The Porphyromonas gingivalis clpB gene is involved in cellular invasion in vitro and virulence in vivo

Authors


  • Present address: Lihui Yuan, Department of Physiology, College of Medicine, University of Florida, Gainesville, FL 32610, USA.

  • Editor: Patrik Bavoil

Correspondence: Ann Progulske-Fox, Department of Oral Biology, Center for Molecular Microbiology, College of Dentistry, University of Florida, PO Box 100424, Gainesville, FL 32610-0424, USA. Tel.: +1 352 846 0770; fax: +1 352 392 2361; e-mail: apfox@dental.ufl.edu

Abstract

ClpB, a component of stress response in microorganisms, serves as a chaperone, preventing protein aggregation and assisting in the refolding of denatured proteins. A clpB mutant of Porphyromonas gingivalis W83 demonstrated increased sensitivity to heat stress, but not to hydrogen peroxide and extreme pHs. In KB cells, human coronary artery endothelial (HCAE) cells and gingival epithelial cells, the clpB mutant exhibited significantly decreased invasion suggesting that the ClpB protein is involved in cellular invasion. Transmission electron microscopic analysis showed that the clpB mutant was more susceptible to intracellular killing than the wild-type strain in HCAE cells. The global genetic profile of the clpB mutant showed that 136 genes belonging to several different cellular function groups were differentially regulated, suggesting that ClpB is ultimately involved in the expression of multiple P. gingivalis genes. A competition assay in which a mixture of wild-type W83 and the clpB mutant were injected into mice demonstrated that the clpB mutant did not survive as well as the wild type. Additionally, mice treated with the clpB mutant alone survived significantly better than those treated with the wild-type strain. Collectively, these data suggest that ClpB, either directly or indirectly, plays an important role in P. gingivalis virulence.

Introduction

Pathogens may encounter multiple stresses while infecting hosts, including elevated temperatures (caused by fever) (Small et al., 1986), oxidative stress (Springer et al., 2001), and pH stress (Horwitz & Maxfield, 1984; Porte et al., 1999). The bacteria must overcome the harsh environment to colonize or invade the host and cause disease. This necessitates the production of stress proteins, which are believed to be bacterial virulence factors.

Stresses can cause cellular proteins to denature and form stable insoluble aggregates that are devoid of biological activity (Jaenicke, 1995). Because misfolding and aggregation of proteins are two of the major threats to all living organisms, bacterial cells have developed an elaborate protection system that includes molecular chaperones. Chaperones bind to denatured proteins and direct the proteins to regain their native tertiary structure, so that protein function can be restored.

Molecular chaperones, most of which are stress inducible, include several distinct families of proteins. ClpB is one member of the Hsp100 family and plays a key role in cell survival during stress. The Hsp100/ClpB chaperone belongs to the AAA+ protein superfamily, which generally drives the assembly and disassembly of protein complexes in an ATP-dependent manner. ClpB belongs to a family of ATPases (Clp ATPases), which are involved in protein degradation and disaggregation. The other members of this family include: ClpA, ClpC, ClpE, ClpL, ClpV, ClpX, and ClpY (Butler et al., 2006). However, the function of ClpB is distinct from that of the other Clp ATPases as it is not involved in protein degradation, but instead is involved in disaggregation of protein complexes, in a cooperative fashion with chaperones DnaK, DnaJ, and GrpE (Motohashi et al., 1999).

In Saccharomyces cerevisiae, ClpB was found to rescue aggregated proteins in cooperation with Hsp70/DnaK (Glover & Lindquist, 1998; Krzewska et al., 2001; Lee et al., 2003). An Escherichia coli clpB mutant was found to contain an increased amount of insoluble proteins, indicating the importance of ClpB in the removal of the heat-aggregated proteins in the cell. In addition, a Helicobacter pylori clpB mutant showed increased thermosensitivity (Allan et al., 1998). A Brucella suis clpB mutant not only showed increased sensitivity to high temperature, but also to other stress conditions such as ethanol stress and acid pH (Ekaza et al., 2001). Mutation of clpB was also found to decrease virulence in both Salmonella typhimurium (Turner et al., 1998) and Listeria monocytogenes (Chastanet et al., 2004). A mutation in clpB also exhibited decreased virulence in Leishmania donovani, both in vivo and in vitro (Clos et al., 2001).

Porphyromonas gingivalis is a gram-negative, anaerobic, black pigmented bacterium that is considered to be one of the major etiologic agents of periodontal disease (Socransky & Haffajee, 1992). Periodontal diseases, including gingivitis and periodontitis, are serious infections that can lead to periapical bone and tooth loss. Porphyromonas gingivalis is found in subgingival plaque in periodontal pockets that have temperatures higher than normal body temperature and varying levels of oxygen (Mettraux et al., 1984; Kung et al., 1990; Meyerov et al., 1991; Fedi & Killoy, 1992; Lindskog et al., 1994; Tanaka et al., 1998). This environment presents the bacteria with adverse conditions that they must overcome in order to establish a niche within the host. Additionally, P. gingivalis has the ability to attach and to invade KB (a human epidermoid carcinoma), epithelial, endothelial, and dendritic cells (Lamont et al., 1992; Duncan et al., 1993; Sandros et al., 1993, 1994; Deshpande et al., 1998; Cutler et al., 1999; Takahashi et al., 2006). The ability of P. gingivalis to invade human coronary artery endothelial (HCAE) cells and coronary artery smooth muscle cells has also been previously reported (Dorn et al., 1999). Moreover, P. gingivalis was found to be internalized in oral epithelial cells in vivo (Rudney et al., 2001). Given the environmental conditions that P. gingivalis encounters in vivo, it is likely that the expression of heat shock proteins and their subsequent assistance in refolding of denatured proteins are important for P. gingivalis survival and virulence.

