models one of the early stages of biofilm development: the recruitment, coadhesion, and accumulation of P. gingivalis cells from the planktonic phase into a sessile biofilm. This process is contingent on interbacterial signaling events and occurs prior to a further increase in number through growth and division (34). x-y and x-z images of the heterotypic P. gingivalis-S. gordonii biofilms are shown in Fig. 4A. S. gordonii cells developed biofilms that extensively covered the glass surface, and cells of parental P. gingivalis, along with those of the clpC mutant, formed discrete microcolony accumulations clearly separated from each other, consistent with previous studies (11). In contrast, while the clpB mutant exhibited the same degree of total accumulation, there were fewer microcolonies with a larger volume (Fig. 4B and C). The clpXP mutant formed more abundant accumulations within the mixed P. gingivalis-S. gordonii biofilm, and total fluorescence analysis verified the enhanced accumulation of the clpXP mutant compared to the parental level (Fig. 4B). Interestingly, the average biofilm heights across the x-z sections showed no increase in vertical accretion for the clpXP mutant (Fig. 4C). The complemented ClpB and ClpXP strains displayed heterotypic biofilm features similar to those of the parental control (Fig. 4D and E). Collectively, these results indicate that ClpB is required to maintain optimal surface area/volume ratios of heterotypic P. gingivalis microcolonies, whereas ClpXP is required for optimal spacing of the P. gingivalis microcolonies.

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FIG. 4. ClpB and ClpXP regulate heterotypic P. gingivalis-S. gordonii biofilms. S. gordonii stained with hexidium iodide (red) was cultured on glass plates. P. gingivalis strains were stained with fluorescein (green) and reacted with the S. gordonii biofilms for 24 h. Colocalized bacteria appear yellow. Mixed-species biofilm accumulations were viewed by confocal microscopy, and fluorescent optical x-y sections were reconstructed with Image J and analyzed with DAIME software. (A) Confocal laser scanning microscopy images of x-y and x-z projections of parental strain 33277 and clp mutants clpB, clpC, and clpXP. Magnification, x40. Total P. gingivalis accumulation was measured by grain area analysis of 268.6- by 268.6-µm x-y sections for 33277, clpB, clpC, and clpXP strains (B) or 33277 with pT-COW (CWT) and CclpXP (D). Average biofilm height of P. gingivalis accumulation was measured across three random x-z sections for 33277, clpB, clpC, and clpXP strains (C) or CWT and CclpB (E). Results for the complemented strains are expressed as percentages of CWT levels. * denotes P values of <0.05 (t test) for comparison to 33277.

The Clp proteins are required for intracellular invasion and survival of P. gingivalis. In the subgingival crevice, P. gingivalis cells will encounter the host epithelial cells that line the gingival tissue and form both a physical barrier to microbial intrusion and an interactive interface that signals the presence of bacteria to the immune system. P. gingivalis cells can survive and replicate within GECs (36), and the organism can be detected within these cells in vivo (10). The role of the Clp proteins in the invasion of, and survival within, primary cultures of GECs was assessed by a standard antibiotic protection assay. All of the clp mutants exhibit significant decreases in intracellular survival compared to that of the parental strain (Fig. 5A). Additionally, complementation successfully restored the invasion phenotype to the parental levels (Fig. 5B). Therefore, the Clp stress response system is essential for optimal invasion of P. gingivalis into GECs.

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FIG. 5. clp mutants are impaired in GEC invasion. GECs (1 x 105) were infected with P. gingivalis strains (1 x 107 bacterial cells) for 2 h. Extracellular bacteria were killed with gentamicin and metronidazole and intracellular bacteria released by lysis of the GECs. Numbers (CFU) of intracellular bacteria were determined by viable counting of the cell lysates. (A) Invasion of 33277, clpB, clpC, and clpXP strains. (B) Invasion of CclpB, CclpC, and CclpXP, expressed as percentages of the invasion level of 33277 with pT-COW (CWT). * denotes P values of <0.0005 (t test) for comparison to 33277.

