Department of Oral Biology, Centre for Oral Health Sciences, Malmö University, Malmö, Sweden1
Department of Oral Biology, Faculty of Dentistry, University of Manitoba, 780 Bannatyne Ave, Winnipeg, Manitoba, CanadaR3E 0W22
Author for correspondence: I. 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: acidurance, starvation, protein secretion, oral streptococci
Abbreviations: ATR, acid tolerance response
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INTRODUCTION |
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Previously, we have demonstrated the induction of an acid tolerance response (ATR) by exponential-phase cultures of S. mutans in response to an acid shock from pH 7·5 to 5·5 that resulted in enhanced survival at low pH, 3·53·0 (Svensäter et al., 1997 ). This exponential-phase ATR required protein synthesis, since survival was abolished in the presence of chloramphenicol. The incubation of cells with 14C-labelled amino acids during the acid shock, followed by protein extraction and PAGE analysis, demonstrated the transient up-regulation of acid-responsive proteins over a 2 h period (Hamilton & Svensäter, 1998
). More recently, 2D electrophoresis has demonstrated the up-regulation of 64 proteins within the first 30 min of a pH change from 7·5 to 5·5, with 49 proteins down-regulated during the same period (Svensäter et al., 2000
). Of the up-regulated proteins, 25 were specific to the acid response, while other proteins were also influenced by alternative stress conditions. These proteins are undoubtedly related to the variety of physiological changes observed with cells of S. mutans following a shift in pH from 7·5 to 5·5, while growing in continuous culture with a glucose limitation (Hamilton & Buckley, 1991
).
Enteric bacteria possess a variety of acid survival systems with the responses differing depending on the growth medium, the stage of growth and other factors (Foster, 1995 ; Lin et al., 1995
; Castanie-Cornet et al., 1999
). The earlier known acid response of Salmonella typhimurium, now known as the pH-dependent exponential-phase ATR (Lee et al., 1994
), is supplemented by at least two other strategies: a pH-independent general stress resistance dependent on the alternative sigma factor RpoS (
s), and an additional pH-dependent stationary-phase ATR. Comparisons between the acid-survival strategies in Sal. typhimurium, Escherichia coli and Shigella flexneri have indicated that all these organisms possessed the RpoS-dependent resistance system, while the latter two organisms possess several acid-resistance systems not present in Sal. typhimurium and requiring components of complex medium, such as glutamate and arginine (Lin et al., 1995
). Recent work with E. coli has shown that cells actually possess three overlapping acid-resistance systems to protect stationary-phase cells in acid environments (Castanie-Cornet et al., 1999
).
Although current evidence indicates that most oral streptococci generate a pH-dependent exponential-phase ATR (Svensäter et al., 1997 ; Hamilton & Svensäter, 1998
), little information is available on the acid tolerance of oral streptococci during very slow growth or in the stationary phase, conditions frequently encountered by bacteria in dental plaque (Brecx et al., 1983
). Unlike enteric bacteria, oral streptococci are relatively inactive metabolically in the stationary phase unless they have synthesized endogenous energy reserves, such as glycogen, in the presence of exogenous carbohydrate. In addition, upon depletion of the energy reserves, the transmembrane pH gradient will dissipate with the intracellular pH assuming the same value as the external pH, which in acidic environments will result in cessation of cellular activity (Hamilton, 1990
). As a consequence, we were interested in whether S. mutans could generate an ATR in the stationary phase and, if so, what factors influenced such a response. For this, we compared the acid tolerance of two freshly isolated and two laboratory strains of S. mutans growing in complex medium at pH 7·5 and 5·5, using survival at pH 3·5 for 3 h as a measure of acid resistance. Unlike the laboratory strains, the freshly isolated strains were shown to possess a pH-dependent stationary-phase ATR and acid resistance was increased by carbon starvation in complex medium. Using the fresh isolate S. mutans H7 as a model system, it was demonstrated that stationary-phase acid tolerance appears to be related to enhanced protein secretion and degradation in the early-stationary phase.
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METHODS |
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Acid tolerance during growth.
To test for pH-dependent and pH-independent stationary-phase ATRs, the acid resistance of cells was tested during normal batch growth. Cells were grown anaerobically in TYEG at pH 7·5 or 5·5 with the culture pH maintained by the addition of KOH. The pH varied by less than ±0·3 units throughout the growth period. Periodically, duplicate culture samples were removed and the cells were subjected to an acid challenge at pH 3·5 for 3 h followed by plating for survivors on trypticase agar. This latter pH is 0·20·5 units above the pH which kills 100% of exponential-phase cells grown at pH 7·5 (Svensäter et al., 1997 ). Rapid acidification was achieved by centrifuging 1·0 ml of the culture suspension in a microfuge at 15000 g for 3 min, washing the cells twice in pre-warmed sterile TYEG buffered at pH 3·5 and resuspending the cells in the same medium prior to incubation at 37 °C. All dilutions were plated in triplicate with the plates incubated at 37 °C for a minimum of 3 d. The percentage of cell survivors at each time point was calculated by comparing the numbers of cells surviving the pH 3·5 challenge and the number of cells in the original culture sample just prior to acidification. The data presented represent the mean of at least three separate determinations with the standard errors calculated by the Statview program for the Macintosh.
