Department of Oral Microbiology, Malmö University, S-21421 Malmö, Sweden1
Department of Oral Biology, University of Manitoba, 780 Bannatyne Ave, Winnipeg, Manitoba, Canada R3E 0W22
Author for correspondence: Ian R. Hamilton. Tel: +1 204 789 3615. Fax: +1 204 789 3948. e-mail: ihamilt{at}cc.umanitoba.ca
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ABSTRACT |
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Keywords: stress, stress proteins, cross-protection, Streptococcus mutans
Abbreviations: 2DE, two-dimensional polyacrylamide electrophoresis; BM, basal medium (the salts, vitamins and amino acids of MM4 medium); IOD, integrated optical density
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INTRODUCTION |
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During short-term exposure to acidic environments, S. mutans maintains pH homeostasis by proton extrusion from the cell via the membrane-associated, proton-translocating ATPase (H+/ATPase) (Bender et al., 1986 ; Hamilton & Buckley, 1991
), and by acid end-product efflux (Carlsson & Hamilton, 1996
; Dashper & Reynolds, 1996
). Sustained growth at pH 5·5, however, results in increased H+/ATPase (Bender et al., 1986
; Belli & Marquis, 1991
; Hamilton & Buckley, 1991
) and glycolytic activity (Hamilton & Ellwood, 1978
), which is accompanied by a lowering of the pH optimum for sugar transport and glycolysis (Hamilton & Buckley, 1991
), and increased lactic acid formation (Hamilton, 1987
). Moreover, unlike the enteric bacteria (Padan et al., 1981
), S. mutans does not maintain a constant intracellular pH during a falling external pH, but exhibits a relatively consistent transmembrane pH gradient (~1·0 unit) sustained by a carbon source (Hamilton, 1986
, 1990
; Hamilton & Buckley, 1991
; Dashper & Reynolds, 1992
). Thus, growth at pH 5·5 results in the induction of metabolic changes that permit the organism to maintain the transmembrane pH gradient at lower pH values (Hamilton, 1986
; Hamilton & Buckley, 1991
; Dashper & Reynolds, 1992
)
Recently, we have demonstrated that exposure of exponential-phase cells of S. mutans LT11 to a pH change from 7·5 to 5·5 resulted in the induction of an acid-tolerance response over a 2 h period that increased cell survival at pH 3·0 (Svensäter et al., 1997 ). The pH change resulted in significant alterations in protein synthesis: pulsing the cells with 14C-labelled amino acids at intervals during the 2 h period, followed by extraction and one-dimensional polyacrylamide gel electrophoresis, revealed the upregulation of 36 proteins, with 25 of these being acid-responsive proteins appearing within the first 30 min of the pH change (Hamilton & Svensäter, 1998
). The synthesis of all but two of the proteins was transient during the 2 h adaptation period. The identity of the proteins is not known, but molecular mass comparisons suggested that both acid-specific proteins (i.e. components of the H+/ATPase) and general stress proteins (i.e. heat-shock proteins) are present in extracts of the acid-induced cells.
The coordinated induction of general and specific proteins by a variety of stress conditions is relatively well characterized in Escherichia coli and Bacillus subtilis, particularly with respect to the heat-shock proteins (Hecker et al., 1996 ; Yura et al., 1993
). A number of stress responses have been identified in Lactococcus lactis and it is clear that there is an overlap in the protective mechanisms induced by various conditions (Rallu et al., 1996
). Carbohydrate-starved stationary-phase cells of L. lactis IL1403 exhibited increased resistance to acid, heat, ethanol and osmotic and oxidative stress (Hartke et al., 1994
), while UV-irradiated cells of the same strain showed enhanced survival against acid, ethanol, hydrogen peroxide and heat (Hartke et al., 1995
). An overlap between the heat-shock and salt responses has also been reported for L. lactis MG1363, with all salt-stress-induced proteins also subject to induction by heat stress (Kilstrup et al., 1997
).
