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Department of Biochemistry and Microbiology, School of Medicine, Loma Linda University, Loma Linda, CA 92350, USA
Correspondence
H. M. Fletcher
hfletcher{at}llu.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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These authors contributed equally to this work.
Present address: Department of Biological Sciences, Oakwood College, Huntsville, AL, USA.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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, 1999; Griffen et al., 1999; Travis et al., 2000). This organism is also associated with other systemic diseases, including atherosclerosis (reviewed by Okuda & Ebihara, 1998; Teng et al., 2002; Kinane & Marshall, 2001). The major virulence factors of P. gingivalis, the gingipains, possess high levels of proteolytic activity and have been the focus of much attention (Genco et al., 1999; Imamura, 2003; Nakagawa et al., 2003). The major gingipains are extracellular and/or cell-associated. The Arg-specific gingipains, RgpA and RgpB, are encoded by the genes rgpA and rgpB respectively, whereas the Lys-specific protease (Kgp) is encoded by one gene, kgp (Nakayama, 2003). There is a gap in our understanding of the regulation and processing of the gingipains. We have reported the post-translational regulation of the gingipains in the vimA-defective mutant P. gingivalis FLL92 (Abaibou et al., 2001). The vimA gene is a part of the bcp-recA-vimA operon. Further, protein–protein interaction studies using the purified rVimA showed that this protein interacts with the gingipains and the HtrA homologue in P. gingivalis (Vanterpool et al., 2006). The role, if any of HtrA in gingipain regulation and virulence in P. gingivalis is unclear.
Protein quality control, which is essential for bacterial survival, is regulated by chaperones and proteases (Lu & McBride, 1994; Goulhen et al., 2003). HtrA (high temperature requirement A) is a periplasmic heat-shock serine protease that functions as a molecular chaperone at low temperatures and has proteolytic activity at elevated temperatures (Kim & Kim, 2002). In several organisms, including bacteria, yeast, plant and humans, HtrA is considered an important factor involved in protein folding and maturation as well as in the degradation of misfolded proteins (Poquet et al., 2000). Inactivation of the htrA gene has been shown to affect the sensitivity of many organisms to thermal and oxidative stress (Pallen & Wren, 1997; Lipinska et al., 1990). Here we report that HtrA physically interacts with the gingipains RgpA, RgpB and Kgp in P. gingivalis. HtrA is also involved in resistance to thermal and oxidative stress in P. gingivalis.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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. P. gingivalis strains were grown in brain heart infusion (BHI) broth (Difco) supplemented with haemin (5 µg ml–1), vitamin K (0.5 µg ml–1) and cysteine (0.1 %). Experiments with hydrogen peroxide were performed in BHI without cysteine. Escherichia coli strains were grown in Luria–Bertani broth. Unless otherwise stated, all cultures were incubated at 37 °C. P. gingivalis strains were maintained in an anaerobic chamber (Coy Manufacturing) in 10 % H2/10 % CO2/80 % N2. Growth rates for P. gingivalis and E. coli strains were determined by measuring OD600. Antibiotics were used at the following concentrations: clindamycin, 0.5 µg ml–1; erythromycin, 300 µg ml–1; carbenicillin, 100 µg ml–1.
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). For plasmid DNA analysis, DNA extraction was performed by the alkaline lysis procedure as previously described (Johnson et al., 2004). For large-scale preparation, plasmids were purified using the Qiagen plasmid maxi kit.
Generation of the htrA-defective mutant P. gingivalis strain.
A 1.5 kb fragment carrying the intact htrA ORF (gene number PG0535, http://www.oralgen.lanl.gov) was amplified by PCR using primers P1 and P2 (Table 2). This fragment was cloned into the pCR2.1-TOPO plasmid vector (Invitrogen) and designated pFLL200. The 1.5 kb fragment was then isolated from pFLL200, digested with BamHI and XbaI and ligated to pUC19 linearized with the same enzymes. The new recombinant plasmid was designated FLL201. Orientation was confirmed by restriction analysis. The ermF-ermAM cassette, which confers erythromycin/clindamycin resistance in E. coli and P. gingivalis, was PCR-amplified from pVA2198 using Pfu turbo (Stratagene) with primers P5 and P6 (Table 2). The amplified fragment was inserted into the MscI restriction site of the htrA gene. The resultant recombinant plasmid, pFLL202, was used as a donor in electroporation of P. gingivalis W83 as previously reported (Vanterpool et al., 2004).