In a previous study, the clpB gene (as annotated in the P. gingivalis database-TIGR with locus ID PG1118) was determined to be up-regulated fivefold in a luxS mutant of P. gingivalis compared with W83 (Yuan et al., 2005). The purpose of this research was to investigate the importance of clpB in P. gingivalis virulence.

Materials and methods

Bacterial and cell culture conditions

Porphyromonas gingivalis wild-type W83 was grown on blood agar plates (BAPs) consisting of 4% (w/v) trypticase soy agar (Difco Laboratories, Franklin Lakes, NJ), 0.5% (w/v) yeast extract (Difco Laboratories), 5% (v/v) sheep blood (Lampire Biological Laboratories, Pipersville, PA), 5 μg hemin mL−1 (Sigma, St Louis, MO), and 1 μg vitamin K1 mL−1 (Sigma). Porphyromonas gingivalis was also cultured in trypticase soy broth (TSB) supplemented with 5 μg of hemin and 1 μg vitamin K1 mL−1. Cells were grown and maintained at 37 °C in an anaerobic chamber (Coy Manufacturing, Ann Arbor, MI) containing an atmosphere of 85% (v/v) N2, 10% (v/v) H2, and 5% (v/v) CO2. The P. gingivalis W83 clpB mutant strain LY2003 was maintained as described for the wild-type strain, except that 5 μg mL−1 of clindamycin (Sigma) was added to the medium to maintain the antibiotic selective pressure.

Mutant construction

PCR was used to generate a 573-bp internal fragment (from position 580 to 1152) of the clpB gene (total gene size is 2592 bp) using the primer clpBF1: 5′-AAAAGGATCCCGACGCACCAAGAACA-3′ and clpBB1: 5′-AAACTGCAGCAGGAACCGCTCCGTAA-3′ (underlined sequences indicate the BamHI and PstI cutting sites, respectively). The DNA fragment was then cloned into a P. gingivalis suicide vector, pVA3000 (Lee et al., 1996), using the BamHI and PstI sites. Escherichia coli strain S17-1 was used to deliver the vector containing the internal fragment into P. gingivalis W83 by conjugation on blood agar plates. Resulting transconjugants were selected on plates containing clindamycin (5 μg mL−1). Homologous recombination of the vector with the chromosome resulted in two copies of the gene that were truncated at either the 5′ or the 3′ end. Southern blot analysis was carried out to confirm the creation of the clpB mutant. An ECL detection kit was used according to the manufacturer's protocol for Southern blot analysis (Amersham Biosciences Corp., Piscataway, NY).

Stress experiments

For all stress-related experiments, the W83 and LY2003 strains were cultured in supplemented TSB with gentamicin 50 μg mL−1 until they reached the mid exponential phase.

Temperature-induced stress

One milliliter of each of W83 and LY2003 cultures was centrifuged at 15 600 g for 2 min. The cell pellets were then washed using 1.0 mL of 0.1 M glycine buffer (pH 7), resuspended in 1.0 mL of the same buffer, and incubated at 50 °C. Aliquots were removed at 0 and 8 min.

Hydrogen peroxide (H2O2)-induced stress

Ten milliliters of each of the W83 and LY2003 cultures were centrifuged at 4000 g for 10 min at 4 °C. The pellets were washed with 10 mL of 0.1 M glycine buffer (pH 7) and centrifuged at 4000 g for 10 min. Then the pellets were resuspended in 0.1 M glycine buffer containing 0.35 mM H2O2. Aliquots were taken at 0, 15 and 30 min.

pH-induced stress

Bacterial cells were treated as described above but pellets were resuspended in 0.1 M glycine buffer (pH ranges from 3 to 10 in 1 U increments). Aliquots were taken at 30 min, 45 min, 1 h, and 2 h.

For all stress experiments, decimal dilutions were performed with the aliquots taken, plated on BAPs and CFU enumerated after 7–10 days incubation. Percent survival was calculated by comparison with the number of CFUs in the inoculum.

