Carbohydrate uptake in the oral pathogen Streptococcus mutans: mechanisms and regulation by protein phosphorylation

Streptococcus mutans is the primary etiological agent of dental caries in man and other animals. This organism and other related oral streptococci use carbohydrates almost exclusively as carbon and energy sources, fermenting them primarily to lactic acid which initiates erosion of tooth surfaces. Investigations over the past decade have shown that the major uptake mechanism for most carbohydrates in S. mutans is the phosphoenolpyruvate (PEP)-dependent phosphotransferase system (PTS), although non-PTS systems have also been identified for glucose and sucrose. Regulation of sugar uptake occurs by induction/repression and inducer exclusion mechanisms in S. mutans, but apparently not by inducer expulsion as is found in some other streptococci. In addition, ATP-dependent protein kinases have also been identified in S. mutans and other oral streptococci, and a regulatory function for at least one of these has been postulated. Among a number of proteins that are phosphorylated by these enzymes, the predominant soluble protein substrate is the general phospho-carrier protein of the PTS, HPr, as had previously been observed in a variety of Gram-positive bacteria. Recent results have provided evidence for a role for ATP-dependent phosphorylation of HPr in the coordination of sugar uptake and its catabolism in S. mutans. In this review, these results are summarized, and directions for future research in this area are discussed.     

Role of HtrA in growth and competence of Streptococcus mutans UA159

We report here that HtrA plays a role in controlling growth and competence development for genetic transformation in Streptococcus mutans. Disruption of the gene for HtrA resulted in slow growth at 37 degrees C, reduced thermal tolerance at 42 degrees C, and altered sucrose-dependent biofilm formation on polystyrene surfaces. The htrA mutant also displayed a significantly reduced ability to undergo genetic transformation. A direct association between HtrA and genetic competence was demonstrated by the increased expression of the htrA gene upon exposure to competence-stimulating peptide. The induction of htrA gradually reached a maximum at around 20 min, suggesting that HtrA may be involved in a late competence response. Complementation of the htrA mutation in a single copy on the chromosome of the mutant could rescue the defective growth phenotypes but not transformability, apparently because a second gene, spo0J, immediately downstream of htrA, also affects transformation. The htrA and spo0J genes were shown to be both individually transcribed and cotranscribed and probably have a functional connection in competence development. HtrA regulation appears to be finely tuned in S. mutans, since strains containing multiple copies of htrA exhibited abnormal growth phenotypes. Collectively, the results reveal HtrA to be an integral component of the regulatory network connecting cellular growth, stress tolerance, biofilm formation, and competence development and reveal a novel role for the spo0J gene in genetic transformation.     

变形链球菌电击转化条件的优化研究

目的为增强对变形链球菌的电击转化效率,探索常用的胞壁弱化剂甘氨酸在电击转化中的加入模式。方法以氨苄青霉素抗性的pGL3 basic质粒作为外源DNA,通过电击转化导入变形链球菌参考株UA159内,并在选择性培养基上筛选阳性转化克隆,以优化筛选出最佳的甘氨酸加入浓度与模式,同时比较了不同电击方案转化效率的差异。结果在变形链球菌对数生长期后加入终浓度为10%的甘氨酸可有效地增强电击转化的效率;而不同的电转电压的选择对于转化效率的影响,在本实验中差异未见显著性。结论研究证实了甘氨酸作为胞壁弱化剂可增强对变形链球菌的转化效率,并优化了对变形链球菌的电击转化方案。     

Modification of Gene Expression and Virulence Traits in Streptococcus mutans in Response to Carbohydrate Availability

The genetic and phenotypic responses of Streptococcus mutans, an organism that is strongly associated with the development of dental caries, to changes in carbohydrate availability were investigated. S. mutans UA159 or a derivative of UA159 lacking ManL, which is the EIIAB component (EIIAB^Man) of a glucose/mannose permease of the phosphoenolpyruvate:sugar phosphotransferase system (PTS) and a dominant effector of catabolite repression, was grown in continuous culture to steady state under conditions of excess (100 mM) or limiting (10 mM) glucose. Microarrays using RNA from S. mutans UA159 revealed that 174 genes were differentially expressed in response to changes in carbohydrate availability (P < 0.001). Glucose-limited cells possessed higher PTS activity, could acidify the environment more rapidly and to a greater extent, and produced more ManL protein than cultures grown with excess glucose. Loss of ManL adversely affected carbohydrate transport and acid tolerance. Comparison of the histidine protein (HPr) in S. mutans UA159 and the manL deletion strain indicated that the differences in the behaviors of the strains were not due to major differences in HPr pools or HPr phosphorylation status. Therefore, carbohydrate availability alone can dramatically influence the expression of physiologic and biochemical pathways that contribute directly to the virulence of S. mutans, and ManL has a profound influence on this behavior.     