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).
Protein concentration determination.
Protein concentration was calculated spectrophotometrically using an Eppendorf Biophotometer according to the manufacturer's instructions (Brinkman).
SDS-PAGE and immunoblot analysis.
SDS-PAGE and transfer to nitrocellulose membranes were as described by Vanterpool et al. (2006). The blots were probed with antibodies against specific protease domains (Olango et al., 2003) or HagA (generously donated by Ann Progulske-Fox, University of Florida). The secondary antibody was immunoglobulin G (heavy plus light chains)–horseradish peroxidase conjugate (Zymed Laboratories). Immunoreactive proteins were detected by using the Western Lighting Chemiluminescence Reagent Plus kit (Perkin-Elmer Life Sciences).
Analysis of P. gingivalis htrA-defective mutant genes by RT-PCR.
Total RNA was extracted from P. gingivalis strains grown to early stationary phase (OD600 1.2) using the RiboPure kit (Ambion). The primers used for RT-PCR analysis were specific for the kgp (primers P3 and P4), htrA (P1 and P2) and sigA genes (P7 and P8) (Table 2
). The RT-PCR reaction (50 µl) contained 1 µg template RNA in the Superscript One-step RT-PCR reaction mix (Invitrogen). Negative controls were RT-PCR in the absence of reverse transcriptase.
Cell fraction preparation.
One-litre cultures of P. gingivalis strains FLL203 and W83 were grown to stationary phase (OD600 1.5) from actively growing cells. Preparations of whole-cell culture, cell-free medium, cell suspension, vesicles and vesicle-free medium were made as previously reported (Olango et al., 2003). The whole-cell culture (WC) fraction is a sample of the culture after the bacterium has been grown to a specific growth phase. This sample has the bacterial cells suspended in the growth medium, and the enzyme activity includes the gingipains that are attached to the bacterial cell surface plus those secreted into the culture medium. After centrifugation, the cell pellet was resuspended, and the enzyme activity in this sample (the cell suspension fraction, CS) represents the gingipains that are attached to the bacterial cell surface. The enzyme activity in the supernatant (the cell-free medium fraction, CF) includes the gingipains that are secreted into the culture medium. Secreted gingipains can either be associated with vesicles or soluble in the culture medium; thus, ultracentrifugation of the cell-free fractions will yield a vesicle pellet (V) and a supernatant of soluble gingipains (VF).
Effect of heat shock on gingipain activity.
Cultures of P. gingivalis strains FLL203 and W83 were grown for 16 h to an OD600 of 1.5; 2 ml samples from these cultures were incubated for 10 min at 55 °C and proteolytic activities were analysed immediately and 30 min after heat shock treatment.
Growth at elevated temperatures and under oxidative stress conditions.
Cultures (100 ml) of P. gingivalis strains FLL203 and W83 were grown at 37 °C for 16 h to an OD600 of 1.5. For growth at elevated temperatures, the cultures were then incubated at 42 °C under anaerobic conditions for 28 h. Growth was determined at intervals of 0, 4, 8, 24 and 28 h as described above. For growth under oxidative stress conditions, cultures were grown in BHI without cysteine in the presence of 0.25 mM hydrogen peroxide. Controls were grown (with cysteine) in the absence of hydrogen peroxide.
Cloning and expression of rHtrA.