Cell cultures

All types of cells were cultured in 75 cm2 flasks at 37 °C in a humidified atmosphere with 5% (v/v) CO2. Confluent monolayers were split by treating with trypsin-versene (Bio Whittaker, East Rutherford, NJ). KB cells (ATCC CCL-17) were maintained in Dulbecco's modified Eagle's medium (DMEM) (ATCC, Manassas, VA) supplemented with 10% (v/v) fetal bovine serum (Invitrogen, Carlsbad, CA) and 100 mg mL−1 of penicillin–streptomycin (Invitrogen). Human coronary artery endothelial (HCAE) cells, a primary cell line (Cambrex, East Rutherford, NJ), were maintained in EBM-2 medium supplemented with EGM-2-MV single-use aliquots (Cambrex). Gingival epithelial (GE) cells (kindly provided by Dr Richard Lamont and obtained from healthy gingival tissues from patients undergoing surgery for removal of impacted third molars) were cultured in keratinocyte basal medium (KBM) supplemented with insulin, triiodothyronine (T3), transferrin, recombinant human epidermal growth factor (rhEGF), epinephrine, bovine pituitary extracts (BPE), and hydrocortisone.

Cell invasion assays

Invasion experiments were carried out in 24-well plates in which 105 cells were plated at the bottom of each well. The bacteria were then added to the wells at an multiplicity of infection of 100 : 1 and an antibiotic protection assay for P. gingivalis was performed as described previously (Lamont et al., 1995). Briefly, the bacteria and the cells were incubated at 37 °C for 90 min, at which time the wells used for the total interaction assay (bacteria that adhered plus those that invaded) were washed with 1 × phosphate-buffered saline (PBS) for three times, followed by lysis of the cells with sterile distilled water, and plating of serial dilutions. For wells used for the invasion assay (number of intracellular bacteria), the external adherent bacteria were killed by the addition of 300 μg mL−1 gentamicin and 200 μg mL−1 metronidazole for 1 h. The cells were then washed with PBS, lysed, and serial 10-fold dilutions plated. The experiments were repeated at least twice, each time using three wells for each bacterial strain.

Data normalization and statistical analysis

The cellular interaction and invasion data were normalized with their inoculums and were analyzed using Student's t-test. Results were considered statistically significant with P value ≤0.05.

Transmission electron microscopy

HCAE cells were infected with the wild-type P. gingivalis or the clpB mutant as described above except for that the bacteria remained in contact with the cells for 6 h. The infected cells were then fixed with 2% glutaraldehyde/2% paraformaldehyde/2 mM CaCl2/0.1 M sodium cacodylate, pH 7.4, and acid phosphatase activity was localized using cytidine 5′-monophosphate and cerium chloride as described by Robinson (1985). Specimens were post fixed in 1% osmium tetroxide in 0.1 M sodium cacodylate, dehydrated, and embedded in Epon 812 resin. Thin sections (60–80 nm) were cut and examined on a JEOL 100CX transmission electron microscope.

RNA preparations

Porphyromonas gingivalis wild-type W83 and the clpB mutant strain, LY2003, were grown in 40 mL of TSB to an OD of 2.0 at 600 nm. The bacterial cells were then collected by centrifugation at 4 °C and processed immediately for RNA extraction. Total RNA was isolated from independent quadruplicate broth cultures with 10 mL of Trizol reagent (Invitrogen). Bacterial RNA was subsequently isolated following the protocol described by the manufacturer. All RNA samples were treated with RNAse-free DNAse I (Ambion, Austin, TX) and purified using the RNeasy kit (Qiagen Inc., Valencia, CA) according to the manufacturer's instructions for the optional DNAse in-column treatment. Reference RNA (refRNA) was isolated from W83 cells grown in TSB to an OD of 0.5, 1.0, 1.5, and 2.0 at 600 nm. This refRNA was further used in the microarray hybridizations. The refRNA was purified as above, aliquoted, and stored at −80 °C until further use. All RNA preparations were tested to confirm the absence of DNA using conventional PCR with primers for the P. gingivalis gene ftn (ferritin – locus number PG1286, http://cmr.tigr.org/tigr-scripts/CMR/shared/GenePage.cgi?locus=PG_1286).

cDNA synthesis and preparation of probes

Reverse transcription reactions were performed according to the protocol provided by The Institute of Genomic Research (TIGR) at http://pfgrc.tigr.org/protocols.shtml with the following minor modifications: Superscript III (Invitrogen) was used as the reverse transcriptase, the amount of RNA used was increased to 5.0 μg, and the molar ratio of aa-dUTP : dTTP used was 2 : 1. Purified W83 and LY2003 cDNAs were then coupled with indocarbocyanine (Cy3)-dUTP, while reference cDNAs were coupled with indodicarbocyanine (Cy5)-dUTP (Amersham).

Microarray hybridizations

Porphyromonas gingivalis W83 microarray glass slides (version 2) were kindly provided by TIGR. The microarrays consisted of 1907 70-mer oligonucleotides representing 1990 ORFs. The full 70-mer complement is printed four times on the surface of each microarray slide. Additional details regarding the arrays are available at http://pfgrc.tigr.org/slide_html/array_descriptions/P_gingivalis_2.shtml. Four individual Cy3-labeled cDNA samples, originating from four different cultures of W83 or LY2003 grown in broth without antibiotics, were hybridized to the arrays along with Cy5-labeled reference cDNA samples, for a total of eight slides. With this approach, dye swap is not necessary. Hybridizations were carried out in an MAUI 4-bay hybridization system (BioMicro Systems Inc., Salt Lake City, UT) for 16 h at 42 °C. The slides were then washed according to TIGR protocols and scanned using a GenePix scanner (Axon Instruments Inc., Union City, CA) at 532 nm (Cy3 channel) and 635 nm (Cy5 channel) with a Cy3 : Cy5 ratio of 1 : 1.

