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1 Department of Oral Biology, College of Dentistry and Center for Molecular Microbiology, University of Florida, Gainesville, FL 32610, USA
2 Department of Anatomy and Cell Biology, College of Medicine and Center for Molecular Microbiology, University of Florida, Gainesville, FL 32610, USA
Correspondence
Ann Progulske-Fox
apfox{at}dental.ufl.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 Physiology, College of Medicine, University of Florida, Gainesville, FL 32610, USA.
The GEO accession number for the microarray data reported in this paper is GSE10027.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). HtrA has a dual function: one function is as a chaperone at lower temperatures, while its proteolytic activity is activated at elevated temperatures (Spiess et al., 1999). When bacteria confront stressful environments, including elevated temperatures or reactive oxygen intermediates, damaged proteins tend to form in the cell. Thus, elimination of the damaged proteins becomes essential for the bacteria to survive. In Escherichia coli, the periplasmic HtrA has been found to act as a stress response protease (Davies & Lin, 1988; Laskowska et al., 1996; Lipinska et al., 1990; Strauch & Beckwith, 1988). In other bacteria, such as Yersinia enterocolitica and Brucella abortus (Elzer et al., 1994; Li et al., 1996), HtrA has been shown to play a role in the response to high temperature and oxidative stress conditions.
HtrA has also been found to be related to virulence in some species. For example, in Y. enterocolitica, Salmonella typhimurium and B. abortus, htrA mutants show decreased virulence compared to the wild-type, indicating the involvement of HtrA in pathogenicity (Chatfield et al., 1992; Elzer et al., 1996; Li et al., 1996). The essential role of HtrA in virulence prompted the use of a Salmonella htrA mutant as a live vaccine (Tacket et al., 2000).
Porphyromonas gingivalis, a Gram-negative, anaerobic, black-pigmented bacterium, is considered to be a primary aetiologic agent of certain types of periodontal diseases (Socransky & Haffajee, 1992). P. gingivalis is found in subgingival plaque in periodontal pockets that have temperatures higher than normal body temperature and varying levels of oxygen (Fedi & Killoy, 1992; Kung et al., 1990; Lindskog et al., 1994; Mettraux et al., 1984; Meyerov et al., 1991; Tanaka et al., 1998). This is an environment in which the bacteria are frequently stressed and one in which they must compete in order to survive.
P. gingivalis can attach to and invade oral epithelial cells (Dorn et al., 2000; Lamont et al., 1995; Sandros et al., 1994). Furthermore, it has been reported to be present in atherosclerotic plaque (Kozarov et al., 2005) and is able to invade human coronary artery endothelial cells (HCAEC) (Dorn et al., 2001). In these environments, P. gingivalis may be subjected to multiple conditions of stress; thus the expression of heat-shock proteins in P. gingivalis should be highly significant for P. gingivalis survival.
A conserved homologue of the htrA gene in P. gingivalis has been identified (http://cmr.tigr.org/tigr-scripts/CMR/shared/AnnotationSearch.cgi, locus number PG0593). In a previous study, the htrA gene was determined to be upregulated in a luxS mutant of P. gingivalis compared to wild-type strain W83 (Yuan et al., 2005). Here we report the construction of an htrA mutant, W83
0593, which has been used to characterize the functions of htrA in relation to stress, cell invasion and virulence in animal models.
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METHODS |
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). Cells were grown and maintained at 37 °C in an anaerobic chamber containing an atmosphere of 85 % (v/v) N2, 10 % (v/v) H2 and 5 % (v/v) CO2. The P. gingivalis W83 htrA mutant, strain W830593, was maintained as described for the wild-type strain, except that 5 µg clindamycin ml–1 was added to the medium to maintain the antibiotic selective pressure.
Mutant construction.
