A universal stress protein of Porphyromonas gingivalis is involved in stress responses and bio¢lm formation Wen Chen, Kiyonobu Honma, Ashu Sharma & Howard K. Kuramitsu Department of Oral Biology, School of Dental Medicine, University at Buffalo, State University of New York, Buffalo, New York, NY, USA
Correspondence: Howard K. Kuramitsu, Department of Oral Biology, School of Dental Medicine, University at Buffalo, State University of New York, Buffalo, New York, NY 14214-3092, USA. Tel.: 11 512 249 5901; fax: 11 716 829 3942; e-mail:
[email protected] Received 27 May 2006; revised 15 July 2006; accepted 18 July 2006. First published online 29 August 2006.
Abstract Porphyromonas gingivalis is recognized as one of the major periodontal pathogens in subgingival plaque, which is implicated in the progression of chronic periodontal disease. We analyzed the role of upsA in P. gingivalis 381 and its uspAdeficient mutant CW301 under various stress conditions. In general, the uspA mutant was less tolerant to a variety of environmental stresses relative to the parental strain. In addition, gene expression of uspA is upregulated during biofilm formation. Biofilm formation of the uspA mutant was also less than that of strain 381. In conclusion, the uspA gene affecting the stress responses of P. gingivalis is required for optimal biofilm formation.
DOI:10.1111/j.1574-6968.2006.00426.x Editor: William Wade Keywords Porphyromonas gingivalis ; universal stress protein (USP); biofilm; stress responses.
Introduction As Porphyromonas gingivalis, along with other pathogenic bacteria in the oral cavity, appears to reside primarily in biofilm structures commonly termed dental plaque (Xie et al., 2000), it is important to understand the molecular basis of biofilm formation by these organisms at the genetic level. Universal stress proteins (USPs) are involved in the stress responses of bacteria and have been recognized as a conserved family (USP family) of bacterial proteins that have also been suggested to be ancestors of the developmentalassociated, DNA-binding, MADS-box proteins of eukaryotes (Mushegian & Koonin, 1996). These proteins are also members of the RecA-dependent DNA protection and repair systems in Escherichia coli (Diez et al., 2000). Considering that biofilm formation is one response to bacterial environmental stress, it would not be surprising if the USP family of proteins played a regulatory role in biofilm formation. Therefore, in the present study, the regulation of the expression of the uspA gene homolog of P. gingivalis was examined. In addition, the gene was inactivated and its role in biofilm formation has been suggested. Finally, this gene was shown to play a role in the stress responses of these organisms.
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Materials and methods Bacterial strains and plasmids All bacterial strains and plasmids are listed in Table 1. Porphyromonas gingivalis strains were maintained anaerobically on blood agar plates containing tryptic soy broth (TSB; Difco Laboratory, Detroit, MI) supplemented with 10% sheep blood (Becton Dickinson and Co., Sparks, MD), hemin (5.0 mg mL1, Sigma-Aldrich, St Louis, MO), menadione (1.0 mg mL1, Sigma-Aldrich) and gentamicin (25 mg mL1, Sigma-Aldrich). Plasmid prtT:Em was constructed in our laboratory previously (unpublished results). Plasmids pUC19 (Invitrogen Corp., Carlsbad, CA), pCR2.1Topo TA cloning vector (Invitrogen) and prtT:Em were maintained in E. coli DH5 in the presence of 50 mg of ampicillin mL1. Escherichia coli S17.1 (Tokuda et al., 1998a) and plasmid pKDCMZ (Nakayama, 1994) were used for conjugation with P. gingivalis 381.