Glucose-depleted stationary phase.
To assess the effect of glucose depletion on acid tolerance, exponential-phase cells grown at pH 7·5 in either TYEG or MADM were rapidly washed and resuspended in the same medium at pH 7·5 and 5·5 without glucose. Following a 2 h adaptation period at 37 °C, duplicate cell samples were removed for plate counts on trypticase agar prior to rapid acidification of the culture to pH 3·5 as described above. In order to determine the rate of acid killing, duplicate samples were removed each hour over a 3 h period and the cultures were diluted and plated for survivors on trypticase agar. As above, the percentage survival was calculated from the cell counts obtained during exposure to pH 3·5 and compared to those of the same samples prior to acidification to pH 3·5. Control cells were incubated in the same medium supplemented with 20 mM glucose. The pH in all experiments varied less than±0·2 units during any incubation period and the data presented represent the mean of at least three separate determinations.
Intracellular glycogen analysis.
The glycogen content of cells was determined during the growth of the test strains in TYE containing 10 mM glucose. Culture samples (10 ml) were removed periodically to a boiling water bath for 10 min followed by centrifugation at 15000 g for 15 min. The boiled cells were washed twice in cold distilled water and resuspended at 0·4 mg dry weight ml-1 and then frozen (-70 °C) until analysed for glycogen. Glycogen was assayed by the method of DiPersio et al. (1974) .
2D gel electrophoresis.
Culture supernatant fractions were filter-sterilized (0·22 µm) and concentrated 10-fold (Ultrafree-MC, 5000 NMWL; Millipore) and the proteins/peptides were separated by 2D electrophoresis essentially as previously described by Svensäter et al. (2000) . The first dimension isoelectric focusing was run on linear 7 cm immobilized pH gradient (IPG) strips (Amersham Pharmacia Biotech) in the pH range 47 and the proteins were separated by 150 V for 1 h, 300 V for 1 h, 600 V for 1 h and 3500 V for 13 h. Following separation, the strips were immediately frozen at -80 °C until the second dimension, SDS-PAGE, could be carried out with 10% polyacrylamide gels using the Mini Protean II system (Bio-Rad). The gels were then silver-stained according to the manufacturer (Amersham Pharmacia Biotech) and scanned with a calibrated UMAX transmission scanner. Spot volumes were determined with BioImage software (version 1.6) on a Sun UltraSparc workstation (Genomic Solutions) and were defined as the sum of the pixel values comprising the protein minus the sum of the background pixel values. A reference gel was chosen and each of the other gels was matched to it selecting anchor proteins on the images and allowing the BioImage software to automatically match the images. Proteins of known molecular mass were used as standards to generate molecular mass values and pI values were deduced from the linearity of the IPG strips.
Zymography.
Proteins in filtered culture supernatants were concentrated (100-fold) by centrifugal filtration (Amicon) and separated on 10% SDS-PAGE gels containing covalently bound gelatin or 12% SDS-PAGE gels containing covalently bound casein (Bio-Rad). Electrophoresis was carried out at 100 V for 2 h at room temperature. The gels were then incubated at room temperature in 2·5% Triton X-100 for 30 min and then placed in a developing buffer (50 mM Tris-base, pH 7·5; 0·2 M NaCl; 5 mM CaCl2; 0·02% Brij-35) overnight at 37 °C. The gels were then stained with 0·5% Coomassie brilliant blue in 40% methanol/10% acetic acid for 1 h and destained with 40% methanol/10% acetic acid. Protease activity was detected as a clear zone against a stained background.
Analytical procedures.
Protein was determined by the method of Bradford (1976) , while glucose was assayed enzymically by the method of Kingsley & Getchell (1960)
.
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RESULTS |
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DISCUSSION |
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One feature not readily assessed in the growth experiments was the nature of the short transitory increase in survivors as the cells entered the stationary phase (Figs 1 and 2
) at a point that coincided with the depletion of glucose. That the effect was due to the depletion of glucose was seen in the artificial stationary-phase experiment which showed with strain H7 (Fig. 3a
) that, while adapted cells were inherently more acid resistant than unadapted cells, the acid tolerance of unadapted and adapted cells was enhanced by the absence of glucose during the acid challenge at pH 3·5 over the 3 h period. No such differential effect was seen with the laboratory strain S. mutans Ingbritt (Fig. 3b
) and strain LT11. Earlier results with S. mutans H7 (Svensäter et al., 2000
) have demonstrated cross-protection of cells to acid killing by prior exposure of cells to starvation conditions. Starvation, induced by exposure of cells to fivefold diluted basal medium, was most protective when the glucose concentration was diluted from 20 to 4 mM, although an enhanced effect over cells adapted in full-strength medium was seen when the diluted medium was devoid of glucose.