The technique to create mutants has been described elsewhere (Bélanger et al., 2007). Briefly, a 573 bp internal fragment of the htrA gene (positions 301–874 of the gene) was generated by PCR using the primer PG0593F (MT), 5'-AAAAGGATCCTCGGAGGCGAATCCAGAAAC-3' (starting at position 301), and PG0593B (MT), 5'-AAACTGCAGACTGCTTGATGTCGGCTACCAC-3' (ending at position 874). Underlined sequences indicate BamHI and PstI sites, respectively. The DNA fragment was then cloned into the P. gingivalis suicide vector pVA3000 (Lee et al., 1996) at the BamHI and PstI sites. E. coli strain S17-1 was used to deliver the vector containing the internal fragment into P. gingivalis W83 by conjugation on blood agar, then the 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, truncated at either the 5' or the 3' end. Southern blot analysis was done to confirm the construction of the htrA mutant, W830593, using an ECL detection kit according to the manufacturer's protocol (Amersham). Growth curves were performed to verify if the insertion of the vector into the P. gingivalis genome would affect the growth of the htrA mutant.
Stress experiments
For all stress-related experiments, strains W83 and W830593 were cultured in supplemented TSB without antibiotics until they reached the mid-exponential phase of growth.
(i) Temperature stress experiments.
One millilitre each of W83 and W830593 cultures was centrifuged at 15 600 g for 2 min, then the cell pellets were washed using 1 ml 0.1 M glycine buffer (pH 7). Cell pellets were then resuspended in 1 ml of the same buffer and incubated at 50 °C. Aliquots were removed at 0 and 8 min.
(ii) Hydrogen peroxide (H2O2)-induced stress.
Ten millilitres each of W83 and W830593 cultures was centrifuged at 4000 g for 10 min at 4 °C, then washed with 10 ml 0.1 M glycine buffer (pH 7). The bacterial pellets were resuspended in 0.1 M glycine buffer containing 0.35 mM H2O2. Aliquots were taken at 0, 15 and 30 min.
(iii) pH stress experiments.
Bacterial cells were treated as described above and the pellets were resuspended in 0.1 M glycine buffer (pH range from 3 to 10 in 1 unit increments). Aliquots were taken at 0, 30 and 45 min and 1 and 2 h.
Decimal dilutions from the above experiments were made and plated onto BAPs. Plates were incubated in an anaerobic chamber for 7–10 days and c.f.u. were enumerated. The percentage survival of the bacteria was calculated using the number of c.f.u. at time 0.
Cell cultures.
KB cells were once thought to be derived from an oral cancer, but in fact they were derived from a glandular cancer of the cervix (Masters, 2002). Nevertheless, KB cells have been used extensively as a model to study host cell interactions with P. gingivalis. HCAEC, a primary cell line (Cambrex), were maintained in EBM-2 medium supplemented with EGM-2-MV single-use aliquots (Cambrex). Primary gingival epithelial cells (GEC) (kindly provided by Dr Richard Lamont) were cultured in KBM medium supplemented with insulin, triiodothyronine, transferrin, recombinant human epidermal growth factor, epinephrine, bovine pituitary extracts and hydrocortisone (Cambrex), as described by Lamont et al. (1995). Cells were cultured in 75 cm2 flasks at 37 °C in a humidified atmosphere with 5 % (v/v) CO2. Confluent monolayers were split by treatment with trypsin-versene. KB cells (ATCC CCL-17) were maintained in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10 % (v/v) fetal bovine serum and 100 mg penicillin/streptomycin ml–1.
Cell invasion assay.
Invasion experiments were done in 24-well plates containing 105 cells per well. P. gingivalis was then added at an m.o.i. of 100 : 1 and a standard antibiotic protection assay was performed as described previously (Lamont et al., 1995; Yuan et al., 2007). The experiments were performed in triplicate, each time using three wells for each strain.
Transmission electron microscopy.
KB and HCAEC were infected for 2.5 h with wild-type P. gingivalis or the htrA mutant as described above. 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.
P. gingivalis wild-type W83 and mutant strain W830593 were grown in 40 ml TSB to an OD600 of 2.0. 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 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) and purified using the RNeasy kit (Qiagen), 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 OD600 of 0.5, 1.0, 1.5 and 2.0. This refRNA was used in the microarray hybridizations. 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 amino acid-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.