Microarray data analysis

Sixteen-bit TIFF single-channel images were stored and later loaded into tigr spotfinder software (http://www.tm4.org/spotfinder.html). The intensity values of each spot were measured and then normalized using LOWESS (LocFit Normalization – mode: global) and Interlog Mean (Interactive Log Mean Centering Normalization) using the default settings, but with the following modification: one bad channel tolerance: generous; followed by in-slide replicate analysis using tigrmidas software (http://www.tm4.org/midas.html). Spots that were flagged as faulty, owing to either low-intensity values or signal saturation, were automatically discarded. Final statistical analysis was carried out using BRB array tools (http://linus.nci.nih.gov/BRB-ArrayTools.html). A total of 1902 genes passed the filtering criteria after the analysis by the brb software. Class prediction was then, determined at P value of 0.005. Therefore, at this significance level, there are possible 9.5 false-positive genes.

Virulence studies

In order to test the mutant strain for alterations in virulence relative to the wild type, a competition assay was carried out using 30 BALB/c mice as described previously (Wu et al., 2002). The mice were injected subcutaneously with a 1 : 1 ratio of P. gingivalis W83 and LY2003 strains as follows: overnight cultures were pelleted and resuspended in PBS so that 2.5 × 109 cells strain−1 in a total volume of 100 μL were injected into each mouse. The surviving bacteria were then recovered from the lesion sites from six mice daily for 5 days. Decimally diluted samples were spread on BAPs with 50 μg mL−1 gentamicin. Colonies that arose after 1 week of anaerobic incubation were replica patched onto BAPs with or without 5 μg mL−1 clindamycin. After incubation for 3 days in the anaerobic chamber, the percentage of clindamycin-resistant colonies (mutant strain) was determined.

In addition to survival in vivo, the virulence of the LY2003 mutant strain was compared with P. gingivalis W83 in a separate experiment in which eight mice in a group of 16 were challenged with subcutaneous injections of each individual bacterial strain alone at a dose of 7.5 × 109 bacteria per mouse. Mice were then examined daily for 5 days to assess their general health status and the number of dead animals. Mouse survival curves were created using graphpad software. The log-rank test was used for statistical analysis and the P value was set at 0.05 (graphpad Software, San Diego, CA).

Results

Heat stress response of W83 and LY2003

Strains W83 and LY2003 were subjected to 50 °C for 8 min. The percentage of survival was then calculated based on the number that survived compared with the inoculum. After 8 min at 50 °C, W83 showed significantly higher survival than did the clpB mutant, LY2003: 23.9% survival for W83, but only 4.3% survival for LY2003 (P=0.02). This result indicates that in P. gingivalis, ClpB is at least partially responsible for survival under conditions of heat stress.

H2O2 stress experiments

W83 and LY2003 strains were also stressed using 0.35 mM H2O2 in 0.1 M glycine buffer for 15 and 30 min. Comparison of the percentage survival of the wild type and the mutant revealed that the clpB mutant LY2003 had decreased survival at both time points (11.7% for LY2003 and 21.9% for W83 at 15 min, 0.8% for LY2003 and 1.9% for W83 at 30 min), but the difference was not statistically significant (P=0.14 at 15 min and P=0.18 for 30 min).

pH stress experiments

W83 and the clpB mutant were stressed at various pH from 3 to 10 at 1 U increments in 0.1 M glycine buffer for 30 min, 45 min, 1 h, and 2 h. The clpB mutant showed no statistical difference in survival at any of the time points tested when compared with the W83 strain (P>0.05, data not shown).

Total interaction and invasion with different cell types

KB cells, primary HCAE cells, and primary human GE cells (Lamont et al., 1995) were used in this study. The E. coli strain MC1061 was used as a noninvasive control (Dorn et al., 1999).

KB cells

Total interaction for this study is defined as the number of adherent plus the number of intracellular (invaded) bacterial cells at the time the host cells were lysed. The results showed that the clpB mutant had a significantly lower total interaction with KB cells than did the wild type, since the total interaction from the clpB mutant LY2003 was five times less than that of W83 (P<0.01) (Fig. 1a).

Figure 1.

 Total interaction with and invasion of various cell types as determined using a standard antibiotic protection assay. Values in the figure represent the means and SDs for triplicate samples of lysates from the infection of 105 cells by 107 bacteria from at least two independent experiments. W83: wild type; LY2003: clpB mutant; MC1061: Escherichia coli strain used as a noninvasive control. (a) Total interaction with KB cells (P<0.01). (b) Invasion of KB cells (P<0.05). (c) Total interaction with HCAE cells (P<0.001). (d) Invasion of HCAE cells (P<0.001). (e) Total interaction with GE cells (P<0.05). (f) Invasion of GE cells (P<0.005). HCAE, human coronary artery endothelial; GE, gingival epithelial.