The overall invasive process can be divided into three broadly defined steps, comprising adherence, internalization, and intracellular survival. To dissect the contribution of the Clp proteins to each of these steps, mutants were tested for adhesion and for internalization capability. Adherence was measured in an ELISA format with P. gingivalis antibodies and GECs that were formalin fixed to prevent internalization (53). All of the clp mutants adhered to GECs to the same level as the parental strain (Fig. 6), indicating that the defect in invasion is not the result of reduced adherence. Interestingly, in one previous study, loss of ClpB resulted in a reduction in adherence and internalization of epithelial cells for strain W83 (74). However, strain W83 is naturally deficient in GEC invasion, with levels over 1 log less than those of strain 33277 or of low-passage clinical isolates (36, 64). Thus, ClpB may play a different role in W83-epithelial cell interactions than in 33277.

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FIG. 6. Adherence to GECs is not affected by clp mutations. Formalin-fixed GECs were reacted with P. gingivalis strains at an MOI of 100 for 30 min. Binding was quantified with specific antibodies and colorimetric substrate reactions in an ELISA format. A fimbria-deficient mutant, fimA (72), was used as a control for reduced adherence. * denotes P values of <0.005 (t test) for comparison to 33277.

Internalization was measured by confocal microscopy with P. gingivalis antibodies and FITC-labeled secondary antibodies. A series of fluorescent optical x-y sections were collected to create digitally reconstructed images throughout the volume of the cell (x-z section and z projection of x-y sections), and total P. gingivalis fluorescent accumulation in the stack of z projections representing whole cells was quantified. P. gingivalis wild-type cells rapidly entered GECs and accumulated in the perinuclear area (Fig. 7A), consistent with previous reports (2). The clpB mutant demonstrated levels of both internalization and intracellular location similar to those of the parent. In contrast, the clpC and clpXP mutants showed significant decreases in entry efficiency. Quantitation of intracellular P. gingivalis levels confirmed the results of the visual inspection (Fig. 7B). Internalization of complemented ClpC and ClpXP strains returned to parental control levels (Fig. 7C). These results suggest that ClpC and ClpXP are involved in the process of entry of P. gingivalis into GECs, whereas ClpB is required for intracellular survival.

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FIG. 7. Differential effects of ClpB, ClpC, and ClpXP on internalization into GECs. Epithelial cells were cultured on glass plates and infected with P. gingivalis strains for 30 min. Bacterial cells were detected with P. gingivalis antibodies and FITC-secondary antibodies (green). The cell volume of the GECs was visualized with tetramethyl rhodamine isocyanate-phalloidin staining for actin (red). Infected cells were viewed by confocal microscopy, and fluorescent optical x-y sections were reconstructed and analyzed with Image J. (A) Confocal laser scanning microscopy images of a 0.2-µm optical x-y section through GECs infected with 33277, clpB, clpC, and clpXP strains. Magnification, x40. Quantitative image analysis of intracellular P. gingivalis fluorescence in the stack of z projections representing whole cells for wild-type (33277), clpB, clpC, and clpXP strains (B) or 33277 with pT-COW (CWT), CclpB, CclpC, and CclpXP strains (C). Intracellular P. gingivalis levels are expressed as percentages of CWT levels for complemented strains. * denotes P values of <0.01 (t test) for comparison to the wild type.

ClpXP controls surface expression of Mfa protein. To gain greater insight into the potential role of the Clp stress response system in the mechanisms of control of the biofilm and invasive properties of P. gingivalis, we investigated the expressions of well-characterized virulence determinants that relate to these phenotypes, namely, the long (FimA) and short (Mfa) fimbriae and the SerB serine phosphatase (37, 52, 64, 72). Total bacterial proteins were extracted and probed with antibodies to FimA, Mfa, and SerB by Western blotting. The blot analysis showed that expression of Mfa was increased in the clpXP mutant (Fig. 8A). No change in expression of FimA or SerB was detected in any of the clp mutants. Furthermore, expression of Mfa was restored to wild-type levels in the complemented strain (Fig. 8B). The relative ratios of Mfa1/FimA in the parental and mutant strains were determined by scanning densitometry (Fig. 8C), which confirmed that Mfa is induced in the ClpXP mutant. To assess whether Mfa expression on the bacterial cell surface was also increased in the clpXP mutant, an ELISA with whole cells and antibodies to Mfa was performed (Fig. 8D). Surface exposure of Mfa was increased in the clpXP mutant compared to that in the parental strain but not in the clpB or clpC mutant strains. An increase in Mfa expression could be the result of an elevated rate of gene transcription or could result from effects on posttranscriptional processing. To begin to distinguish between these possibilities, we compared mfa mRNA levels in the parent and mutant strains by using quantitative real-time RT-PCR. The results showed no difference in mfa gene transcription (Fig. 8E), suggesting that the role of ClpXP in restricting Mfa protein exposure occurs at the protein processing level.

