Multiple stress responses in Streptococcus mutans and the induction of general and stress-specific proteins

Gunnel Svensäter1, Bodil Sjögreen1 and Ian R. Hamilton2

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


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The authors have previously demonstrated that Streptococcus mutans shows an exponential-phase acid-tolerance response following an acid shock from pH 7·5 to 5·5 that enhances survival at pH 3·0. In this study the response of S. mutans H7 to acid shock was compared with the responses generated by salt, heat, oxidation and starvation. Prior induction of the acid-tolerance response did not cross-protect the cells from a subsequent challenge by the other stresses; however, prior adaptation to the other stresses, except heat (42 °C), protected the cells during a subsequent acid challenge at pH 3·5. Starvation by fivefold dilution of the basal medium (BM) plus fivefold reduction of its glucose content increased the numbers of survivors 12-fold, whereas elimination of glucose from fivefold-diluted BM led to a sevenfold enhancement compared to the control cells; this indicated a relationship between the acid and starvation responses. The stress responses were further characterized by comparing the 2D electrophoretic protein profiles of exponential-phase cells subjected to the various stress conditions. Cells were grown to exponential phase at pH 7·5 (37 °C) and then incubated for 30 min under the various stress conditions in the presence of 14C-labelled amino acids followed by cell extraction, protein separation by 2D gel electrophoresis and image analysis of the resulting autoradiograms. Using consistent twofold or greater changes in IOD% as a measure, oxidative stress resulted in the upregulation of 69 proteins, 15 of which were oxidation-specific, and in the downregulation of 24 proteins, when compared to the control cells. An acid shock from pH 7·5 to 5·5 enhanced synthesis of 64 proteins, 25 of them acid-specific, while 49 proteins exhibited diminished synthesis. The dilution of BM resulted in the increased formation of 58 proteins, with 11 starvation-specific proteins and 20 showing decreased synthesis. Some 52 and 40 proteins were enhanced by salt and heat stress, with 10 and 6 of these proteins, respectively, specific to the stress. The synthesis of a significant number of proteins was increased by more than one, but not all stress conditions; six proteins were enhanced by all five stress conditions and could be classified as general stress proteins. Clearly, the response of S. mutans to adverse environmental conditons results in complex and diverse alterations in protein synthesis to further cell survival.

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


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Bacteria in the biofilms on teeth (dental plaque) are subjected to cycles of acid shock resulting from the rapid formation of acid end-products generated during the metabolism of dietary carbohydrate by the acidogenic oral microflora. The rate of acid formation in human plaque, as measured by in vivo pH telemetry, shows that the intake of carbohydrates can lower the plaque pH from 7 to 4 in as little as 3 min depending on the age of the biofilm and the concentration of the carbohydrate (Imfeld & Lutz, 1980 ; Yamada et al., 1980 ; Jensen et al., 1982 ). Furthermore, the frequent ingestion of sugar is associated with a lowering of the ‘resting’ plaque pH and an increase in dental caries. Early studies (Stephan, 1944 ) demonstrated that the microflora associated with high caries activity was not only capable of rapid plaque acidification following the ingestion of sugar, but was also tolerant to the low resting plaque pH. Thus, it is not surprising that acid tolerance is an important ‘virulence’ property associated with cariogenic bacteria, such as Streptococcus mutans, Lactobacillus species and certain non-mutans streptococci (Bowden, 1991 ; Sansone et al., 1993 ; van Houte et al., 1996 ).

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.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Bacterial strain and media.
S. mutans H7 was isolated from an approximal caries lesion at pH 5·0 and maintained anaerobically in complex liquid medium containing (per litre): tryptone (10 g), yeast extract (5 g) and 20 mM glucose buffered with 40 mM phosphate/citrate buffer as previously described (Svensäter et al., 1997 ). Comparative studies on cross-protection and 14C-labelled amino acid incorporation into cellular protein during stress were carried out with cells growing anaerobically in a minimal medium (MM4) containing six amino acids (glutamate, serine, cysteine, valine, leucine, asparagine) to which was added various amount of glucose (Hamilton & Svensäter, 1998 ). In the context of this paper, the basal medium (BM) refers to the salts, vitamins and amino acids of MM4 medium.