The difference between the two strains with respect to invasion was even greater. The number of intracellular LY2003 was 60-fold less than the number of intracellular W83; 0.005% of the LY2003 inoculum and 0.3% of the W83 inoculum invaded the KB cells (P<0.05) (Fig. 1b).

HCAE cells

The total interaction of LY2003 with HCAE cells was three times less than that of W83 (Fig. 1c), as only 0.08% of the LY2003 inoculum was recovered compared with 0.24% of the W83 strain (P<0.001). Similar to invasion of KB cells, the difference in invasion of HCAE cells was even greater than that obtained from the total interaction assay; LY2003 invaded 15 times less than did W83 (P<0.001) (Fig. 1d).

GE cells

The total interaction of W83 with GE cells produced results similar to those obtained using HCAE cells in that the wild-type interaction was twice as much as LY2003 (P<0.05) (Fig. 1e). Most significantly, invasion by W83 and LY2003 showed the greatest difference in GE cells; LY2003 invaded 194 times less than W83 (P<0.005) (Fig. 1f).

Microscopy

After 6 h of infection, both P. gingivalis W83 (Fig. 2a) and the clpB mutant (Fig. 2b) were found within HCAE cell vacuoles. The presence of autophagic vacuoles and autolysosomes in the infected HCAE cells suggested that both bacterial strains were capable of promoting autophagy. W83 was localized predominantly in vacuoles that contained multiple bacteria but lacked the lysosomal enzyme, acid phosphatase (Fig. 2a). However, the clpB mutant was found in vacuoles containing acid phosphatase and appeared to be in various stages of degradation (Fig. 2b).

Figure 2.

 Internalized Porphyromonas gingivalis in HCAE cells. HCAE cells were infected with P. gingivalis W83 (a) or the clpB mutant (b) for 6 h. Porphyromonas gingivalis could be found in vacuoles that either contained (black arrow) or lacked (white arrow) acid phosphatase. Nascent autophagic vacuoles (AV) that not had yet acquired acid phosphatase and mature autolysosomes (AL) that contained acid phosphatase were observed in cells infected with either wild-type or mutant P. gingivalis. Scale bar=1 μm. HCAE, human coronary artery endothelial.

Microarray experiments

The microarray experiments indicated that a total of 136 genes (ORFs) were differentially regulated in the clpB mutant at a significance level of P<0.005. Eighty-three genes were up-regulated and 53 genes were down-regulated. The genes were grouped into classes according to cellular roles as established in the TIGR P. gingivalis genome database (Table 1). Thus, the absence of ClpB significantly altered the expression of a large number of genes in P. gingivalis. Examples of these are genes involved in cell envelope composition, such as biosynthesis and degradation of surface polysaccharides and lipopolysaccharides, transport of proteins and lipoproteins. Additionally, the hemagglutinin HagB (PG1972), an established virulence factor involved in the adherence of P. gingivalis to HCAE cells (Song et al., 2005), was down-regulated in the mutant (2.3-fold). Interestingly, the heat shock proteins GrpE (PG1775) and DnaJ (PG1776) were also down-regulated in the ClpB mutant (13.4- and 11.0-fold, respectively). Moreover, a chaperone belonging to the FKBP family, SlyD, (PG1315) was found to be down-regulated (8.9-fold). However, two other members of this chaperone family, FkpA (PG0709) and trigger factor (PG0762), were found to be up-regulated (4.0- and 10.3-fold, respectively). Additionally, genes ompH1 (PG0192), ompH2 (PG0193), and tetR (PG1240) were down-regulated in the mutant. Interestingly, grpE (PG1775), dnaJ (PG1776), slyD (PG1315), ompH1 (PG0192), ompH2 (PG0193), and tetR (PG1240) have all been previously reported to be up-regulated during contact of P. gingivalis with epithelial cells (Hosogi & Duncan, 2005). In addition, a hypothetical protein (PG0419), also described as up-regulated upon contact of P. gingivalis with epithelial cells (Hosogi & Duncan, 2005), was down-regulated in the mutant. A summary of differentially regulated genes in the clpB mutant is shown in Table 2. No polar effects were observed due to the clpB inactivation, because no change in the expression of flanking genes could be found after analysis of the microarray data.

Table 1.   Genes (ORFs) differentially regulated in the Porphyromonas gingivalis clpB mutant as determined by microarray analysis
Gene categoriesNumber of genes
Up-regulatedDown-regulated
  1. The genes were grouped into functional classes based on their regulation pattern. The change in expression for all listed genes is a minimum of twofold difference at a significance level of P<0.005.

