Acid Tolerance Response Research Articles

Chitosan Nanoparticles Affect the Acid Tolerance Response in Adhered Cells of Streptococcus mutans

In this study we evaluated the effect of chitosan nanoparticles on the acid tolerance response (ATR) of adhered Streptococcus mutans. An ATR was induced by exposing S. mutans to pH 5.5 for 2 h and confirmed by exposing the acid-adapted cells to pH 3.5 for 30 min, with the majority of cells appearing viable according to the LIVE/DEAD® technique. However, when chitosan nanoparticles were present during the exposure to pH 5.5, no ATR occurred as most cells appeared dead after the pH 3.5 shock. We conclude that the chitosan nanoparticles tested had the ability to hinder ATR induction in adhered S. mutans.

Helicobacter pylori Exhibits a Fur-Dependent Acid Tolerance Response

Helicobacter pylori colonizes the acid environment of the gastric mucosa. Like other enteric bacterial pathogens, including Salmonella enterica, which must survive a brief exposure to that environment, H. pylori displays a rapid response to subtle changes in pH, which confers an increased ability to survive at more extreme acidic pH. This two-step acid tolerance response (ATR) requires de novo protein synthesis and is dependent on the function of the global regulatory protein Fur. We have explored the physiological bases of the ATR in H. pylori. Proteomic analysis of phenotypes of H. pylori and fur mutant strains show that subtle pH changes elicit significant changes in the pattern of proteins synthesized. A loss-of-function mutation in the fur gene, obtained by insertion of an antibiotic resistance cassette, indicated that Fur regulates the expression of a fraction of H. pylori proteins. A subset of proteins is involved in the ATR and confer a negative ATR phenotype.

The Acid Tolerance Response of Salmonella spp.: An adaptive strategy to survive in stressful environments prevailing in foods and the host

One of the best studied Salmonella spp. adaptive responses is the Acid Tolerance Response (ATR), responsible for bacterial survival under extreme acid conditions, and well characterized for Salmonella enterica serovar Typhimurium. This review covers the numerous studies carried out in the last two decades describing the influence of different environmental parameters on the S. Typhimurium ATR, including the complex molecular mechanisms implicated and involvement of the ATR in virulence. The review also deals with the food safety implications of this adaptive response. Finally, the limited data available regarding the ATR of other Salmonella enterica serovars is also discussed.

Effect of glutathione on growth of the probiotic bacterium Lactobacillus reuteri

Glutathione (GSH) is an abundant nonprotein thiol that plays numerous roles within the cell. Previously, we showed that Lactobacillus salivarius has the capacity to mount a glutathione-mediated acid-tolerance response. In the present work we provide evidence of a requirement for GSH by Lactobacillus reuteri and have studied the role of GSH during cell growth. Medium supplementation with 0.5 mM GSH as the sole sulfur source enhanced cell growth, resulting in an increase in glucose consumption, and increased cell GSH and protein contents compared with levels seen in the absence of supplementation. Moreover, L. reuteri showed enhanced amino acid consumption when grown with 0.5 mM GSH. These findings indicate that glutathione is a nutrient for bacterial growth.

Cyclopropanation of Membrane Unsaturated Fatty Acids Is Not Essential to the Acid Stress Response of Lactococcus lactis subsp. cremoris

Cyclopropane fatty acids (CFAs) are synthetized in situ by the transfer of a methylene group from S-adenosyl-L-methionine to a double bond of unsaturated fatty acid chains of membrane phospholipids. This conversion, catalyzed by the Cfa synthase enzyme, occurs in many bacteria and is recognized to play a key role in the adaptation of bacteria in response to a drastic perturbation of the environment. The role of CFAs in the acid tolerance response was investigated in the lactic acid bacterium Lactococcus lactis MG1363. A mutant of the cfa gene was constructed by allelic exchange. The cfa gene encoding the Cfa synthase was cloned and introduced into the mutant to obtain the complemented strain for homologous system studies. Data obtained by gas chromatography (GC) and GC-mass spectrometry (GC-MS) validated that the mutant could not produce CFA. The CFA levels in both the wild-type and complemented strains increased upon their entry to stationary phase, especially with acid-adapted cells or, more surprisingly, with ethanol-adapted cells. The results obtained by performing quantitative reverse transcription-PCR (qRT-PCR) experiments showed that transcription of the cfa gene was highly induced by acidity (by 10-fold with cells grown at pH 5.0) and by ethanol (by 9-fold with cells grown with 6% ethanol) in comparison with that in stationary phase. Cell viability experiments were performed after an acidic shock on the mutant strain, the wild-type strain, and the complemented strain, as a control. The higher viability level of the acid-adapted cells of the three strains after 3 h of shock proved that the cyclopropanation of unsaturated fatty acids is not essential for L. lactis subsp. cremoris survival under acidic conditions. Moreover, fluorescence anisotropy data showed that CFA itself could not maintain the membrane fluidity level, particularly with ethanol-grown cells.