In the current study, we were interested in the relationship between the acid-tolerance response generated by S. mutans and the response of the organism to other adverse conditions, such as, salt, heat, starvation and oxidative stress. Two approachs were taken: an examination of whether reciprocal cross-protection exists between these various stresses, and a comparison of the stress-induced proteins formed under the various conditions. For the latter study, cells were exposed to stress in the presence of 14C-labelled amino acids and the protein extracts subjected to two-dimensional polyacrylamide electrophoresis (2DE) and autoradiography. Comparative analysis revealed three groups of proteins: general stress proteins enhanced under all conditions, those specific to the particular stress, and those induced by more than one, but not all, stress conditions.
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METHODS |
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Adaptation conditions.
Cells were grown to mid-exponential phase in MM4 with 20 mM glucose at pH 7·5; the cells were rapidly concentrated by centrifugation (5000 g for 5 min) and washed in sterile saline (0·16 M NaCl), and aliquots (~108 cells ml-1) were inoculated into 5 ml fresh MM4 medium with 20 mM glucose buffered with 40 mM phosphate/citrate buffer under the following conditions: (a) pH 5·5, (b) 42 °C, (c)+0·2 M NaCl, and (d)+2 mM H2O2. Also, three starvation conditions were tested: S-1, 20% BM+20 mM glucose; S-2, 20% BM+4 mM glucose; and S-3, 20% BM without glucose. Both unadapted control and test cells were incubated at pH 7·5 and 37 °C, except (a) and (b), respectively. Unless otherwise specified, incubation was for 2 h and the pH of the suspensions did not change by more than ±0·2 pH units.
Challenge conditions.
Adapted and unadapted exponential-phase cells were harvested and rapidly washed in sterile saline by centrifugation (5000 g for 5 min), then added at a constant cell concentration (~108 cells ml-1) to 5 ml fresh MM4 medium and incubated for 2 h under the following conditions: (a) pH 3·5, (b) 50 °C, (c)+2 M NaCl, (d)+15 mM H2O2, and the three starvation conditions listed above. Both unadapted control and test cells were incubated at pH 7·5 and 37 °C, except (a) and (b), respectively. Incubation was for 2 h and the pH of the suspensions did not change by more than ±0·2 pH units.
Cross-protection protocols.
Cross-over experiments were undertaken to determine whether (i) the prior induction of the acid-tolerance response by exponential-phase cells of S. mutans H7, achieved by incubating pH 7·5-grown cells at pH 5·5 for 2 h, would affect survival under challenge with the other stresses, and (ii) the prior adaptation to the other stresses would affect the ability of the cells to survive a 2 h exposure in MM4 medium buffered at pH 3·5. Following exposure to the stress, cell samples were diluted and plated on trypticase agar (g l-1: trypticase, 10; yeast extract, 2; Na2CO3, 2; NaCl, 5; glucose, 2; agar, 10; buffered at pH 7·2). The percentage of cell survivors was calculated by comparison with viable cell counts of the culture suspension just prior to challenge. The data shown are the means of at least three determinations±standard error.
14C labelling of stress proteins.