  2. ORF, open reading frame.

Amino acid biosynthesis10
Biosynthesis of cofactors, prosthetic groups, and carriers31
Cell envelope63
Cellular processes01
Central intermediary metabolism20
Conserved hypothetical and hypothetical protein2320
DNA metabolism21
Energy metabolism121
Fatty acid and phospholipid metabolism30
Mobile and extrachromosomal element functions43
Protein fate34
Protein synthesis70
Purines, pyrimidines, nucleosides, and nucleotides33
Regulatory functions22
Signal transduction00
Transcription15
Transport and binding proteins42
Unknown function77
Table 2.   Examples of differentially regulated genes in the Porphyromonas gingivalis clpB mutant as determined by microarray analysis
Locus*Gene*Common name*Cellular role*Fold expressionParametric value
PG0192ompH1Cationic outer membrane proteinCell envelope−5.50.000257
PG0193ompH2Cationic outer membrane proteinCell envelope−11.54.9 × 10−5
PG0350 Internalin-related proteinUnknown function+12.00.001643
PG0419 Conserved hypothetical proteinHypothetical proteins−2.90.003167
PG0709fkpAFKBP-type peptidylprolyl isomeraseProtein fate+4.00.001707
PG0762 Trigger factorProtein fate+10.31.0 × 10−6
PG1240tetRTranscriptional regulator −4.00.00085
PG1315slyDFKBP-type peptidylprolyl isomeraseProtein fate−8.90.004202
PG1775grpEHeat-shock proteinProtein fate−13.40.000566
PG1776dnaJHeat-shock protein 40Protein fate−11.01.0 × 10−6
PG1777 Conserved hypothetical proteinHypothetical proteins−9.90.001034
PG1778 Conserved hypothetical proteinHypothetical proteins−40.32.1 × 10−5
PG1779 Conserved hypothetical proteinHypothetical proteins−39.81.0 × 10−5
PG1972hagBHemagglutinin proteinPathogenesis−2.30.001893
PG2173omp28Outer membrane lipoproteinCell envelope+5.40.001523

Competition assay

The in vitro growth rates of W83 and LY2003 were found to be the same (data not shown). This indicated that integration of the vector into the P. gingivalis chromosome did not adversely affect the growth rate of the clpB mutant. The mutant was then tested for alterations of virulence properties compared with the wild type in a mouse abscess model. A group of 30 BALB/c mice 7–8-weeks old was injected subcutaneously with a 1 : 1 ratio of wild type vs. the mutant. Each day after injection and continuing for 5 days, six mice from each group were sacrificed and the subcutaneous area with the bacterial mixture was aspirated. CFU were then enumerated by plating serial dilutions. For day 1 and day 2 after the injections, LY2003 comprised 60% and 65.4%, respectively, of the total bacteria recovered (Fig. 3). The Student's t-test showed that the differences were statistically significant. But starting from day 3, the percentage of LY2003 dropped to 30.4%, 33%, and 35.4% for subsequent days. Differences were found to be statistically significant compared with the inoculum, which was set at 50%. These data indicated that LY2003 was much less able to survive beginning at day 3 in mice than the wild-type strain.

Figure 3.

 Virulence of the clpB mutant LY2003 compared with the wild-type W83 strain in a competition assay. One group of 30 BALB/c mice was injected subcutaneously with a 1 : 1 ratio of Porphyromonas gingivalis strain LY2003 and W83. The surviving bacteria were then recovered from the lesion sites from six mice in each group daily for 5 days. The percentage of mutant cells from the total number of cells recovered was then determined.

Virulence study

In a second set of experiments using a mouse abscess model, two groups of eight mice were challenged with either W83 or LY2003. The mice were then examined daily for 5 days to record the number of dead animals. In addition, six mice treated with W83 died by day 3 but only one mouse in the group infected with LY2003 died by day 3 (Fig. 4). The results remained the same through day 5. A log rank test was used for the statistical analysis of the survival curve. This test generates a P value testing the null hypothesis that the survival curves are identical in the overall populations. This analysis indicated the difference of survival between the W83- and LY2003-treated mice was significant (P=0.03). This result clearly indicated that, compared with W83, LY2003 has significantly reduced virulence in vivo.

Figure 4.

 The percentage survival of the mice treated with Porphyromonas gingivalis W83 and the clpB mutant LY2003. All mice (eight mice per group) were checked daily for survival. The graph was created with graphpad prism software and the significant analysis on the survival data was carried out using log rank test, P=0.03.

Discussion

In the periodontal pocket, P. gingivalis survives various environmental stresses during the progression of the periodontal disease (Kung et al., 1990; Meyerov et al., 1991; Fedi & Killoy, 1992; Lindskog et al., 1994). Among others, this pathogen must be able to survive and persist under heat stress. Consequently, heat-shock proteins, such as ClpB, may allow P. gingivalis to better survive in the inflamed periodontal pocket. In this study, it was shown that ClpB is important in P. gingivalis cellular invasion in vitro and P. gingivalis virulence in vivo. It was also demonstrated that ClpB ultimately may participate in the regulation of several genes in P. gingivalis.