P. 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-labelled cDNA samples originating from four different cultures of W83 or W830593 grown in broth without antibiotics were hybridized to the arrays along with Cy5-labelled reference cDNA samples, for a total of eight slides. With this approach, dye swap is not necessary. Hybridizations were carried out in a MAUI 4-bay hybridization system (BioMicro Systems) for 16 h at 42 °C. The slides were then washed according to TIGR protocols and scanned using a GenePix scanner (Axon) at 532 (Cy3 channel) and 635 nm (Cy5 channel) with a Cy3/Cy5 ratio of 1 : 1.
Microarray data analysis.
16-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 with the following modification: one bad channel tolerance, generous; followed by in-slide replicate analysis using TIGR MIDAS software (www.tm4.org/midas.html). Spots that were flagged as faulty, due 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 BRB software. Class prediction was then determined at a P value of 0.005. Therefore, at this significance level, there were 9.5 possible false-positive genes.
Virulence studies.
(i).
To test the mutant strain for alterations in virulence relative to the wild-type, a competition assay was carried out using 18 BALB/c mice (7–8 weeks old) as described previously (Wu et al., 2002). The mice were injected subcutaneously with a 1 : 1 ratio of P. gingivalis strains W83 and W830593. The surviving bacteria were then recovered from the lesion sites from six mice daily for 3 days. Decimally diluted samples were spread onto BAPs with 50 µg gentamicin ml–1. Colonies that arose after 1 week of anaerobic incubation were replica-patched onto BAPs with or without 5 µg clindamycin ml–1. After incubation for 3 days in the anaerobic chamber, the percentage of clindamycin-resistant colonies (mutant strain) was determined.
(ii).
The virulence of the htrA mutant strain compared with P. gingivalis W83 was tested 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 7.5x109 bacteria per mouse. Mice were then examined daily for 3 days to assess both their general health status and the number of dead animals.
Data normalization and statistical analysis.
The cellular interaction and invasion data were normalized with their inoculums and were analysed using Student's t-test. The mouse abscess model data were assessed using Kaplan–Meyer survival curves. Normality and equality of variances of the competition assay data were assessed using the Kolmogorov–Smirnov and Levene tests, respectively. A one-way ANOVA was used to assess the effect of the number of days on the data. When appropriate, multiple pairwise comparisons were done using the Holm–Sidak method. Results were considered to be statistically significant with a P value 
0.05. All DNA microarrays used in this work comply with the MIAME guidelines and the complete dataset has been deposited in the GEO online database under the provisional accession number GSE10027.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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0593 were compared, but no difference was observed (data not shown). This indicated that integration of the vector into the P. gingivalis chromosome did not adversely affect the growth rate of strain W830593 under the growth conditions tested.
Heat stress experiments
W83 and W830593 were treated at 50 °C for 8 min. The resulting percentage survival was then calculated based on comparison of the surviving number of bacteria to the inoculum number. After 8 min at 50 °C, although the percentage survival of W830593 (27.3 %) was lower than that of W83 (32.9 %), the difference was not great enough to reach a statistically significant level (P>0.05).
Hydrogen peroxide stress experiments
W83 and W830593 were stressed using 0.35 mM hydrogen peroxide in 0.1 M glycine buffer for 15 and 30 min; then the percentage survival was compared between the wild-type and the mutant. The htrA mutant W830593 survival rate decreased when compared to that of W83 at both time points, 20.8 versus 6.2 % at 15 min and 2.1 versus 0.2 % at 30 min, respectively. A statistically significant difference was observed between the strains at both time points (P=0.04 and 0.03, respectively). These data indicate that, in P. gingivalis, htrA is involved in resistance to hydrogen peroxide stress.
pH stress experiments
The htrA mutant W830593 showed no difference in terms of survival compared to W83 at any of the tested pH values (pH 3–11 in 1 unit increments) at any of the time points tested (data not shown).
Total interaction and invasion of cell lines
KB cells, primary HCAEC and primary human GEC were used in this study. The E. coli strain MC1061 was used as a non-invasive control (Dorn et al., 1999).