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 4–7 (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 4–7 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 4–7 (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 30–60 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 12–14% 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) .


   RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Cross-protection
An adaptive response to one stress can often lead to cross-protection against another stress (Hartke et al., 1994 ; Leyer & Johnson, 1993 ); hence we were interested in the relationship of the acid response in S. mutans H7 to other stress responses. We therefore tested whether prior adaptation to acid (pH 7·5 to 5·5) would affect survival under challenge with other stresses. Incubation of pH 7·5-grown exponential-phase cells at pH 5·5 for 2 h resulted in the expected induction of the exponential-phase acid-tolerance response, with fourfold more survivors at pH 3·5 than for control cells maintained at pH 7·5 (data not shown). However, with exception of heating at 50 °C, which significantly reduced cell numbers at pH 3·5, adaptation to acid stress had no significant effect on survival when cells were challenged with 2 M NaCl or 15 mM H2O2, or with fivefold diluted BM with 20, 4 or 0 mM glucose (data not shown).

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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Fig. 1. Effect of prior adaptation to other stresses on the survival of S. mutans cells following a subsequent challenge at pH 3·5 for 2 h. With the exception of the control cells (Con), maintained at pH 7·5 in 100% BM+20 mM glucose, cells were exposed to the other stresses for 2 h prior to the acid challenge. Stress conditions: Acid, pH 5·5; Salt, 0·2 M NaCl; Oxid, 2 mM H2O2; Heat, 42 °C. Starvation conditions: S-1, 20% BM+20 mM glucose; S-2, 20% BM+4 mM glucose; S-3, 20% BM without glucose. All incubations except that for the heat stress were at 37 °C.

 
2DE analysis
The stress responses in S. mutans H7 were further characterized by comparing the expression of proteins in exponential-phase cells during the initial 30 min period immediately following the application of stress by acid, salt, H2O2, heat and a fivefold dilution of BM (starvation condition S-1). Just prior to the application of each stress condition, the growth medium was supplemented with 14C-labelled amino acids and incubation continued for 30 min before protein synthesis was arrested with chloramphenicol, and the cells extracted and the proteins separated by 2DE. The resultant gels were then subjected to autoradiography and image analysis. Duplicate experiments were compared and proteins whose expression was enhanced at least twofold were identified for each of the conditions. As seen in Fig. 2B and Table 1, exposure of cells to 2 mM H2O2 resulted in the enhanced synthesis of 69 proteins compared to the control cells maintained at pH 7·5 (37 °C) (Fig. 2A), 15 of which were specific to oxidative stress. Acid stress, induced by a pH change from 7·5 to 5·5, resulted in the enhanced synthesis of 64 proteins, 25 of which were acid-specific (Fig. 2C), while fivefold dilution of BM resulted in the enhanced synthesis of 58 proteins, including 11 specific to starvation (Fig. 2D). Salt stress, generated by the addition of 0·2 M NaCl, enhanced the synthesis of 10 salt-specific proteins out of a total of 52 proteins (Fig. 2E), while increasing the temperature to 42 °C enhanced the synthesis of 40 proteins, 6 of which were heat-specific (Fig. 2F).



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Fig. 2. Autoradiograms of 2DE protein profiles of exponential-phase cells of S. mutans H7 growing at pH 7·5 with 20 mM glucose and rapidly subjected to various stress conditions for 30 min in the presence of 14C-labelled amino acids followed by extraction, separation by 2DE and autoradiography. Stress conditions: A, control (cells maintained at pH 7·5 and 37 °C); B, oxidative (2 mM H2O2); C, acid (pH 7·5 to 5·5); D, starvation (fivefold dilution of BM); E, salt (0·2 M NaCl); F, heat (37 °C to 42 °C). All triangles indicate proteins enhanced twofold or more; solid triangles () are general stress proteins and divided triangles () indicate stress-specific proteins. pI scales are given at the top of each panel and molecular mass (kDa) on the left.