ClpB contributes to thermotolerance, but not resistance to oxidative and pH stresses

In this study, a decrease in thermotolerance by the clpB mutant strain, LY2003, suggests that ClpB is at least partially responsible for the survival of P. gingivalis. Decreased thermotolerance in clpB mutants has also been identified in E. coli, Cyanobacterium synechococcus, B. suis, and H. pylori (Squires et al., 1991; Eriksson & Clarke, 1996; Allan et al., 1998; Ekaza et al., 2001).

Protein aggregates are formed in the cytoplasm under heat exposure. Microarray data obtained in this study demonstrated that other genes involved in the recovery of protein aggregates, specifically grpE and dnaJ, are also down-regulated in the clpB mutant, suggesting that the decreased thermotolerance observed in the clpB mutant may be related to the absence or diminution of this chaperone machinery. Thus, ClpB, with its ability to rescue stress-damaged proteins from an aggregated state, is important for the survival of P. gingivalis under heat stress. Significantly, the down-regulation of grpE and dnaJ may indicate that, at least in P. gingivalis, there is a coordinate regulation of the expression of these genes and clpB. Further studies are necessary to determine the details of this mechanism.

In B. suis, a clpB mutant showed increased sensitivity to ethanol stress and acidic pH. However, the increased sensitivity to hydrogen peroxides in B. suis required simultaneous inactivation of both clpB and clpA (Ekaza et al., 2001). No clpA homolog in the P. gingivalis W83 genome database (http://cmr.tigr.org/tigr-scripts/CMR/GenomePage.cgi?org=gpg) could be found, although other family members of the ATPase family (clpC, clpP and clpX) were identified. Additionally, it was found in L. monocytogenes that ClpB is not involved in tolerance to heat, salt, detergent, puromycin, or cold, even though its synthesis is inducible by heat shock (Chastanet et al., 2004). Recently, McKenzie et al. (2007) showed that the expression of clpB was not altered when P. gingivalis W83 was exposed to H2O2 (McKenzie & Fletcher, 2007). These data suggest that ClpB may play different roles in the stress response in different bacterial species. The data presented here demonstrated that the P. gingivalis ClpB is not involved in resistance to H2O2 and extreme pHs, but is involved in tolerance to heat stress.

Several genes in P. gingivalis, such as sod, ahpC-F, rubrerythrin, and oxyR, have been shown to be involved in protection against oxidative stress (Lynch & Kuramitsu, 1999; Sztukowska et al., 2002; Diaz et al., 2004, 2006). An extensive search of the microarray data did not identify any change in the expression of these genes. In addition, a microarray study of the P. gingivalis response to H2O2 showed that several stress-related proteins, including grpE and dnaJ, but not ClpB, were up-regulated under these conditions (McKenzie & Fletcher, 2007). However, these data indicate that grpE and dnaJ are down-regulated in the clpB mutant (Table 2). Thus, it is proposed that for short periods of stress, the inactivation of ClpB likely does not alter the oxidative stress response in P. gingivalis, as bacteria may express other genes to overcome the oxidative stress for a short period. Further studies are necessary to more fully characterize the P. gingivalis response to oxidative stress.

ClpB protein is involved in host cell adhesion and invasion

Listeria monocytogenes clpC mutant showed strongly reduced cell invasion in cultured hepatocyte and epithelial cell lines. This can be explained by the reduced expression of the virulence factors InlA, InlB, and ActA, which are responsible for attaching to and entering into cells (Nair et al., 2000). In Yersinia enterocolitica, ClpB also appeared to play a role in invasion of human laryngeal epithelial cells (Hep-2). Also, the expression of important virulence factors, including invasin and flagellin, was decreased in a clpB mutant (Badger et al., 2000). Invasin, an outer membrane protein, is required for efficient translocation of the bacteria from the intestinal lumen to the Peyer's patches (Pepe & Miller, 1993). The decreased expression of invasin of Y. enterocolitica may induce impaired invasion.

Although there is scarce information on ClpB and host-cell interactions in P. gingivalis, other members of the Clp ATPase family (clpC, clpX and clpP) were found to be up-regulated when P. gingivalis was incubated in cell culture medium collected from GE cell cultures (Zhang et al., 2005). Also, the ability of a clpP mutant to invade GE cells was reduced by 50% when compared with the parent strain ATCC 33277, although the mutant was not reduced in its ability to adhere compared with the parent strain (Zhang et al., 2005). These results indicate that the ability of the clpB mutant to adhere and invade three cell types, including KB, HCAE, and GE cells, was severely impaired compared with the parent strain. These microarray data showed that HagB, an adhesin of P. gingivalis for host cells (Song et al., 2005), was down-regulated in the clpB mutant, suggesting that the lower adhesion of the mutant and consequently a lower recovery of internalized mutant cells, when compared with the wild-type strain, may be due, at least partially, to the low expression of hagB. However, the differences observed do not appear to be due to a potential defect in FimA expression, as its expression remained unaltered in the clpB mutant as determined by microarray analysis. Also, negative staining of the clpB mutant corroborates the expression of FimA (data not shown). Western blot analyses also reveal that the ClpB mutation does not alter the FimA expression (R. Lamont, pers. commun.).