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FIG. 8. Expression of Mfa is increased in the absence of ClpXP. (A) Whole cells of P. gingivalis strains were lysed and blotted with antibodies to FimA, Mfa, or SerB. (B) Blot of complemented CclpXP strain with FimA and Mfa antibodies. (C) Scanning densitometric analysis of blots showing ratio of Mfa/FimA. (D) ELISA of formalin-fixed whole cells of wild-type (WT; 33277), clpB, clpC, and clpXP strains probed with Mfa or FimA antibodies. P. gingivalis mutants deficient in FimA (FimA) or Mfa (Mfa) were used as a control for antibody specificity. * denotes P values of <0.05 (t test) for comparison to the WT. (E) Quantitative real-time RT-PCR of mfa and fimA mRNA levels in WT and ClpXP-deficient strains.

Differential expression of Mfa modulates P. gingivalis invasion. The Mfa fimbrial subunit protein is essential for heterotypic biofilm formation by P. gingivalis (37, 52). However, the function of Mfa in invasion of GEC by P. gingivalis has not been investigated. As the clpXP mutant expresses higher levels of Mfa and exhibits reduced invasion, we hypothesized that Mfa may be detrimental to invasion, and thus, an Mfa-deficient mutant would invade GEC more efficiently. Hence, the relative levels of invasion of mfa-null mutant and complemented mutant strains (52) were tested. As shown in Fig. 9, in the absence of Mfa, invasion of P. gingivalis was significantly enhanced, and this effect was reversed in the complemented strain. These results suggest that the defect in invasion exhibited by the clpXP mutant is a consequence of the higher levels of surface Mfa expression.

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FIG. 9. Invasion of GECs is enhanced in the absence of Mfa. GECs (1 x 105) were infected with P. gingivalis strains (1 x 107 bacterial cells) for 2 h. Mfa is an mfa mutant, and CMfa is the mutant complemented with the wild-type (WT) mfa gene (52). Extracellular bacteria were killed with gentamicin and metronidazole, and intracellular bacteria released by lysis of the GECs. Numbers (CFU) of intracellular bacteria were determined by viable counting of the cell lysates. Results are expressed as percentages of input bacteria that were recovered intracellularly. * denotes P values of <0.01 (t test) for comparison to the WT (33277).

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Clp proteolytic complexes, consisting of a proteolytic core and associated ATPases, are a highly conserved bacterial stress response system. In addition, Clp proteins are required for the optimal expression of virulence in a number of pathogens (5, 17, 22). Despite overall homology, the numbers and types of Clp ATPases vary among organisms, and different species appear to utilize the individual components of the Clp system according to their unique environmental requirements. The oral anaerobic opportunistic pathogen P. gingivalis possesses the ClpP proteolytic subunit along with the ATPases ClpB, ClpC, and ClpX. To investigate the role of the Clp system in the stress responses and expression of virulence-associated phenotypes of the organism, mutants were constructed and complemented and the resulting strains tested for growth under a variety of stress conditions, the ability to form biofilms, and the capacity for invasion of epithelial cells.