The assays for the induction of the various stress responses followed the adaptation conditions above with the exception that only starvation condition S-1 (20% BM+20 mM glucose) was tested. Cells were grown to mid-exponential phase (OD600 0·6; the stationary phase OD600 was 1·2) in MM4 with 20 mM glucose at pH 7·5, centrifuged and resuspended in glucose- and buffer-free MM4. The cell suspension was added to the different adaptation media to give a concentration of 5x108 cells ml-1 in a total volume of 2·5 ml. Immediately after addition of the cell suspension, 50 µCi (1850 kBq) of a 14C-labelled amino acid mixture (Amersham) was added to the adaptation medium and incubation carried out for 30 min. Then 1 mg chloramphenicol was added to stop protein synthesis, and the cell suspension was rapidly cooled in ice. The suspensions were centrifuged at 15000 g for 10 min; the cells were washed twice and resuspended in 0·5 ml 10 mM Tris/HCl, pH 6·8, with 1 mM EDTA and 5 mM MgSO4. The cells were stored at -80 °C until used. The frozen cells were thawed, centrifuged and resuspended in 0·5 ml lysis buffer containing 8 M urea, 2% (v/v) Nonidet P-40, 62 mM DTT and 2% (v/v) Pharmalyte, pH range 47 (Pharmacia Amersham Biotech). Proteins were extracted by vortexing 0·5 ml cell samples in lysis buffer with 0·2 mm glass beads (1:1, v/v) six times for 30 s with cooling between vortexing. The samples were centrifuged at 2500 g for 5 min to remove the beads. The bead-free supernatant was centrifuged at 15000 g for 15 min at 4 °C and the resultant cell-free extracts were frozen until used.
2DE.
The first-dimension isoelectric focusing was performed using the Multiphor II horizontal electrophoresis apparatus connected to an EPS 3500 XL Power Supply and Multitemp II thermostatic circulator (Amersham Pharmacia Biotech). Immobiline DryStrip linear immobilized pH gradient (IPG) gel strips (18 cm) with a pH range of 47 were rehydrated in 330 µl sample buffer (SB) containing 8 M urea, 2% (v/v) Nonidet P-40, 10 mM DTT and 2% (v/v) Pharmalyte, pH range 47 (Pharmacia Amersham Biotech). Rehydration was carried out overnight at room temperature in a reswelling casette with strips covered with silicone oil to avoid evaporation. The reswollen strips were rinsed with deionized water and carefully blotted with wet filter paper to remove excess rehydration solution. The strips were placed into the DryStrip kit on the Multiphor II and electrode strips loaded with 300 µl deionized water. The 14C-labelled samples were diluted in the above SB buffer and applied in sample cups under silicone oil at the anodic end of the gel strip. The same amount of sample radioactivity (500000 c.p.m.) was loaded on each gel, corresponding to 3060 µg protein. The proteins were focused overnight at 15 °C under a protective layer of silicone oil using 150 V for 1 h, 300 V for 3 h, 600 V for 3 h, 1200 V for 12 h and 3500 V for 18 h. After focusing, the IPG gel strips were immediately frozen between plastic films at -80 °C.
The second-dimension electrophoresis was carried out essentially as described by Laemmli (1970) with 1214% polyacrylamide gradient gels (185x200x1·0 mm). Immediately before SDS-PAGE, the IPG gel strips were shaken gently in 10 ml 50 mM Tris/HCl (pH 6·8), 2% (w/v) SDS and 26% (v/v) glycerol for 2x15 min. DTT (25 mg) was added to the first equilibration solution, and iodoacetamide (450 mg) and bromophenol blue to the second. The IPG strips were quickly loaded on top of the gel and overlaid with 1% (w/v) molten agarose (Bio-Rad). The separation was performed at 10 °C overnight at 40 mA until the bromophenol blue dye front reached the bottom of the gels. The gels were fixed in 40% methanol and 7·5% acetic acid for 45 min, vacuum-dried at 60 °C for 3 h and then subjected to autoradiography.
The apparent isolelectric point and molecular mass of protein spots were determined relative to those of known protein standards. The pI standards were purchased from Amersham Pharmacia Biotech (carbamylated carbonic anhydrase and glyceraldehyde-3-phosphate dehydrogenase) and co-electrophoresed with bacterial proteins in some of the first-dimension gels. The gels containing pI standards were silver stained according to the procedure recommended by the manufacturer before autoradiography. 14C-labelled molecular mass standards were obtained from Amersham (myosin, 220 kDa; phosphorylase b, 97·4 kDa; bovine serum albumin, 66 kDa; ovalbumin, 46 kDa; carbonic anhydrase, 30 kDa; trypsin inhibitor, 21·5 kDa; and lysozyme, 14·3 kDa) and run alongside the second-dimension gels.