Effect of acid tolerance response (ATR) on attachment of Listeria monocytogenes Scott A to stainless steel under extended exposure to acid or/and salt stress and resistance of sessile cells to subsequent strong acid challenge

The aim of this study was to investigate the potential effect of adaptive stationary phase acid tolerance response (ATR) of Listeria monocytogenes Scott A cells on their attachment to stainless steel (SS) under low pH or/and high salt conditions and on the subsequent resistance of sessile cells to strong acid challenge. Nonadapted or acid-adapted stationary-phase L. monocytogenes cells were used to inoculate ( ca. 10 8 CFU/ml) Brain Heart (BH) broth (pH 7.4, 0.5% w/v NaCl) in test tubes containing vertically placed SS coupons (used as abiotic substrates for bacterial attachment). Incubation was carried out at 16 °C for up to 15 days, without any nutrient refreshment. L. monocytogenes cells, prepared as described above, were also exposed to low pH (4.5; adjusted with HCl) or/and high salt (5.5% w/v NaCl) stresses, during attachment. On the 5th, 10th and 15th day of incubation, cells attached to SS coupons were detached (through bead vortexing) and enumerated (by agar plating). Results revealed that ATR significantly ( p < 0.05) affected bacterial attachment, when the latter took place under moderate acidic conditions (pH 4.5, 0.5 or 5.5% w/v NaCl), with the acid-adapted cells adhering slightly more than the nonadapted ones. Regardless of acidity/salinity conditions during attachment, ATR also enhanced the resistance of sessile cells to subsequent lethal acid challenge (exposure to pH 2 for 6 min; pH adjusted with either hydrochloric or lactic acid). The trend observed with viable count data agreed well with conductance measurements, used to indirectly quantify remaining attached bacteria (following the strong acid challenge) via their metabolic activity. To sum, this study demonstrates that acid adaptation of L. monocytogenes cells during their planktonic growth enhances their subsequent attachment to SS under extended exposure (at 16 °C for up to 15 days) to mild acidic conditions (pH 4.5), while it also improves the resistance of sessile cells to extreme acid treatment (pH 2). Therefore, the ATR of bacterial cells should be carefully considered when applying acidic decontamination strategies to eradicate L. monocytogenes attached to food processing equipment.

Acid Induced Outer Membrane Phenotype in Salmonella typhi-a Gene Product of fliC

Salmonella enterica has been reported to have evolved different strategies to subvert normal host cellular functions which allow it to enter into and proliferate within the host cells. Although low pH of stomach is a formidable barrier to infections, but majority of the Enterobacteriaceae family members exhibit strong innate resistance to low pH by expressing certain gene products involved in acid tolerance responses (ATR). In this brief report, we have isolated and characterized an over expressed outer membrane protein of Salmonella enterica serovar Typhi Ty2 grown under acidic conditions (pH 4.5). The protein band was excised and subjected to peptide mass fingerprinting (MALDI-TOF). The fragments obtained were BLAST using MASCOT search engine and it was identified as flagellin (fliC) gene product (53.2 kDa) consisting of 506 amino acid residues. Up-regulation of flagellin protein may influence the functional capacity of Salmonella enterica during the course of infection. Further, an attempt to knockout this gene may help in confirming the exclusive role of fliC gene product in the biological implications such as apoptosis, inflammation, cytokine production, adhesion and invasion which would help in better understanding of the host-pathogen interactions.