Oligonucleotide primers (P1 and P2) specific for the ORF of the htrA gene were synthesized and used for PCR amplification. The amplified fragment carrying the htrA ORF was cloned into pTrcHis2-TOPO expression plasmid (Invitrogen) carrying coding for a C-terminal His-tag. The recombinant plasmid (designated pFLL137) was then transformed into competent E. coli TOP10F' cells (Invitrogen). Orientation of the htrA gene was determined by restriction endonuclease digestion. The nucleotide sequence of the insert was determined to rule out the occurrence of mutations. E. coli carrying the htrA ORF was grown in Luria–Bertani broth to exponential phase (OD600 0.7) in the presence of 50 µg carbenicillin ml–1. IPTG was added at exponential phase and the cells were incubated at 37 °C with shaking for an additional 6 h. Cells were harvested by centrifugation and lysed by sonication. Cell membrane and debris were then harvested by centrifugation. The supernatant and pellet were analysed for the presence of the rHtrA protein. The presence of the poly-histidine tag was confirmed using the GelCode 6xHis Protein Tag kit according to the manufacturer's instructions (Pierce).
Protein–protein interactions.
The E. coli cells carrying the expressed rHtrA were lysed and the rHtrA was purified using Ni-NTA magnetic beads. The purified rHtrA protein was then incubated for 30 min with magnetic beads coated with Ni-NTA. Beads were washed four times with wash/interaction buffers (50 mM NaH2PO4, 300 mM NaCl, 50 mM imidazole and 0.005 % Tween 20). Cell lysates from P. gingivalis W83 grown to stationary phase were then incubated for 30 min with the with rHtrA-attached Ni-NTA magnetic beads. As a negative control, the lysates were incubated with the magnetic beads without the recombinant protein. After incubation with cell lysates, unbound proteins were eliminated by extensive washing in wash buffer. Proteins were eluted off the beads under denaturing conditions (1x lithium dodecyl sulfate sample buffer, heated to 90 °C for 5 min). Eluates were then analysed by SDS-PAGE and immunoblot analysis as described above.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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-haemolytic phenotype.
Confirmation of inactivation of htrA by PCR analysis
Chromosomal DNA from six randomly chosen clindamycin/erythromycin-resistant colonies and the wild-type W83 strain was analysed by PCR to confirm the inactivation of the htrA gene. If the htrA gene was interrupted by the ermF-ermAM cassette, a 3.5 kb fragment was expected to be amplified using htrA primers P1 and P2. In addition, a 2.1 kb fragment should be amplified from the mutant using the ermF-ermAM primers. The expected 3.5 kb (using htrA primers) and 2.1 kb (using erythromycin primers) fragments were observed only in the erythromycin-resistant strains, not in the wild-type (Fig. 1a). To further confirm the absence of the htrA transcript in the erythromycin-resistant mutant, DNase-treated RNA from P. gingivalis FLL203 and the wild-type was subjected to RT-PCR. As shown in Fig. 1(b), no htrA transcript was detected from the mutant, in contrast to the wild-type W83. Using kgp-specific primers (Table 2) as a control, the expected 0.8 kb fragment was amplified from all the P. gingivalis strains. No amplified fragment was observed from similar reactions in the absence of reverse transcriptase. These results indicated that the insertional inactivation of the chromosomal htrA gene with the 2.1 kb ermF-ermAM antibiotic-resistance cassette was successful. One mutant designated P. gingivalis FLL203 was randomly chosen for further study. The wild-type and the htrA-defective mutant FLL203 showed a similar generation time of 3 h (data not shown). To rule out any polar effect on the gene downstream of htrA, DNase-treated RNA from FLL203 was further analysed by RT-PCR. Using specific primers (P7 and P8; Table 2) for the downstream sigA gene, a fragment of the predicted size was amplified from both the htrA-defective mutant and the wild-type (Fig. 1c).
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). The greatest sensitivity was observed during the stationary growth phase (Fig. 2b).