(i) 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. In terms of total interaction, P. gingivalis W830593 was not found to have any statistically significant difference compared with W83 in KB cells (P>0.05) (Fig. 1a). However, the htrA mutant was found to invade KB cells better than the wild-type strain since the number of recovered W830593 cells was approximately six times that of W83 (P<0.001) (Fig. 1b). This difference was statistically significant, indicating that in KB cells W830593 is more invasive than W83.
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), while the htrA mutant W830593 was found to be more invasive than W83 (P<0.001) (Fig. 1d), since the number of intracellular W830593 cells was four times that of W83 in GEC.
(iii) HCAEC.
The total interaction of W830593 with HCAEC was significantly lower than W83 (P=0.001) (Fig. 1e), but no difference in the numbers of internalized bacteria (invasion) (P>0.05) (Fig. 1f) was observed.
Microscopy
After 2.5 h of infection, both P. gingivalis W83 (Fig. 2a) and the htrA mutant (Fig. 2b) were found within vacuoles in HCAEC. The presence of autophagic vacuoles and autolysosomes in the infected HCAEC suggested that both bacterial strains were capable of promoting autophagy. Both W83 and the htrA mutant were localized predominantly in vacuoles which contained multiple bacteria but lacked the lysosomal enzyme acid phosphatase.
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0593 (Table 1) as was gingipain Kgp/HagD (PG1844), a well known P. gingivalis virulence factor. In addition, 83 hypothetical proteins, 24 cell-envelope genes, 5 regulatory genes and 11 transport and binding proteins genes were differentially regulated in the htrA mutant. Among these are cell-envelope genes related to various cellular processes, including adhesion to host cells and degradation of surface proteins, and thus these genes may relate to bacterial virulence. It is noteworthy that there were no polar effects observed as a result of the htrA gene inactivation, as no alteration in the expression of flanking genes could be found after analysis of the microarray data.
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0593. For 3 days after injection, six mice were sacrificed each day and the subcutaneous abscess contents were analysed. During the first 2 days of sampling, the percentage of recovered W830593 was higher than the number of recovered W83 (58.5 versus 41.5 % for the first day and 57.3 versus 42.7 % for the second day) (Fig. 3). However, the percentage of recovered W830593 dropped to 28.4 % on the third day, and it was significantly lower compared to day 1 (P=0.0008) and day 2 (P=0.001), indicating a decreased survival ability of W830593 over a longer time period compared to W83.
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0593 at a dose of 7.5x109 c.f.u. per mouse. The general health status of the mice treated with W83 and W830593 was the same for days 1 and 2 as all showed diffused lesions on their bodies and some were unable to walk normally. On day 3, 6/8 mice died in the W83 group while 3/8 in the W830593 group died. However, this difference was not statistically significant (P=0.143). |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Kung et al., 1990; Lindskog et al., 1994; Meyerov et al., 1991). The role of HtrA under different stress conditions has been shown for several bacterial species, although the relevance is not universal among all the species tested.
Environmental pH plays an important role in the survival of human pathogens. For P. gingivalis, resistance to various pH conditions is necessary for the bacteria to survive in oral cavities and periodontal pockets where the pH is known to vary considerably (Bickel & Cimasoni, 1985; McDermid et al., 1988). In this study, the P. gingivalis htrA mutant was not affected by changes in pH. The unchanged survival rate between the wild-type and the htrA mutant under different pH values indicates that other gene products are probably responsible for resistance to pH stress in P. gingivalis.
The importance of htrA for survival and/or growth at elevated temperatures varies according to the bacterial species (Biswas & Biswas, 2005; Brondsted et al., 2005; Lipinska et al., 1989; Stack et al., 2005). Even though its role is critical for several species, it is not important for Salmonella typhimurium and Staphylococcus aureus (Johnson et al., 1991; Rigoulay et al., 2005). Our data indicate that the P. gingivalis htrA also is not involved in the response to heat shock, at least at 50 °C. However, in another study, a P. gingivalis htrA mutant was found to grow more slowly than the wild-type at 42 °C over a period of 24 h (Roy et al., 2006). Thus, P. gingivalis may have different mechanisms for dealing with heat shock (50 °C) versus heat adaptation (42 °C) and these data indicate that htrA is likely to play a role in long-term adaptation to elevated temperature, but not in short-term adaptation.