Enzymatic characterization of acid tolerance response (ATR) during the enhanced biohydrogen production process from Taihu cyanobacteria via anaerobic digestion

Enhancement of biohydrogen production via anaerobic digestion from Taihu cyanobacteria (blue algae) after acid stress on anaerobic sludge, and the enzymatic characterization of the acid tolerance response (ATR) during the enhanced biohydrogen production process were investigated in this study. Comparing to those of the control, biohydrogen accumulation and hydrogen content increased by 1.9 and 1.7 times, when 12.5 and 7.5 g/L of acid stress on anaerobic sludge were performed respectively. Other than that, activities of hydrolytic enzymes, such as β-glucosidase, BAA-proteolytic enzyme and phosphatase were all improved during the enhanced biohydrogen process after appropriate acid stress. Significantly, activity of glutamate decarboxylase (GAD), the main microbial ATR stimulated by excessive acids, was increased consistently with the biohydrogen accumulation. Therefore, acid stress might be a practical approach to improving the biochemical traits of the anaerobic sludge. In turn, improved hydrolysis of organic substances would help the anaerobic sludge better survive excessive organic acids, and then enhance biohydrogen production from Taihu cyanobacteria.

Amino acids improve acid tolerance and internal pH maintenance in Bacillus cereus ATCC14579 strain

This study investigated the involvement of glutamate-, arginine- and lysine-dependent systems in the Acid Tolerance Response (ATR) of Bacillus cereus ATCC14579 strain. Cells were grown in a chemostat at external pH (pH e) 7.0 and 5.5. Population reduction after acid shock at pH 4.0 was strongly limited in cells grown at pH 5.5 (acid-adapted) compared with cells grown at pH 7.0 (unadapted), indicating that B. cereus cells grown at low pH e were able to induce a marked ATR. Glutamate, arginine and lysine enhanced the resistance of unadapted cells to pH 4.0 acid shock of 1-log or 2-log populations, respectively. Amino acids had no detectable effect on acid resistance in acid-adapted cells. An acid shock at pH 4.0 resulted in a marked drop in internal pH (pH i) in unadapted cells compared with acid-adapted cells. When acid shock was achieved in the presence of glutamate, arginine or lysine, pH i was maintained at higher values (6.31, 6.69 or 6.99, respectively) compared with pH i in the absence of amino acids (4.88). Acid-adapted cells maintained their pH i at around 6.4 whatever the condition. Agmatine (a competitive inhibitor of arginine decarboxylase) had a negative effect on the ability of B. cereus cells to survive and maintain their pH i during acid shock. Our data demonstrate that B. cereus is able to induce an ATR during growth at low pH. This adaptation depends on pH i homeostasis and is enhanced in the presence of glutamate, arginine and lysine. Hence evaluations of the pathogenicity of B. cereus must take into account its ability to adapt to acid stress.

Changes in ffh, uvrA, groES and dnaK mRNA Abundance as a Function of Acid-Adaptation and Growth Phase in Bifidobacterium longum BBMN68 Isolated from Healthy Centenarians

The acid adaption is commonly used as a strategy to enhance the acid tolerance of bifidobacteria. However, the acid tolerance response (ATR) mechanism elicited by this method is unclear. Real-time relative-quantitative PCR was applied to analyze the changes in the expressions of ffh, uvrA, groES, and dnaK involved in the ATR after acid-adaptation in Bifidobacterium longum BBMN68 in different growth phases. BBMN68 was cultured at a constant neutral pH during the whole growth phase. Without acid-adaptation, the survival ratios at the lethal pH 3.0 were 0.25% and 17% in the exponential and stationary phases, respectively. The genes ffh, uvrA, groES, and dnaK were significantly higher in the stationary phase than in the exponential phase. The results indicated that although there was no acid stress, the acid tolerance of cells was elevated from the exponential phase into stationary phase. After acid-adaptation at pH 5.0 for 120 min, the survival ratios of BBMN68 in the exponential and stationary phases were increased to 2.5 and 31%, respectively. In the exponential phase, ffh, uvrA groES, and dnaK were significantly decreased after acid-adaptation. In the stationary phase, after acid-adaptation for 15, 60, and 120 min, the genes uvrA, groES, and dnaK were significantly decreased, whereas, ffh was significantly up-regulated at 15 min, and then suppressed at 60 and 120 min after acid-adaptation. The results represented that the ATR in B. longum was different from other bacteria, and ffh may be the transient acid gene.