Biofilm-associated gene expression by real-time PCR The expression of potential biofilm-related genes was examined by real-time PCR. The following PCR primers were 2006 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved
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Table 1. Bacteria and plasmids Bacterial strain or plasmid
Description
References
Porphyromonas gingivalis FDC 381 Porphyromonas gingivalis CW301 Porphyromonas gingivalis CW311 Escherichia coli DH5 Escherichia coli S17.1 pUC19 prtT:EM pCR-2.1-TOPO pKDCMZ mpCR-/usp:EM pKusp:lacZ
P. gingivalis wild-type strain, which was isolated at Foysyth Institute P. gingivalis uspA-inactivated strain. EMr P. gingivalis UspA:LacZ fusion protein express strain. CMr, Host strain of gene operation. Helper strain for triparental conjugation Cloning vector for gene operation Vector has EM cassette TOPO TA cloning vector Suicide gene integration vector for P. gingivalis upsA gene inactivated vector by EM cassette insertion upsA::lacZ fusion protein expression vector
Lab strain This study This study Invitrogen Tokuda et al. (1998) Invitrogen Lab collection Invitrogen Nakayama (1994) This study This study
used to detect each gene. uspA: 5 0 -GTG ACC CTG GCT ATT CAT TC-3 0 and 5 0 -GGA CTG ATA TAG ACG TGC-3 0 ; fimA: 5 0 -GTG GTA TTG AAG ACC AGC-3 0 and 5 0 -AAT GTG ATT ACC CTC TCC-3 0 , mfa1: 5 0 - TTT GGT CGG AGC ATT GCT CT-3 0 ; and 5 0 -GCC GAC AGC AGA ATT AAC CT-3 0 . Real-time PCR was performed on cDNA isolated from planktonic and biofilm samples after adjusting OD at 260 nm to 0.1. Real-time PCR was carried out using the above primers, iQ SYBR green Supermix (Bio-Rad Lab) and an iCycler thermal cycler equipped with the MyiQ real-time PCR detection system as per the manufacturer’s recommendations (BioRad Lab). The relative expression levels of each gene transcript were then calculated by normalizing the levels of each specific RNA with the levels of 16S rRNA gene. By normalizing the Ct values for specific genes to the total amount of 16S rRNA gene, all samples were compared, the relative fold change in the samples were calculated using the DDCt method described in the MyiQ real-time PCR detection system (BioRad Laboratories) and each gene expression level in biofilm cells was compared with planktonic cells from the results of these calculations.
Construction of P. gingivalis uspA -deficient mutant CW301 and uspA::lacZ fusion protein expressing strain CW311 The uspA homolog sequence of P. gingivalis W83 was identified by searching the Institute for Genomic Research database (http://www.ncbi.nlm.nih.gov) with the amino acid sequence of the E. coli uspA gene. A pair of primers, 5 0 GCA CAA GCG TTT ATC CAT CG-3 0 and 5 0 -CAT TTG CCG GAT TCA TCA GG-3 0 , was designed based upon the sequence of the uspA gene of P. gingivalis W83. Inactivation of the uspA gene from P. gingivalis 381 was accomplished by insertion of an Em cassette (Fletcher et al., 1995) into the BamHI and KpnI sites, which are 213 and 685 bp downstream, respectively, from the ATG initiation codon of uspA. Erythromycin-resistant transformants would grow only as a result of a double-crossover recombination. In order to confirm that the Em cassette was inserted into the predicted 2006 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved
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sites within the uspA gene on the chromosome of strain 381, Southern blot analysis was carried out as described previously (Chen et al., 2002). Twenty-one erythromycinresistant colonies were obtained. One of them was chosen for further study and designated CW301. The effects described in this study relative to the uspA mutant cannot be polar effects as Northern blot analysis indicates that the uspA gene is transcribed as a monocistronic gene (data not shown). For estimation of protein expression of UspA, a uspA:: lacZ fusion protein expressing strain was constructed as follows: plasmid pSBL20, constructed in our laboratory (Hiratsuka et al., 1998), was digested with BamHI to yield a fragment containing the promotorless lacZ gene. This fragment was inserted into the BamHI site within the uspA gene to yield an in-frame fusion construct, mpCR/uspA::lacZ. The shuttle vector pKDCMZ (Nakayama, 1994) was digested with PstI and KpnI to allow for insertion of a PstI–KpnI fragment from mpCR/uspA::lacZ containing the uspA::lacZ fragment, to yield pKuspA::lacZ. Plasmid pKuspA:lacZ containing the lacZ chimeric gene was then transformed into E. coli S17.1 with selection on Luria–Bertani agar (Difco Laboratory) plates containing chloramphenicol. Conjugation between the constructs and P. gingivalis 381 was carried out as described previously (Tokuda et al., 1998b). Colonies were screened by Southern blot analysis for preliminary confirmation of the correct construction (Chen et al., 2002). One was named CW311 and selected for further study.
Measurement of b-galactosidase activities Porphyromonas gingivalis parental strain 381 and the uspA:: lacZ fusion protein expressing strain were harvested and subjected to toluene treatment (Karunakaran et al., 1997). The toluenized cells were assayed for b-galactosidase activity as previously described by Karunakaran et al. (1997) with ONPG (o-nitrophenyl-b-D-galactoside) as the substrate, and the resultant enzyme activities were expressed as Miller units FEMS Microbiol Lett 264 (2006) 15–21
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(MU). All assays were performed in triplicate and repeated at least three times.