In response to H2O2, the htrA mutant was more sensitive than the wild-type when treated with H2O2 for 15 and 30 min. Roy et al. (2006) demonstrated that the growth of a P. gingivalis htrA mutant was adversely affected by H2O2 over a period of 28 h. Therefore, htrA in P. gingivalis appears to play a role in the oxidative stress response. Previous reports have indicated that several genes, such as sod, ahpC–F, oxyR and the gene encoding rubrerythrin are involved in the response of P. gingivalis to oxidative stress (Diaz et al., 2004, 2006; Lynch & Kuramitsu, 1999; Sztukowska et al., 2002). Our microarray data did not show any change in the expression of these genes. However, a separate microarray study of the P. gingivalis response to H2O2 showed that stress-response-related genes grpE, dnaJ, htpG and groEL were upregulated under these conditions (McKenzie & Fletcher, 2007). Our data demonstrate that all these genes, as well as clpB, were downregulated in the htrA mutant, suggesting that the lower oxidative stress tolerance observed for the htrA mutant may be due to downregulation of the chaperone machinery. In a previous study, we report that the mutation of clpB in P. gingivalis also downregulated several genes involved in the stress response, but not htrA. The clpB mutant was sensitive to heat, but not to oxidative and pH stress (Yuan et al., 2007). Thus, several proteins appear to be involved in the molecular mechanism of the oxidative stress response in P. gingivalis, but the details of their action remains to be elucidated.
The relevance of HtrA to cell invasion by P. gingivalis seems to be cell-type dependent, as the invasion rate of the htrA mutant with epithelial cells (KB and GEC) was increased when compared to the parent strain while no difference was observed with endothelial cells (HCAEC). This might be related to the fact that P. gingivalis does not utilize the same trafficking mechanisms within different cell types. For example, in epithelial cells, internalized P. gingivalis cells are found freely in the cytoplasm or within single-membrane-bound vacuoles (Belton et al., 1999; Duncan et al., 1993; Houalet-Jeanne et al., 2001; Lamont et al., 1995; Njoroge et al., 1997), while in endothelial cells, P. gingivalis is internalized in phagosomes and later sequestrated by the autophagy machinery (Dorn et al., 2001). In this study, the htrA mutant induced an autophagic response within the host HCAEC as shown previously for P. gingivalis (Dorn et al., 2001). Both wild-type and mutant cells were found with similar cell morphology in autophagic vacuoles. These results are consistent with the data obtained in the invasion assays in which similar numbers of internalized bacteria were recovered for both wild-type and mutant. These different invasion strategies suggest that P. gingivalis may have a different genetic profile when invading epithelial and endothelial cells, and may gain entry into the different cell types by diverse mechanisms.
In a previous study with HEp-2 cells, which P. gingivalis does not invade, htrA, htpG (PG0045), groEL (PG0520), dnaK (PG1208), grpE (PG1775), PG2167 and PG0419 were all been shown to be upregulated when P. gingivalis was in contact with the HEp-2 cells (Hosogi & Duncan, 2005). Our microarray data demonstrated that these genes are downregulated in the htrA mutant, suggesting that they may not be relevant to internalization and survival of internalized P. gingivalis in epithelial cells. Ultimately, the higher recovery of mutant cells compared to wild-type cells may be due to changes in the expression of other genes affected by the mutation in htrA. Since there are 140 genes upregulated in the htrA mutant, particularly genes involved in cell envelope composition (15), transport and binding of proteins (9), genes of unknown function (25) and hypothetical proteins (35), it is certainly possible that other unknown invasion/virulence-related genes contributed to the increased invasion observed in epithelial cells. In addition, no difference in the expression of fimA, another relevant P. gingivalis adhesin, was observed, as confirmed by the microarray data and negative staining of the htrA mutant (data not shown).