Greater differences in the invasion assays than in the adhesion assays were observed between the mutant and the wild-type strain, suggesting that ClpB may also be involved in internalization and/or the survival of P. gingivalis inside the host cells. It is also likely that the changes in expression of other genes affected by the mutation in clpB are also responsible for the lower recovery of internalized mutant cells than wild-type bacteria. For example, grpE (PG1775), dnaJ (PG1776), slyD (PG1315), ompH1 (PG0192), ompH2 (PG0193), tetR (PG01240) and a hypothetical protein (PG0419) have all been shown to be up-regulated in P. gingivalis when in contact with Hep-2 cells (Hosogi & Duncan, 2005). In this study, the expression of each of these genes was down-regulated in the clpB mutant, thus the lower numbers of internalized mutant cells may be due to the altered expression of one or more of these genes.

These data indicate that ClpB likely plays an important role in entry/trafficking within different host cell types. This could be explained by the fact that P. gingivalis uses different mechanisms for intracellular trafficking in these cell types. It has been shown that P. gingivalis is found both free in the cytoplasm or within single membrane bound vacuoles in KB cells (Duncan et al., 1993; Njoroge et al., 1997; Houalet-Jeanne et al., 2001). In GE cells, P. gingivalis accumulates freely in the cytosol, more specifically in the perinuclear area (Lamont et al., 1995; Belton et al., 1999). However, internalized P. gingivalis are found within autophagosomes in HCAE cells (Dorn et al., 2001).

In this study, both P. gingivalis W83 and the clpB mutant induced an autophagic response within the host HCAE cells. However, widespread degradation of the mutant bacteria was observed throughout the infected cells when compared with the wild-type strain. These results are consistent with the data obtained in the invasion assays with the clpB mutant in which fewer bacteria are recovered compared with the wild-type strain and suggests that the clpB mutant is defective in trafficking and is more readily killed intracellularly. The exact mechanisms of P. gingivalis invasion/survival remain to be elucidated.

ClpB is involved in virulence in vivo

In the mice competition assay, the number of the clpB mutant cells recovered from the animals decreased beginning on the third day. This suggests that the functional loss caused by the clpB mutation is phenotypically complemented by the wild-type strain, at least during the first two days, since both strains were injected into the animals as a mixture. When the wild type and clpB mutant were injected separately into two groups of mice, the clpB mutant was less virulent than the wild type. Overall, the clpB mutant does not survive as well as W83. This suggests that ClpB plays a role in P. gingivalis virulence in vivo.

Collectively, the animal data suggest that ClpB is important for the survival and virulence of P. gingivalis in vivo. In addition, the clpB mutant invaded significantly less than did wild-type W83 in three different cell types, suggesting that this mutant may have a decreased ability to invade cells in vivo. Thus the decreased virulence observed in mice could also be due to the decreased invasion ability of the mutant. As observed in these microarray data, the altered expression of multiple genes in the clpB mutant may explain the decreased survival and virulence of the mutant. The altered expression of HagB in the mutant could indicate that the mutant may have its survival compromised in vivo, since Lee et al. (1996) have shown that HagB is expressed during the course of P. gingivalis infection in mouse abscess models (Lee et al., 1996). Moreover, the down-regulation of stress response proteins such as grpE (PG1775), dnaJ (PG1776), and slyD (PG1315) may contribute to the overall lower virulence of the clpB mutant. Alternatively, the reduced virulence of the clpB mutant may result from a more efficient host response against the mutant. Several genes had altered expression in the clpB mutant with potential modifications in the cell envelope. Thus, the host–mutant bacteria interactions and, consequently, the host inflammatory and innate immune responses may be altered. Several studies have shown that inactivation of P. gingivalis virulence factors may differentially influence innate immune and inflammatory responses in vitro and in vivo (Gibson et al., 2006; Kishimoto et al., 2006; Ohno et al., 2006; Shelburne et al., 2007). Further characterizations of the host–mutant bacteria interactions are necessary to clarify the low virulence of the clpB mutant in vivo.

In conclusion, it was shown that disruption of clpB in P. gingivalis altered the expression of multiple genes affecting several different cellular functions, suggesting that ClpB is ultimately involved in the expression of numerous P. gingivalis genes. These data also demonstrate that ClpB in P. gingivalis is responsible for resistance to temperature stress but not to other stress conditions tested (H2O2 and unfavorable pH). The clpB mutant showed a large decrease in the ability to invade different cell types, indicating that ClpB plays an important role during host–pathogen interactions. In addition, the animal experiments performed clearly demonstrated that the clpB gene is important for the virulence of this pathogen in vivo.

Acknowledgements

This work was supported by the University of Florida Center for Molecular Microbiology and NIH grants NIDCR DE 007496 and DE 013545. Debra Akin is thanked for transmission electron microscopy assistance, Nathan Nemecek for technical help, and Henry Baker (Microarray Core Facility at the University of Florida) for microarray assistance. Thanks are also due to The Institute for Genomic Research (Rockville, MD) for kindly providing the microarray slides.

Authors' contribution

L.Y. and P.H.R. contributed equally to this article.

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