While all the major families of HSPs are present in P. gingivalis, the functionality of these proteins in resistance to thermal stress has yet to be defined. In this study, we demonstrated that ClpC and ClpXP were required for optimal growth of P. gingivalis at a high temperature. ClpC and ClpP have also been shown to be required for growth of S. aureus at a high temperature (16), and ClpXP is important for survival of S. aureus under osmotic, oxidative, and cold stress. Loss of ClpXP did not affect resistance to oxidative stress in P. gingivalis; however, as a strict anaerobe, P. gingivalis possesses multiple mechanisms for responding to oxidative stress, including those involving superoxide dismutase, alkyl hydroperoxide reductase, rubrerythrin, HtrA, Dps, and the manganese transporter FeoB2 (28, 46, 49, 50, 56, 65), some of which are controlled by the transcriptional regulator OxyR (15). Hence, with multiple redundancy, a mutational analysis may not reveal individual contributions to oxidative-stress resistance. Loss of ClpB did not affect heat tolerance in P. gingivalis 33277, although ClpB does play role in adaptation to thermal stress in E. coli, S. aureus, and L. monocytogenes (7, 16, 62). However, in addition to HSPs, universal stress protein A (UspA) (8) and the periplasmic serine protease HtrA (56) have been shown to contribute to heat tolerance in P. gingivalis. UspA and HtrA could thus compensate for loss of ClpB or allow ClpB to perform more exclusive roles in P. gingivalis, as is discussed further below.

The transition of bacteria from planktonic to biofilm mode often involves multiple adaptive responses, including stress responses. In the absence of ClpC and ClpXP, monospecies biofilm formation by P. gingivalis was enhanced, in terms of both total accumulation and biofilm height. While the current study did not address the in vivo consequences of the in vitro increase in biofilm formation, the activities of ClpC and ClpXP can be predicted to regulate the biofilm-forming potential of P. gingivalis in vivo. Indeed, control of biofilm development in bacteria involves complex multilevel regulatory pathways that can either stimulate an increase in biomass or limit or stabilize biofilm accumulation according to environmental conditions. For example, in S. aureus, mutation of clpP increases biofilm formation (16). In contrast, the transcription factor RpoS limits P. aeruginosa biofilm depth (23, 68), and RpoS production is regulated at multiple levels, including transcription, translation, and proteolysis, in response to stress conditions (66). Activities controlled by LuxS, the AI-2 synthase, also repress biofilm formation by Staphylococcus epidermidis and Helicobacter pylori (9, 71). In P. gingivalis, control of biofilm accumulation has also been shown to require the leucine-rich surface protein InlJ (6), the cytoplasmic low-molecular-weight tyrosine phosphatase PtpA, and FtsH, an integral membrane zinc metallopeptidase (60). Similar to ClpC and ClpX, FtsH is an AAA+ ATPase that is universally conserved in bacteria (26). Thus, the activities of a range of ATPase-dependent proteases in P. gingivalis are important for optimizing biofilm potential. The extent to which this potential is realized will depend on other bacterial, environmental, and contextual factors.

In vivo, oral biofilms are complex multispecies communities that develop through a variety of coadhesive, nutritional, metabolic, and signaling interactions among the constituent organisms (27, 54). P. gingivalis accumulates into heterotypic biofilm colonies with S. gordonii as a result of interbacterial attachment mediated by the long and short fimbriae of P. gingivalis, which interact with streptococcal GAPDH and Ssp surface proteins, respectively (43, 52). Binding of the short fimbrial protein Mfa to SspB provides the stimulus for biofilm accumulation, and this interaction is driven by an approximately 27-amino-acid binding domain of SspB termed BAR (4, 14). The binding and biofilm-inducing characteristics of BAR are dictated by the electrostatic and hydrophobic properties of a motif comprising the amino acid sequence NITVK (12). Similar to what was found for the monospecies biofilms, loss of ClpXP resulted in more abundant mixed P. gingivalis biofilms. Notably, the clpXP operon is not regulated at the transcriptional level in P. gingivalis-S. gordonii biofilms (60); hence, normal levels of these proteins are generally sufficient to control biofilm development. In contrast, loss of ClpB, while not significantly affecting total P. gingivalis accumulation, resulted in fewer, larger microcolonies. ClpB, which is thought not to associate with ClpP, may therefore have a more specific role in regulation of biofilm accumulation in the presence of S. gordonii. Considered together, these results indicate that unrestricted biofilm accumulation may be detrimental to P. gingivalis. The biofilms that accumulate in the oral cavity will experience fluctuations in nutrient availability and oxygen tension. Bacterial inhabitants, therefore, will be maximally competitive if they can optimize oxygen exposure and nutrient availability throughout their biofilm structures. Such constraints would provide for optimal biofilm proportions that vary among organisms and for the consequent development of pathways, such as those mediated by the Clp system in P. gingivalis, to ensure that biofilms do not exceed the optimal size.