A regulatory role of htrA in virulence gene expression has been previously identified in P. gingivalis. Roy et al. (2006) demonstrated that an htrA-deficient mutant had decreased Arg-gingipain activity after heat-shock treatment. In Staphylococcus aureus, the expression of 
, β and 
haemolysins, in addition to several other virulence factors, were affected by an htrA mutation (Rigoulay et al., 2005). Lyon & Caparon (2004) demonstrated that a mutation of htrA in Streptococcus pyogenes affected the production of the virulence factors SpeB, cysteine protease and the streptolysin S haemolysin. Mutation of htrA has also been shown to affect biofilm formation in Listeria monocytogenes (Wilson et al., 2006) and Streptococcus mutans (Biswas & Biswas, 2005).
In vivo, an L. monocytogenes htrA mutant has been shown to have attenuated virulence in mice compared to the wild-type (Stack et al., 2005; Wilson et al., 2006; Wonderling et al., 2004). Similar results have been observed with htrA mutations in several other bacterial species (Brondsted et al., 2005; Farn & Roberts, 2004; Ibrahim et al., 2004). This probably reflects a requirement for adaptation to an unfavourable and/or changing environment, such as that encountered during disease progression in a host. In the mouse competition assay, the htrA mutant did not survive as well in the mice compared to the wild-type. In addition, when the wild-type and the htrA mutant were injected separately into the mouse abscess model, half as many mice infected with the htrA mutant died as those infected with the wild-type strain. Collectively, these animal data suggest that htrA plays a role in P. gingivalis survival and virulence in vivo.
Gene profiling data demonstrated that there are a total of 253 differentially regulated genes in the htrA mutant, indicating that knocking down the expression of htrA directly or indirectly affects the expression of many other genes. A total of 24 cell-envelope-related genes were differentially regulated in the htrA mutant. Cell-envelope genes are an important class that are likely to be involved in bacterial pathogenesis. They generally function as a dynamic interface between the bacteria and the environment, maintaining the cell structure, protein transport, cellular adhesion and binding to other substrates. It has been shown that the knockout of htrA affects the expression of surface proteins in other bacterial species. For example, Biswas & Biswas (2005) demonstrated that the expression of glucosyltransferases, fructosyltransferases and other surface proteins was affected by an htrA mutation in Streptococcus mutans. Stack et al. (2005) demonstrated that htrA expression in L. monocytogenes is dependent upon the LisRK sensor-kinase, a system known to respond to changes in the integrity of the cell envelope. In addition, capsule formation has been shown to be affected by an htrA mutation in Klebsiella pneumoniae (Cortes et al., 2002). Of significance, our data showed that the htrA mutation altered the expression of transcription regulators PG0173, PG1007 and PG1040, and also PG1431, a regulator of the LuxR family, suggesting that HtrA ultimately may participate in the regulation of several genes in P. gingivalis. Finally, the microarray data were obtained from in vitro cultures of W83 and the htrA mutant, and may not therefore precisely reflect the situation that occurs during invasion and survival in vivo. A more direct analysis using RNA obtained from invading/infecting P. gingivalis may provide additional information as to why the htrA mutant invades epithelial cells better than the wild-type strain and help to explain its low virulence in vivo.
In conclusion, we have demonstrated that htrA in P. gingivalis is responsible for resistance to hydrogen peroxide stress, but not to other stress conditions tested (high temperature and unfavourable pH). The disruption of htrA in P. gingivalis altered the expression of multiple genes, affecting several different cellular functions. Significantly, the downregulation of the chaperone machinery in the htrA mutant may indicate that HtrA in P. gingivalis may play a central role in the oxidative stress response. The htrA mutant did not show a difference in terms of total interaction with two epithelial cell types, and invaded the latter cell types better than the wild-type. In addition, the animal experiments performed demonstrated that the htrA gene is probably involved in the virulence of this pathogen in vivo.
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ACKNOWLEDGEMENTS |
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Edited by: P. E. Kolenbrander
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Received 15 November 2007;
revised 18 December 2007;
accepted 19 December 2007.
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