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). In cells grown at 42 °C for 8 h or 28 h, Rgp activity was increased by approximately 20 % and 10 % respectively in the mutant compared to the wild-type strain (Fig. 3b). In contrast to the parent strain at a similar cell density, there was greater Rgp activity in the HtrA-defective mutant grown at 42 °C (Fig. 3c). The ability of P. gingivalis FLL203 to recover from heat shock was also evaluated. After a 10 min incubation at 55 °C, both the wild-type and the mutant strains were further incubated for 24 h at 37 °C. A slightly longer lag phase was observed in the htrA-defective mutant in comparison to the wild-type (data not shown). To determine if the gingipains are affected by brief incubation at elevated temperatures in the htrA-defective mutant, P. gingivalis W83 and FLL203 were incubated at 55 °C for 10 min. At 37 °C, there was no significant difference in Rgp activity between the mutant and the wild-type (data not shown). However, immediately after heat treatment, Rgp activity in the wild-type was elevated by 7 % in comparison to the untreated control. Interestingly, the Rgp activity in the htrA-defective mutant was decreased by 13 % in comparison to the untreated control. Rgp activities determined 30 min after heat treatment were similar for the wild-type and FLL203 (data not shown).
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) and the htrA-defective mutant (Fig. 4b) was very similar under normal growth conditions. However, the distribution of the Rgps appeared to be slightly altered by incubations at 42 °C. There appeared to be approximately 30 % less cell-associated Rgp activity in the htrA-defective mutant (Fig. 4d) in comparison to the wild-type (Fig. 4c). Very little alteration in Kgp activity was observed (data not shown). Immunoblot analysis of extracellular fractions demonstrated the absence of RgpA and RgpB immunoreactive bands in the htrA-defective mutant (data not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Katsuragi et al., 2003; Wolff et al., 1997) would suggest that factors that are important in oxidative and temperature stress would be critical for the survival of P. gingivalis. In many organisms, HtrA is known to play a regulatory role in response to environmental signals and is protective against oxidative and temperature stress (Cortes et al., 2002; Foucaud-Scheunemann & Poquet, 2003; Lipinska et al., 1990; Lyon & Caparon, 2004; Noone et al., 2000; Pallen & Wren, 1997; Poquet et al., 2000). HtrA is also involved in the biogenesis of bacterial virulence factors. Thus its inactivation results in attenuated virulence (Cortes et al., 2002; az-Torres & Russell, 2001; Lyon & Caparon, 2004). In our study, we further evaluated the role of HtrA in P. gingivalis. We have demonstrated that the htrA-defective mutant is more sensitive to elevated temperature than the wild-type. In another study, L. Yuan & A. Progulske-Fox (unpublished) demonstrated that the survival of the htrA-defective mutant is not significantly affected by a 50 °C heat shock. Collectively, these data may suggest that HtrA may play a role in long-term adaptation to prolonged elevated temperature, as demonstrated in this study. This could have implications for growth in the periodontal pocket, where there is usually increased temperature due to inflammation.
In addition to the altered growth of the htrA-defective mutant at elevated temperature, there was an increase in gingipain activity in the mutant in comparison to the wild-type. The decreased cell density of the htrA-defective mutant at elevated temperature compared to the wild-type may indicate that the level of gingipain activity is significant. Our results indeed indicated that at a similar cell density, there was significantly more gingipain activity in the mutant than in the wild-type. Collectively, these results could suggest an involvement of HtrA in gingipain regulation at elevated temperatures. We are currently investigating the molecular changes that occur with the gingipains under prolonged growth at 42 °C. Prolonged growth of P. gingivalis at temperatures above 37 °C has previously been observed to result in a significant decrease in gingipain activity (Amano et al., 1994; Forng et al., 2000; Lynch & Kuramitsu, 1999; Murakami et al., 2004a, b; Percival et al., 1999). Under these environmental conditions, it is likely that the decrease in proteolytic activity could be correlated with a downregulation in the expression of the gingipain genes (Percival et al., 1999). HtrA and other members of this family, including DegS and DegP, have a stress sensor function and are associated with the periplasmic side of the bacterial membrane (Day & Hinds, 2002; Krojer et al., 2002; Pallen & Wren, 1997). The stress sensor function is modulated via the PDZ domain and initiates a cascade of events that results in the induced expression of several genes critical to the stress response (Day & Hinds, 2002; Krojer et al., 2002; Pallen & Wren, 1997). A comparison of the P. gingivalis HtrA homologue with those of other known organisms reveals a PDZ domain and an N-terminal hydrophobic region, indicating membrane association (http://www.tigr.org) (van & Hendriks, 2003; Pallen & Wren, 1997). The mechanism for the regulatory role of HtrA in the expression of the gingipain genes in P. gingivalis is unknown. Other virulence genes in P. gingivalis are known to be downregulated at elevated temperature. However, it is unclear if a common mechanism is involved in their regulation. This is the subject of further investigation in our laboratory.