P. gingivalis is a facultative intracellular pathogen that can survive and replicate inside GECs. An intracellular location may protect the organism from immune activity and from antibiotics and may thus provide a reservoir of P. gingivalis that could lead to disease recrudescence. The invasion process is a multistep procedure that requires fimbria-mediated attachment of P. gingivalis to integrin receptors, subsequent signal transduction events that lead to cytoskeletal remodeling, and entry of the organism into the cytoplasm (36, 38, 72). Around 40% of the expressed proteome of P. gingivalis is differentially regulated as the organism adapts to an intracellular location (75). Among the regulated proteins are ClpC and ClpXP, which show elevated expression in the presence of GEC components (75). Here, we found that the absence of ClpB, ClpC, or ClpXP significantly impaired the invasive capacity of P. gingivalis. While adhesion of all the mutant strains was normal, clpC and clpXP mutants were defective in entry into GECs. Thus, Clp proteolytic activity may be required for processing of proteins that are involved in the internalization process. A similar situation occurs with Salmonella, which requires the ClpXP protease to degrade FlhD and FlhC, master regulators of the synthesis of flagellum that are involved in entry into epithelial cells (58, 63). In contrast, ClpB seems to play no role in the entry of P. gingivalis into epithelial cells but is important for intracellular survival. Similarly, S. aureus cells lacking the ClpB chaperone are unable to replicate intracellularly in bovine T cells (16). ClpB is not directly involved in protein degradation (70); rather, in cooperation with DnaK/DnaJ, ClpB solubilizes protein aggregates (20, 45). Thus, the importance of ClpB for intracellular survival and for maintaining heterotypic biofilm architecture may be related to its ability to disaggregate and reactivate aggregated protein effectors.

The short fimbriae of P. gingivalis are 0.1 to 0.5 µm in length and are composed predominantly of the Mfa protein subunit. Mutants of P. gingivalis that lack Mfa show impaired formation of both monospecies biofilms (42) and heterotypic biofilms with S. gordonii (52). Consistent with this, in the absence of ClpXP, expression of Mfa was increased. Thus, the higher levels of homotypic and heterotypic biofilm formation in the clpXP mutant may result from elevated surface exposure of Mfa. Conversely, as loss of Mfa enhanced invasion, the increased expression of Mfa in the clpXP mutant could be the cause of the reduced invasion by this strain. We speculate that more abundant Mfa protein on the cell surface may disrupt physical parameters of FimA fimbrial surface presentation and impede the ability of the FimA fimbriae to activate their cognate integrin receptors. Upregulation of Mfa expression was not accompanied by an increase in mfa transcriptional activity. Hence, one possible mechanism by which ClpXP restricts Mfa expression may be through degradation of an inhibitor of Mfa synthesis or transport.

 
This work was supported by grants DE12505, DE11111, and DE14605 from the NIDCR.

 
* Corresponding author. Mailing address: Department of Oral Biology, College of Dentistry, University of Florida, Gainesville, FL 32610-0424. Phone: (352) 392-5067. Fax: (352) 392-2361. E-mail: rlamont{at}dental.ufl.edu

Published ahead of print on 7 December 2007.

Present address: Department of Periodontics, Dental Branch, University of Texas Health Sciences Center at Houston, Houston, TX.

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Journal of Bacteriology, February 2008, p. 1436-1446, Vol. 190, No. 4
0021-9193/08/$08.00+0     doi:10.1128/JB.01632-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

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