Autoradiography.
The dried gels were exposed to X-ray film (Hyperfilm b-max, Amersham) for 14 d and films developed with Kodak D19.
Image analysis.
Analysis of digitized images, including spot finding, quantification and matching, was done by using Visage 2-D Electrophoresis Image Analysis software version 4.6 J (Millipore) on a Sun Sparc work station. A reference autoradiogram was chosen and each of the other autoradiograms was matched to it using the BioImage software. The matching procedure consisted of manually selecting 20 tiepoints or anchor proteins on the images and then allowing the machine to automatically match the images. Proteins of known molecular mass were then used as standards to generate pI and molecular mass values for all the spots in the matched gels by interpolation and extrapolation. The integrated optical density (IOD) of each protein, expressed as a percentage of the total area of blackening of the film attributed to proteins (IOD%), was calculated along with pI and molecular mass for each individual spot. Each stress experiment was carried out at least twice with different cell cultures, and only spots showing a consistent change in IOD% of at least twofold were taken into account.
Analyses.
Protein was determined by the method of Bradford (1976) with the appropriate concentration of lysis buffer in the standards as described by Fey et al. (1997)
.
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RESULTS |
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The converse of the above experiment was also undertaken to determine whether prior adaptation to the other stresses would augment or diminish cell survival after a subsequent 2 h acid challenge at pH 3·5. As seen in Fig. 1, as expected the pH 5·5-adapted cells (Acid) showed increased survival over the pH 7·5 control cells. Also, prior exposure to the other stresses tested, except a temperature increase to 42 °C (Heat) and the addition of 2 mM H2O2 (Oxid), stimulated survival at pH 3·5 compared to the control cells. Particularly noticeable was the effect of starvation conditions with cells pre-incubated in 20% BM containing 4 mM glucose (S-2) showing a 12-fold stimulation of survival. Even cells incubated in 20% BM without glucose (S-3) showed a sevenfold increase in survivors over the control cells incubated in 100% BM with 20 mM glucose (Con). This demonstrates a clear relationship between starvation and acid tolerance.
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DISCUSSION |
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In this study, the reproducibility of the gels, as determined by the repeated analysis (more than six times) of control and acid-stressed cell extracts, was substantially improved by the use of the precast Immobiline pH strips and by making the extraction buffer compatible with that used in the first-dimension isoelectric focusing separation. The gels in Fig. 2 were generated with a pH range of 47 in the first dimension and routinely resulted in the separation of 420575 proteins with the various stress conditions. Preliminary studies with first-dimension separation over the pH range 610 revealed only a total of 1520 basic proteins, none of which were enhanced during acid shock from pH 7·5 to 5·5. This indicates that the 69 proteins enhanced twofold or more by acid in Fig. 2
constitute the major acid-responsive proteins in the organism. Although the cell extracts from the other stress conditions were not subjected to first-dimension separation in the pH 610 range, the relative paucity of basic proteins in the organism suggests that the vast majority were observed in this study.
In examining the protein profiles in Fig. 2, one is struck by relatively few general stress proteins (six) enhanced twofold or more by all five stress conditions, although the number of stress-specific proteins ranged from 15% to 39% of the enhanced proteins and a significant number were induced by two to four conditions. Raising the threshold to only those proteins enhanced fivefold or more (Table 3
) eliminated 66% of the proteins in the twofold enhanced group and reduced the general stress proteins from six to two (proteins 118b and 141). A higher proportion of stress-specific proteins (37 of 67, or 55%) survived this analysis, the highest number being associated with acid (19 ASPs) and oxidative stress (12 OxSPs). Furthermore, analysis of the 18 proteins whose synthesis was diminished fivefold or more (Table 4
) revealed that most of these proteins (14) were specific to the stress applied. These experiments permit the analysis of alterations in the relative rate of synthesis of particular proteins compared to all other proteins and do not attempt to assess the absolute synthetic rate of these proteins. Furthermore, we are assuming that this approach identifies proteins (enzymes) whose upregulation is indicative of increased metabolic activity in the cells under the particular stress and which may, therefore, be more important than the downregulated proteins (Bloomberg, 1997
). This, of course, may be an incorrect assumption.