 

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Table 1. Analysis of the proteins in the 2DE gels in Fig. 2 enhanced or diminished twofold or more by oxidative, acid, starvation, salt or heat stress

 
Comparative analysis of the 2DE gels in Fig. 2 indicated that the synthesis of six proteins was upregulated by all five stress conditions; these could be considered ‘general’ stress proteins. In addition, there were a considerable number of proteins (16–32) enhanced by two, three or four of the stress conditions (Table 2). In order to focus on the most significant protein changes, further analysis was restricted to only those proteins demonstrating fivefold or greater increases in labelling under the stress condition when compared to the same spot in the non-stressed control cells using the IOD% analysis (see Methods) (Table 3). Using this threshold, more proteins (30) were enhanced fivefold or more by acidification than by the other stress conditions, with 19 proteins (63%) specific to acid alone (ASPs). Of the ASPs, the most significant increases in synthesis occurred with proteins 510, 501 and 502, which showed 43-, 36- and 21-fold increases, respectively, in labelling during the 30 min incubation. H2O2-induced oxidative stress generated 25 proteins at this threshold, including 12 specific proteins (OxSPs), with the greatest incorporation of 14C in proteins 200, 106 and 210. Starvation and salt stress each enhanced the synthesis of 13 proteins; however, only one protein was specific to starvation (171) and one to salt (209). The temperature increase to 42 °C resulted in 15 upregulated proteins, with four being specific to heat (HSPs); the most significant increase (54-fold) was with protein 210. Interestingly, protein 210 was also enhanced by oxidative (25-fold) and salt (13-fold) stress.


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Table 2. Number of proteins in Fig. 2 enhanced twofold or more by one stress condition which are also enhanced by a second condition

 

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Table 3. Proteins upregulated fivefold or more in exponential-phase cells of S. mutans H7 under conditions of stress

 
Further analysis of the 2DE gels revealed 18 proteins whose expression was reduced fivefold or more by the various stress conditions, with the majority (14) specific to the particular stress condition (Table 4). Eight proteins were downregulated by salt and heat stress, while oxidation, acid and starvation stress resulted in the diminished synthesis of three proteins, two proteins and one protein, respectively. Notable changes were shown by the specifically repressed proteins 43 (HSRP), 83 (OxSRP) and 108 (SaSRP), with reductions of 21-, 43- and 29-fold, respectively.


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Table 4. Proteins downregulated fivefold or more in exponential-phase cells of S. mutans H7 under conditions of stress

 

   DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Our previous results with S. mutans LT11 demonstrated that an acid shock from pH 7·5 to 5·5 induced a response in exponential-phase cells that resulted in enhanced survival following incubation at a pH (3·0) that killed control cells maintained at pH 7·5 (Svensäter et al., 1997 ). This acid-tolerance response required 2 h to complete. Subsequent pulse–chase experiments with 14C-labelled amino acids, followed by one-dimensional polyacrylamide electrophoresis and autoradiography, revealed the enhanced formation of 36 proteins following acidification when compared to control cells maintained at pH 7·5 (Hamilton & Svensäter, 1998 ). Some 25 proteins appeared within the first 30 min and all but two of the 36 proteins were synthesized transiently, a response similar to that seen with Salmonella typhimurium (Foster, 1993 ). Preliminary experiments employing 2DE with S. mutans LT11 indicated that the protein profiles for control and acid-stressed cells were similar to those seen in Fig. 2 for S. mutans H7 in this study (data not shown), suggesting that the ability to give complex inducible responses to various stresses is a common characteristic of this species. S. mutans H7 was selected for these studies because it was a fresh strain isolated from a caries lesion at low pH (unpublished results) and, unlike S. mutans LT11, had no previous history of genetic manipulation (Tao et al., 1993 ).

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 4–7 in the first dimension and routinely resulted in the separation of 420–575 proteins with the various stress conditions. Preliminary studies with first-dimension separation over the pH range 6–10 revealed only a total of 15–20 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 6–10 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 {alpha} 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 {gamma} subunit (41 kDa) could be associated with either protein 135 or protein 511, while the {delta} (27 kDa) and {epsilon} (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 ).


   ACKNOWLEDGEMENTS
 
We would like to thank Elke Greif (Winnipeg) and Ulla-Britt Larsson (Malmö) for their excellent technical assistance. This study was supported by grants to I.R.H. from the Medical Research Council of Canada (MT-3546) and to G.S. from the Medical Research Council of Sweden (K97-24X-12266-01).


   REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
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Received 20 May 1999; revised 6 September 1999; accepted 24 September 1999.