In this study, the gingipain activity in response to heat shock appeared to be different from the response of the organism grown at elevated temperature for an extended period. Decreased gingipain activity was observed in the htrA-defective mutant, in contrast to the wild-type, which showed an increase in activity. It is possible that during heat shock, denaturation of the gingipains could lead to decreased gingipain activity. In the wild-type, it is likely that the presence of the HtrA protein may stabilize the gingipains. HtrA is also known to have a chaperone function, and could play a role in the protection of the gingipains under these conditions, although we cannot rule out the possibility of other heat-shock proteins being involved. Preliminary results from our laboratory have demonstrated that in protein–protein interaction experiments with rHtrA, this protein can interact with the gingipains as well as HagA (data not shown). These findings are currently being confirmed. In addition, HtrA can interact with the VimA protein, which can bind to the gingipains and regulate their activity (Vanterpool et al., 2006). Collectively, these data could further support a direct involvement of HtrA in the regulation of gingipain activity. It is likely that a complex of proteins stabilize the gingipains at elevated temperature, although HtrA is not essential for the maturation/activation of the gingipains under normal conditions. We cannot rule out the possibility that HtrA may regulate the expression of other heat-shock proteins that would be missing in the htrA-defective mutant. Several heat-shock proteins in P. gingivalis, including GroES, GroEL, DnaK and HtpG, have been documented (Yamazaki et al., 2004; Yoshida et al., 1999; Goulhen et al., 2003; Maeda et al., 1994).
In addition to having elevated temperatures, the periodontal pocket is an oxidative environment due to the presence of reactive oxygen species (Chapple, 1997; Katsuragi et al., 2003; Sculley & Langley-Evans, 2002). Consistent with reports of other organisms (Ibrahim et al., 2004; Mutunga et al., 2004; Wonderling et al., 2004), our data also suggest that the htrA-defective mutant is more susceptible to hydrogen peroxide than the wild-type. This was also observed in a similar study by L. Yuan & A. Progulske-Fox (unpublished). Given the multiple mechanisms of oxidative stress resistance in P. gingivalis, the relative significance of HtrA in this process is unclear. The virulence potential of the htrA-defective mutant was altered compared to that of the wild-type (L. Yuan & A. Progulske-Fox, unpublished).
In conclusion, we can envision a scenario in P. gingivalis where the HtrA protein may be important for regulation of gingipain activity in the inflammatory microenvironment of the periodontal pocket. A specific mechanism for this interaction will be the subject of further study.
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ACKNOWLEDGEMENTS |
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Received 19 May 2006;
revised 17 July 2006;
accepted 21 July 2006.
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, FLL203 control; 
, FLL203 treated; 
, W83 control, 
, W83 treated. (b) Difference in OD600 between treated and untreated cultures at exponential (E) and stationary (S) phase. The data in both panels are means±SE of three independent experiments.
-benzoyl-DL-arginine-p-nitroanilide (BApNA) were tested in whole cell culture (WC), cell suspension (CS), cell free medium (CF), vesicles (V) and vesicle-free (VF) fractions of the P. gingivalis strains (see Methods for details). (a) W83 grown at 37 °C, (b) FLL203 grown at 37 °C, (c) W83 grown at 42 °C, (d) FLL203 grown at 42 °C.