Characterization of the Streptococcus sobrinus acid-stress response by interspecies microarrays and proteomics

Streptococcus mutans and Streptococcus sobrinus are considered the primary organisms responsible for human dental caries. The ability to generate acids and to adapt to low pH conditions is directly associated with the cariogenic potential of these bacteria. To survive acidic conditions, both species have been shown to mount an acid-tolerance response (ATR). However, previous characterization of the S.sobrinus ATR identified critical differences in the mechanisms of acid adaptation between S.mutans and S.sobrinus. Here, interspecies microarray and proteomic approaches were used to identify novel, previously unrecognized genes and pathways that participate in the S.sobrinus acid-stress response. The results revealed that, among other things, metabolic alterations that enhance energy generation and upregulation of the malolactic fermentation enzyme activity constitute important acid-resistance properties in S.sobrinus. Some of these acid adaptive traits are shared by S.mutans and might be considered optimal targets for therapeutic treatments designed to control dental caries.

CadC RegulatescadandtdcOperons in Response to Gastrointestinal Stresses and Enhances Intestinal Colonization ofKlebsiella pneumoniae

To explore the mechanisms that enable Klebsiella pneumoniae to survive in the gastrointestinal tract, we characterized the roles of the cad and tdc operons during the bacterial response to acid and bile stress. The transcriptional units of the cad and tdc loci were identified by reverse-transcription polymerase chain reaction (RT-PCR). Transcriptional regulation of the cad and tdc operons under either acid or bile stress was analyzed by real-time RT-PCR. The ability of the mutant strains to colonize the gastrointestinal tract was examined through competition with the wild-type strain during intragastric infection of mice. The cad and tdc operons were single transcriptional units. The NTUH-K2044 wild-type strain displayed an acid tolerance response to inorganic acid and was able to survive in bile encountered in vivo. Altered transcription of the cad and tdc operons by acid depended on CadC. Under either bile stress or acidic conditions, the transcriptional regulation of the tdc operon depended on TdcA. Both the cadCBA and the tdcABCDE mutant strains were impaired in their ability to colonize the mouse intestine, compared with that of the wild-type strain. These results suggest that the cad and tdc loci in K. pneumoniae are involved in the adaptation of the organism to gastrointestinal stress and intestinal colonization.

Physiological and Transcriptional Response ofLactobacillus caseiATCC 334 to Acid Stress

This study investigated features of the acid tolerance response (ATR) in Lactobacillus casei ATCC 334. To optimize ATR induction, cells were acid adapted for 10 or 20 min at different pH values (range, 3.0 to 5.0) and then acid challenged at pH 2.0. Adaptation over a broad range of pHs improved acid tolerance, but the highest survival was noted in cells acid adapted for 10 or 20 min at pH 4.5. Analysis of cytoplasmic membrane fatty acids (CMFAs) in acid-adapted cells showed that they had significantly (P < 0.05) higher total percentages of saturated and cyclopropane fatty acids than did control cells. Specifically, large increases in the percentages of C(14:0), C(16:1n(9)), C(16:0), and C(19:0(11c)) were noted in the CMFAs of acid-adapted and acid-adapted, acid-challenged cells, while C(18:1n(9)) and C(18:1n(11)) showed the greatest decrease. Comparison of the transcriptome from control cells (grown at pH 6.0) against that from cells acid adapted for 20 min at pH 4.5 indicated that acid adaption invoked a stringent-type response that was accompanied by other functions which likely helped these cells resist acid damage, including malolactic fermentation and intracellular accumulation of His. Validation of microarray data was provided by experiments that showed that L. casei survival at pH 2.5 was improved at least 100-fold by chemical induction of the stringent response or by the addition of 30 mM malate or 30 mM histidine to the acid challenge medium. To our knowledge, this is the first report that intracellular histidine accumulation may be involved in bacterial acid resistance.