Stress responses Long-term survival in stationary phase Porphyromonas gingivalis 381 and its uspA mutant CW301 were grown to early stationary phase and further incubated in TSB medium anaerobically at 37 1C for several days. Viable cell counts were then determined from day 1 to day 8. Samples for each day were isolated in triplicate. After plating, the plates were routinely incubated anaerobically at 37 1C for 7 days before the viable colonies were counted. The colonies were counted and compared between the wild type and mutant (Farewell et al., 1996). Temperature stress Early stationary phase cultures of each P. gingivalis strain were inoculated into TSB medium and incubated at 43, 37, 30 or 25 1C for 24 or 48 h under anaerobic conditions. Bacterial cell growth at each temperature was then measured at 600 nm. Hydrogen peroxide sensitivity The sensitivity to hydrogen peroxide was estimated as follows: P. gingivalis strains were grown to the mid-log phase (OD600 nm = 0.5) and adjusted to an OD600 nm of 1.0. The P. gingivalis strains were then exposed to a twofold diluted series of H2O2 (0–1280 mM) for 20 min at 37 1C aerobically. The treated bacterial cells were recovered and inoculated onto TS agar plates for evaluation of the number of surviving CFUs. Bacterial cell survival rates for planktonic and biofilm P. gingivalis cells after H2O2 exposure were also calculated in a similar manner.
Biofilm formation assay Static biofilm formation of P. gingivalis strains was examined in 96-well polystyrene plates (Becton Dickinson, Franklin Lakes, NJ) as described previously (Chen et al., 2002). Biofilm formation was scored as the absorbance of CV-stained biofilms at an optical density at 595 nm (OD595 nm) divided by the absorbance of total growth (including biofilm cells and planktonic cells) at OD595 nm. Biofilm formation was also estimated under various stress concentrations. A flow-cell system for biofilm formation was also carried out as described previously with some modifications (Chen et al., 2002). Briefly, flow-cell chambers (Stovall Life Science Inc., Greensboro, NC) were set up in an anaerobic chamber. The flow cells were washed overnight with sterilized distilled water and with 1 : 3 diluted TSB for 30 min. The flow system was stopped and late-log phase cultures with the same OD600 nm (0.5 mL) of P. gingivalis 381 and mutant CW301 were injected into each track. Bacteria were permitted to attach statically for 3 h. After that, the flow cells were returned to their original orientations and a flow of 1/3 TSB (200 mL min1) was begun. After 36 h of flow, one flow cell of each strain was removed from the chamber for staining with BacLight Live/Dead dye (Molecular Probes, Eugene, OR). 0.5 mL of a 1 : 1 mixture of the two dyes was diluted 1000-fold in distilled water and added to the cells for 10 min. After removal of the unbound dye, the cells were observed with a Leica TCS 4D confocal microscope (Leica Laser Technik, Heidelberg, Germany).
Statistical analysis Strain comparison for biofilm formation and MIC calculations were analyzed using Student’s t-test.
Results Regulation of the uspA gene
Sensitivity to tetracycline Tetracycline sensitivity of P. gingivalis strains was estimated by a modification of the protocol previously described by Miyake et al. (1995). Briefly, P. gingivalis strains were incubated to the mid-log phase and adjusted to an OD of 0.1 at 595 nm. Tetracycline was dissolved in TSB medium and twofold dilutions from 100 mg mL1 to 50 ng mL1 were added to the wells of a 96-well cell culture plate. The P. gingivalis cells were then inoculated into the tetracycline solutions with 1/10 volumes of inocula and incubated for 8 days anaerobically. The minimum inhibitory concentration (MIC) of tetracycline was defined as the lowest concentration of tetracycline that produced complete inhibition of growth. FEMS Microbiol Lett 264 (2006) 15–21
An ORF homologous to the E. coli uspA genes appeared to be upregulated during biofilm formation by P. gingivalis DNA microarray analysis (data not shown). Therefore, to confirm the upregulation of this gene during biofilm formation in vitro, real-time PCR was carried out (Fig. 1). The results confirmed the increased expression of the uspA gene during biofilm formation (1280-fold) and also indicated that the two fimbrial structural genes fimA and mfa1, coding for the major and minor subunit proteins, respectively, appeared to be upregulated approximately 45-fold during this process. Interestingly, expression levels of the fimA and mfa1 fimbrial structural genes were 70 times lower than upsA gene expression after 48 h of biofilm formation (Fig. 1). Nevertheless, these results suggest a role for the 2006 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved
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1.00E+00
10 000
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1
*
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*
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0.1 fimA
mfa1
uspA
* 1.00E-05
Fig. 1. Determination of gene expression levels of three biofilmassociated genes in biofilm cells and planktonic cells of Porphyromonas gingivalis strain 381 by real-time PCR. P o 0.01.