Currently, the identity of the various proteins upregulated under the various stress conditions is not known; however, the nature of the stress does point to a number of possible proteins associated with physiological responses. For example, prolonged acidification of S. mutans cells results in the increased specific activity of the membrane F1 H+/ATPase involved in proton efflux during pH homeostasis (Belli & Marquis, 1991 ; Hamilton & Buckley, 1991
). It is conceivable that the synthesis of the membrane F1 ATPase subunits, previously isolated by Sutton & Marquis (1987)
, could be upregulated during the initial acid-shock period. Examination of Table 3
suggests that the
subunit (58 kDa) might be protein 357, enhanced 8-fold, while the ß subunit (52 kDa) might be protein 503 or protein 504, upregulated 13- and 8-fold, respectively. The
subunit (41 kDa) could be associated with either protein 135 or protein 511, while the
(27 kDa) and
(18 kDa) subunits might be associated with proteins 120 and 88, respectively.
The cross-protection experiments with S. mutans H7 demonstrated that a shift in pH from 7·5 to 5·5 did not protect cells against challenges by salt, oxidation, heat and starvation. Interestingly, the reverse situation, in which the cells were subjected to a 2 h adaptation to the five stress conditions followed by exposure to a challenge pH of 3·5 for 2 h, resulted in varying levels of cross-protection, except for heat shock and oxidative stress (Fig. 1). The other stresses, however, promoted survival at pH 3·5, with particularly notable protection (12-fold) afforded by diluting the basal medium (BM) fivefold and decreasing the glucose from 20 to 4 mM (starvation condition S-2). Even in the absence of a carbon source (starvation condition S-3), a sevenfold increase in survivors was observed. Since BM was comprised of salts, vitamins and amino acids, it is likely that, in addition to the carbon source, the concentration of key peptides and/or amino acids may be an important factor in the acid-tolerance response in S. mutans (Gale & Epps, 1942
; Hersh et al., 1996
).
The overlap between acid and starvation responses is well known and considerable information is now available on the responses to various nutrients by E. coli (Nyström, 1993 ; VanBogelen et al., 1990
), Sal. typhimurium (Spector & Foster, 1993
), B. subtilis (Bernhardt et al., 1997
) and L. lactis (Hartke et al., 1994
, 1995
, 1996
). The acid protection afforded to S. mutans by the starvation response (Fig. 1
) has particular significance in the dental plaque environment. Depending on the age of plaque biofilm, the ingestion of dietary carbohydrate can result in a rapid reduction in pH to values near pH 4, and this pH can be sustained for some time at sugar concentrations above 1% (Yamada et al., 1980
). Thus, one can speculate that as the plaque pH and carbohydrate concentration decrease, the cells receive at least two signals to induce the acid response: one triggered by acid and the other by the concentration of the carbon source. The combined effect would generate a level of acid tolerance greater than that achieved by either signal alone. The response to reductions in the available peptide/amino acid supply is less clear. The reduction in the amino acid pool is probably responsible for the starvation response seen in Fig. 2C
since amino acids are known to influence pH homeostasis (Gale & Epps, 1942
; Hersh et al., 1996
). Furthermore, it is not yet known whether carbon and amino acid deprivation induce separate responses, although work with other bacteria suggests that the induction of specific proteins is regulated by individual nutrient components (Nyström, 1993
; Spector & Foster, 1993
).
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ACKNOWLEDGEMENTS |
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Received 20 May 1999;
revised 6 September 1999;
accepted 24 September 1999.