Effect of transient acid stress on the proteome of intestinal probiotic Lactobacillus reuteri

We report the acid tolerance response and changes in the level of protein expression of probiotic Lactobacillus reuteri subjected to transient (1.5 h) acid stress at pH 3.0. Sixteen acid-responsive proteins were identified by peptide mass fingerprinting including members of five broad functional categories: metabolism, transcription/translation, DNA replication/repair, transport and binding proteins, and pH homeostasis and stress responses. This work can provide some new and relevant information on the inducible mechanisms underlying the capacity of probiotic L. reuteri to tolerate acid stress.

Glutathione-mediated response to acid stress in the probiotic bacterium, Lactobacillus salivarius

Lactobacillus salivarius, a probiotic bacterium, encounters acidic conditions in its passage through the gastrointestinal tract of human and animal hosts. We studied the effect of a rapid downshift in extracellular pH from 6.5 to 4 on cell growth. The maximum growth rate was higher in low pH medium with glutathione supplementation than without. Cells developed a GSH-mediated acid-tolerance response and, when grown with 0.5 mM GSH, reached a higher final density than with other conditions. These findings suggest that the increased growth rate is caused by uptake of GSH which acts as a nutrient source as well as having protective functions, allowing for continued growth.

Role of GlnR in Acid-Mediated Repression of Genes Encoding Proteins Involved in Glutamine and Glutamate Metabolism inStreptococcus mutans

The acid tolerance response (ATR) is one of the major virulence traits of Streptococcus mutans. In this study, the role of GlnR in acid-mediated gene repression that affects the adaptive ATR in S. mutans was investigated. Using a whole-genome microarray and in silico analyses, we demonstrated that GlnR and the GlnR box (ATGTNAN(7)TNACAT) were involved in the transcriptional repression of clusters of genes encoding proteins involved in glutamine and glutamate metabolism under acidic challenge. Reverse transcription-PCR (RT-PCR) analysis revealed that the coordinated regulation of the GlnR regulon occurred 5 min after acid treatment and that prolonged acid exposure (30 min) resulted in further reduction in expression. A lower level but consistent reduction in response to acidic pH was also observed in chemostat-grown cells, confirming the negative regulation of GlnR. The repression by GlnR through the GlnR box in response to acidic pH was further confirmed in the citBZC operon, containing genes encoding the first three enzymes in the glutamine/glutamate biosynthesis pathway. The survival rate of the GlnR-deficient mutant at pH 2.8 was more than 10-fold lower than that in the wild-type strain 45 min after acid treatment, suggesting that the GlnR regulon participates in S. mutans ATR. It is hypothesized that downregulation of the synthesis of the amino acid precursors in response to acid challenge would promote citrate metabolism to pyruvate, with the consumption of H(+) and potential ATP synthesis. Such regulation will ensure an optimal acid adaption in S. mutans.

Characterization of mleR, a positive regulator of malolactic fermentation and part of the acid tolerance response in Streptococcus mutans

BackgroundOne of the key virulence determinants of Streptococcus mutans, the primary etiological agent of human dental caries, is its strong acid tolerance. The acid tolerance response (ATR) of S. mutans comprises several mechanisms that are induced at low pH and allow the cells to quickly adapt to a lethal pH environment. Malolactic fermentation (MLF) converts L-malate to L-lactate and carbon dioxide and furthermore regenerates ATP, which is used to translocate protons across the membrane. Thus, MLF may contribute to the aciduricity of S. mutans but has not been associated with the ATR so far.ResultsHere we show that the malolactic fermentation (mle) genes are under the control of acid inducible promoters which are induced within the first 30 minutes upon acid shock in the absence of malate. Thus, MLF is part of the early acid tolerance response of S. mutans. However, acidic conditions, the presence of the regulator MleR and L-malate were required to achieve maximal expression of all genes, including mleR itself. Deletion of mleR resulted in a decreased capacity to carry out MLF and impaired survival at lethal pH in the presence of L-malate. Gel retardation assays indicated the presence of multiple binding sites for MleR. Differences in the retardation patterns occurred in the presence of L-malate, thus demonstrating its role as co-inducer for transcriptional regulation.ConclusionThis study shows that the MLF gene cluster is part of the early acid tolerance response in S. mutans and is induced by both low pH and L-malate.