1.00E-06 Day 1
Day 3
Day 4
Day 6
Day 7
Day 8
Fig. 2. Measurement of the long-term stationary phase survivability of Porphyromonas gingivalis 381 and uspA mutant CW301. Viable bacterial cells were determined from day 1 to day 8. P o 0.05.
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P. g. FDC 381
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P.g. CW301
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fimbrial genes in biofilm formation under these conditions. These fimbrial genes are thought to be involved in an early stage of biofilm formation by cell surface attachment, coaggregation (Lamont et al., 2002) or autoaggregation (Murakami et al., 1996). As inflamed periodontal tissues are apparently associated with elevated temperatures (Meyerov et al., 1991), it was of interest to determine whether increased temperatures affected UspA expression. For estimation of gene expression of upsA in P. gingivalis at the translational level, a uspA::lacZ fusion protein expressing strain CW311 was constructed and evaluated by b-galactosidase assays at various temperatures. The expression of UspA appeared to be similar at 25 1C (20.33 MU) compared with at 37 1C (19.43 MU). In addition, a further increase in temperature to 42 1C did not result in additional induction of UspA expression (17.77 MU). The expression of UspA was also compared in biofilm and planktonic cells. These results confirmed the results using transcription analysis of mature biofilms and suggested that uspA was upregulated during the formation of biofilms by P. gingivalis (data not shown).
1 0.8 0.6 0.4 0.2 0 24 h
48 h
43 °C
24 h
48 h 37 °C
24 h
48 h 30 °C
24 h
48 h 25 °C
Role of the uspA gene in stationary phase survivability and heat resistance in P. gingivalis
Fig. 3. Determination of Porphyromonas gingivalis growth responses to temperature stress. Each P. gingivalis strain was incubated under various temperatures for 24 or 48 h. Bacterial cell growth was measured at OD600 nm. P o 0.05.
The growth rate of mutant CW301 was similar to that of wild-type 381 in TSB medium under standard growth conditions. One general environmental stress that bacteria are subjected to is that produced during cultivation into the stationary phase. Therefore, using a relatively long-term survival assay (Farewell et al., 1996), strains 381 and CW301 were grown to the early stationary phase and cell viability was determined following prolonged incubation (Fig. 2). The results for the first 4 days in the stationary phase showed decreased viabilities for both strains. How-
ever, the cell viabilities of CW301 were c. 10%, 1%, 0.1% and 0.001% of that of wild-type 381 at days 4, 5, 6 and 7, respectively. For estimation of the relative sensitivity of the uspA mutant to heat stress, strains 381 and CW301 were cultured at 25, 30, 37 and 43 1C for 24–48 h. In the first 24 h, the uspA mutant showed significantly slower growth than wild-type strain 381 at 30 and 43 1C (Fig. 3). The mutant also showed significantly reduced growth compared with the parental
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strain at 30 and 25 1C after 48 h of culture. Interestingly, these two strains did not show significant growth difference at 37 1C, the normal temperature of the human oral habitat. These results suggest that the uspA gene may play a role in the growth of P. gingivalis at suboptimal temperatures.
P. gingivalis uspA mutant sensitivity to oxidative stress Porphyromonas gingivalis strains were exposed to H2O2 for estimation of survival to oxidative stress. The uspA mutant survival rate was drastically decreased relative to the wildtype strain to 16.75% after exposure to 20 mM H2O2 (Fig. 4a). This result indicated that the H2O2 sensitivity of P. gingivalis was increased following upsA inactivation. Porphyromonas gingivalis H2O2 sensitivity was also compared for biofilm and planktonic cells. A comparison of the
H2O2 sensitivities of P. gingivalis 381 biofilms showed greater resistance than planktonic cells (Fig. 4b). A similar trend was observed for uspA mutant CW301 (Fig. 4c). The H2O2 sensitivity of CW 301 was also comparable to that of the parental strain in biofilms in the presence of a range of the oxidant but no significant differences were observed (data not shown). These results are compatible with a role for UspA in the sensitivity of P. gingivalis to H2O2, and suggests that this effect is more pronounced in planktonic relative to biofilm cells.