Carbohydrate-starved cells of Lactobacillus lactis IL1403 exhibit enhanced resistance to acid, heat, ethanol, osmotic and oxidative stress with this cross-protection occurring progressively with the onset of stationary phase (Hartke et al., 1994 ). Unlike the development of the ATR in exponential-phase cells of L. lactis (Rallu et al., 1996
), the stationary-phase response was independent of protein synthesis since it was not abolished, but enhanced, by chloramphenicol or rifamycin. The use of transposon mutagenesis with L. lactis MG1363 has suggested a link between acid tolerance and the stringent response since a number of acid-resistant mutants had defects in the biosynthetic pathway for the stringent response factor (p)ppGpp (Rallu et al., 1996
). Since (p)ppGpp is a key pleiotropic regulator of gene expression and survival in stationary phase (Nyström, 1993
), it is conceivable that the stringent response may also be a factor in the regulation of stress in S. mutans and other oral streptococci.
The enhanced acid resistance of S. mutans H7 and BM71 in complex medium (TYE) compared to the low amino acid defined medium (MADM) clearly differentiates these strains from S. mutans LT11 (Fig. 4). One assumes that differences in the metabolism of proteins, peptides and amino acids by the former organisms are central to this enhanced resistance. The appearance of proteolytic activity with cells of strain H7 during the transition from exponential to stationary phase (Fig. 5
), and the evidence of protein/peptide generation in the culture medium during the exponential phase with subsequent utilization during early-stationary phase (Fig. 6
), support this contention. The appearance of a 55 kDa protease in S. mutans H7, using gelatin as a substrate, confirms an earlier report of such activity by Harrington & Russell (1994)
. As to the extracellular proteins/peptides, preliminary experiments indicate that a majority of the proteins in the culture medium seen in Fig. 6
are secreted by S. mutans H7 into the medium mainly during the mid-exponential phase (O. Björnsson & G. Svensäter, unpublished results). On-going mass spectrometric analysis, using peptide mass fingerprints for protein identification, indicates that the 60 kDa chaperonin DnaK and the glycolytic enzyme enolase are secreted in a manner similar to that recently reported for Streptococcus pyogenes (Chaussee et al., 2001
). Extracellular proteins are known to be important virulence factors and while information is emerging as to the regulation of their expression, less is known about their fate and whether such proteins can be utilized to enhance acid tolerance.
Work with E. coli and Shigella flexneri has identified acid-resistance systems protecting cells to pH 2·5 and requiring glutamate or arginine during the low pH challenge with the arginine-acid survival system in E. coli involving arginine decarboxylase (Lin et al., 1995 ). More recently, a glutamate decarboxylase alkalinization cycle was identified in E. coli to protect cells from cytoplasmic acidification (Hersh et al., 1996
), refining the early observations of Gale & Epps (1942)
. While there is relatively little specific information on the role of amino acids and peptides in acid resistance of S. mutans, oral bacteria are known to utilize salivary proteins for growth (Cowman et al., 1979
; De Jong et al., 1984
) and the uptake of arginine-containing peptides by mixed oral bacteria utilizing glucose has been shown to stimulate pH increases over that observed with glucose alone (Kleinberg et al., 1976
). As the cells enter the stationary phase and the exogenous glucose becomes depleted, an energy source is important for transport processes, consequently the utilization of endogenous carbon reserves, such as glycogen, becomes crucial to cell physiology. This energy source, the principal endogenous energy source for S. mutans (Hamilton, 1976
), is also essential for the maintenance of pH homeostasis by the extrusion of proton via the H+/ATPase (Hamilton & Buckley, 1991
). Thus the increased accumulation of glycogen by S. mutans H7 compared to strain LT11 (Table 1
) would give the former organism a selective energy advantage as the cells entered the stationary phase of growth.
Clearly the current results, coupled with those on the multiple stress response of S. mutans H7 (Svensäter et al., 2000 ), indicate a strong regulatory link between the acid stress and carbon/nitrogen starvation responses in the organism. In comparing the multiple stress response in S. mutans H7, it could be shown that the fivefold dilution of a defined medium resulted in the up-regulation of 58 proteins, 11 of which were specific to starvation; 20 additional proteins exhibited diminished synthesis. Acid shock from pH 7·5 to 5·5, on the other hand, resulted in the up-regulation of 64 proteins and the down-regulation of 49 proteins with 25 specific to the acid response. Of particular interest was the fact that 25 of those proteins that showed enhanced synthesis were common between the acid and starvation responses, and a number of these were associated with enzymes of the glycolytic pathway (unpublished results). Starvation-induced stress resistance is a common feature of both Gram-positive and Gram-negative bacteria with significantly more known about the response in enteric bacteria (Matin, 1991
). The pH-independent general stress resistance in Gram-negative bacteria, such as Sal. typhimurium and E. coli, requires the growth-phase-dependent transcriptional factor
s, the product of the rpoS gene (Hersh et al., 1996
; Lin et al., 1995
). While
s has not been found in Gram-positive bacteria, Bacillus subtilis is known to possess a regulon controlled by the alternative sigma factor
B, regulating 60 general stress proteins activated by various stresses and on entry into the stationary phase (Hecker et al., 1996
; Bernhardt et al., 1997
).
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ACKNOWLEDGEMENTS |
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Received 2 April 2001;
revised 5 June 2001;
accepted 15 June 2001.