Characterization of a glutamate transporter operon, glnQHMP, in Streptococcus mutans and its role in acid tolerance.

Glutamate contributes to the acid tolerance response (ATR) of many Gram-negative and Gram-positive bacteria, but its role in the ATR of the oral bacterium Streptococcus mutans is unknown. This study describes the discovery and characterization of a glutamate transporter operon designated glnQHMP (Smu.1519 to Smu.1522) and investigates its potential role in acid tolerance. Deletion of glnQHMP resulted in a 95% reduction in transport of radiolabeled glutamate compared to the wild-type UA159 strain. The addition of glutamate to metabolizing UA159 cells resulted in an increased production of acidic end products, whereas the glnQHMP mutant produced less lactic acid than UA159, suggesting a link between glutamate metabolism and acid production and possible acid tolerance. To investigate this possibility, we conducted a microarray analysis with glutamate and under pH 5.5 and pH 7.5 conditions which showed that expression of the glnQHMP operon was downregulated by both glutamate and mild acid. We also measured the growth kinetics of UA159 and its glnQHMP-negative derivative at pH 5.5 and found that the mutant doubled at a much slower rate than the parent strain but survived at pH 3.5 significantly better than the wild type. Taken together, these findings support the involvement of the glutamate transporter operon glnQHMP in the acid tolerance response in S. mutans.

The Acid Tolerance Response Alters Membrane Fluidity and Induces Nisin Resistance in Listeria monocytogenes.

The ability of L. monocytogenes cells to adapt to a variety of stressors contributes to its growth in a wide range of foods. The present study examines the effect of acid and of the acid tolerance response (ATR) on membrane fluidity and on the organism's resistance to acid and to the bacteriocin nisin. When ATR was induced in wild-type cells, these cells also became resistant to nisin. ATR(+) cells also had lower membrane rigidities than control ATR(-) cells that had not been subjected to the acid tolerance response. However, cells that were genetically resistant to nisin did not show any significant (P<0.05) change in rigidity when grown in the presence of nisin. These studies suggest that the use of acid and nisin for L. monocytogenes control in ready-to-eat foods may be compromised if cross-resistance emerges.

Cultural conditions required for the induction of an adaptive acid-tolerance response (ATR) in Sinorhizobium meliloti and the question as to whether or not the ATR helps rhizobia improve their symbiosis with alfalfa at low pH

Sinorhizobium meliloti associates with Medicago and Melilotus species to develop nitrogen-fixing symbioses. The agricultural relevance of these associations, the worldwide distribution of acid soils, and the remarkable acid sensitivity of the microsymbiont have all stimulated research on the responses of the symbionts to acid environments. We show here that an adaptive acid-tolerance response (ATR) can be induced in S. meliloti, as shown previously for Sinorhizobium medicae, when the bacteria are grown in batch cultures at the slightly acid pH of 6.1. In marked contrast, no increased tolerance to hydrogen ions is obtained if rhizobia are grown in a chemostat under continuous cultivation at the same pH. The adaptive ATR appears as a complex process triggered by an increased hydrogen-ion concentration, but operative only if other--as yet unknown--concomitant factors that depend on the culture conditions are present (although not provided under continuous cultivation). Although the stability of the ATR and its influence on acid tolerance has been characterized in rhizobia, no data have been available on the effect of the adapted state on symbiosis. Coinoculation experiments showed that acid-adapted indicator rhizobia (ATR+) were present in >90% of the nodules when nodulation was performed at pH 5.6, representing a >30% increase in occupancy compared with a control test. We show that the ATR represents a clear advantage in competing for nodulation at low pH. It is not yet clear whether such an effect results from an improved performance in the acid environment during preinfection, an enhanced ability to initiate infections, or both conditions. The practical use of ATR+ rhizobia will depend on validation experiments with soil microcosms and on field testing, as well as on the possibility of preserving the physiology of ATR+ bacteria in inoculant formulations.