The uspA gene alters the antibiotic sensitivity in P. gingivalis The tetracycline MIC for strain 381 was c. 0.28 mg mL1. The MIC against mutant CW301 was o 0.035 mg mL1 following 8 days of incubation. Therefore, the uspA gene appears
(a) 100
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Fig. 4. Porphyromonas gingivalis resistance to H2O2 exposure. (a) Porphyromonas gingivalis strains were exposed to twofold serial dilutions of H2O2 (0–1280 mM) for 20 min at 37 1C aerobically before inoculation onto TS blood agar plates. P o 0.05. (b and c) Bacterial cell survival rates for planktonic or biofilm cells of P. gingivalis strains following H2O2 exposure were also calculated. P o 0.05. (b) Comparison of the survival of biofilm or planktonic P. gingivalis 381 cells with different concentrations of H2O2. (c) Biofilm cells of mutant CW301 were compared with planktonic CW301 cells.
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Fig. 5. Biofilm phenotype analysis by CLSM. Porphyromonas gingivalis strains formed biofilms after 36 h of incubation in the flow-cell system as described in the text. Biofilms of each strain were stained with BacLight Live/Dead stain (Syto 9 and propidium iodide) and analyzed by CLSM. (a) P. gingivalis 381 and (b) P. gingivalis uspA mutant CW301.
to affect the sensitivity of P. gingivalis to antimicrobial agents such as tetracycline.
Role of the uspA gene in P. gingivalis biofilm formation Biofilm formation by P. gingivalis 381 and its uspA mutant were evaluated under both static and flow-cell conditions. Strain CW 301 showed significantly less biofilm formation than parental strain 381 under static growth conditions (data not shown). Porphyromonas gingivalis biofilm morphology was also evaluated by confocal laser scanning microscopy (CLSM) following biofilm formation under flow-cell conditions. In agreement with the results from the static biofilm assays, P. gingivalis 381 formed more abundant biofilms with bacterial cell clusters than mutant CW301 under flowing conditions (Fig. 5a). In contrast, the upsA mutant attached to the glass surface as a monolayer and did not appear to form microcolonies or cell clusters as did the parental strain (Fig. 5b). Therefore, these results suggested that the uspA gene, either directly or indirectly, influences biofilm formation in P. gingivalis.
Discussion The role of the uspA homolog in the physiology of P. gingivalis 381 as a universal stress protein was confirmed in this study. Based upon preliminary microarray assays, it was suggested that the strain 381 uspA gene might be upregulated during biofilm formation. This was confirmed in the present investigation utilizing real-time RT-PCR analysis (Fig. 1). In addition, the utilization of a uspA::lacZ construct confirmed that such upregulation also occurred at the translational level, consistent with upregulation at the transcriptional level during transformation from the planktonic to the biofilm cellular status, although the magnitude of the changes detected was not great under static biofilm conditions. As biofilm formation may be a form of cellular adaptation to environmental stress, it is not surprising that 2006 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved
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the uspA gene appeared to be upregulated during this developmental process. The uspA gene appears to be involved in the resistance to a variety of environmental stresses as the uspA mutant grew slower than the parental strain at suboptimal temperatures and in the presence of tetracycline. This also indicates the adaptation of the organism to the normal temperature of the human oral cavity. Furthermore, the long-term survivability of the uspA mutant was significantly less than the parent strain 381 (Fig. 2). This further indicates a role for the uspA gene in resistance to the environmental stresses produced during the stationary phase of bacterial growth. The uspA mutant is also significantly more sensitive than its parental strain to H2O2 concentrations greater than 20 mM (Fig. 4a). In addition, biofilms of strains 381 and CW301 showed greater resistance to H2O2 than their respective planktonic cells (Fig. 4b and c). On the other hand, biofilms of both parental strain 381 and its uspA mutant showed sensitivities similar to H2O2 (data not shown). It is recognized that bacteria in biofilms are more resistant to various chemical agents, antibiotics or host immune factors than planktonic cells (Lewis, 2001). The present results clearly indicated that biofilm formation by the uspA mutant is significantly attenuated relative to its parental strain 381 in both static and flow-cell systems. It is likely that biofilm formation is an important factor in the resistance of some organisms to environmental stress. Therefore, the present results suggest that the upsA gene is an important factor in P. gingivalis biofilm formation, at least in vitro. Whether or not this is also the case in vivo requires further investigation. In conclusion, we have demonstrated that the uspA gene is associated with P. gingivalis resistance to a variety of environment stresses as predicted by its homology to other uspA gene homologs. In addition, we demonstrate, for the first time, that this stress protein can also influence biofilm formation by P. gingivalis in vitro. This further supports the hypothesis that biofilm formation may be a form of bacterial response to environmental stress. FEMS Microbiol Lett 264 (2006) 15–21
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Acknowledgement This study was supported in part by NIH Grant DE08293 and a contract from Colgate-Palmolive.
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