Galactose Metabolism by Streptococcus mutans

The galK gene, encoding galactokinase of the Leloir pathway, was insertionally inactivated in Streptococcus mutans UA159. The galK knockout strain displayed only marginal growth on galactose, but growth on glucose or lactose was not affected. In strain UA159, the sugar phosphotransferase system (PTS) for lactose and the PTS for galactose were induced by growth in lactose and galactose, although galactose PTS activity was very low, suggesting that S. mutans does not have a galactose-specific PTS and that the lactose PTS may transport galactose, albeit poorly. To determine if the galactose growth defect of the galK mutant could be overcome by enhancing lactose PTS activity, the gene encoding a putative repressor of the operon for lactose PTS and phospho-β-galactosidase, lacR, was insertionally inactivated. A galK and lacR mutant still could not grow on galactose, although the strain had constitutively elevated lactose PTS activity. The glucose PTS activity of lacR mutants grown in glucose was lower than in the wild-type strain, revealing an influence of LacR or the lactose PTS on the regulation of the glucose PTS. Mutation of the lacA gene of the tagatose pathway caused impaired growth in lactose and galactose, suggesting that galactose can only be efficiently utilized when both the Leloir and tagatose pathways are functional. A mutation of the permease in the multiple sugar metabolism operon did not affect growth on galactose. Thus, the galactose permease of S. mutans is not present in the gal, lac, or msm operons.     

Galactose metabolism by Streptococcus mutans

The galK gene, encoding galactokinase of the Leloir pathway, was insertionally inactivated in Streptococcus mutans UA159. The galK knockout strain displayed only marginal growth on galactose, but growth on glucose or lactose was not affected. In strain UA159, the sugar phosphotransferase system (PTS) for lactose and the PTS for galactose were induced by growth in lactose and galactose, although galactose PTS activity was very low, suggesting that S. mutans does not have a galactose-specific PTS and that the lactose PTS may transport galactose, albeit poorly. To determine if the galactose growth defect of the galK mutant could be overcome by enhancing lactose PTS activity, the gene encoding a putative repressor of the operon for lactose PTS and phospho-beta-galactosidase, lacR, was insertionally inactivated. A galK and lacR mutant still could not grow on galactose, although the strain had constitutively elevated lactose PTS activity. The glucose PTS activity of lacR mutants grown in glucose was lower than in the wild-type strain, revealing an influence of LacR or the lactose PTS on the regulation of the glucose PTS. Mutation of the lacA gene of the tagatose pathway caused impaired growth in lactose and galactose, suggesting that galactose can only be efficiently utilized when both the Leloir and tagatose pathways are functional. A mutation of the permease in the multiple sugar metabolism operon did not affect growth on galactose. Thus, the galactose permease of S. mutans is not present in the gal, lac, or msm operons.     

Malolactic fermentation by Streptococcus mutans

Streptococcus mutans and certain other oral lactic-acid bacteria were found to have the ability to carry out malolactic fermentation involving decarboxylation of L-malate to yield L-lactic acid and concomitant reduction in acidity. The activity was inducible by L-malate in S. mutans UA159 growing in suspensions or biofilms. The optimal pH for the fermentation was c. 4.0 for both suspensions and biofilms, although the pH optimum for malolactic enzyme in permeabilized cells of S. mutans UA159 was close to 5.5. Although malate did not serve as a catabolite for growth of S. mutans, it did serve to protect the organism against acid killing and to maintain ATP pool levels during starvation. Alkalinization associated with malolactic fermentation resulted in pH rise or increased need to add standardized HCl solution to maintain a set pH value in pH-stat experiments. The net conclusion is that malate has the potential to be effective for alkalinization of dental plaque, although the fermentation is sensitive to fluoride and triclosan, which are commonly added to oral care products.     

Development of competence for genetic transformation of Streptococcus mutans in a chemically defined medium

Streptococcus mutans develops competence for genetic transformation in response to regulatory circuits that sense at least two peptide pheromones. One peptide, known as CSP, is sensed by a two-component signal transduction system through a membrane receptor, ComD. The other, derived from the primary translation product ComS, is thought to be sensed by an intracellular receptor, ComR, after uptake by oligopeptide permease. To allow study of this process in a medium that does not itself contain peptides, development of competence was examined in the chemically defined medium (CDM) described by van de Rijn and Kessler (Infect. Immun. 27:444, 1980). We confirmed a previous report that in this medium comS mutants of strain UA159 respond to a synthetic peptide comprising the seven C-terminal residues of ComS (ComS(11-17)) by increasing expression of the alternative sigma factor SigX, which in turn allows expression of competence effector genes. This response provided the basis for a bioassay for the ComS pheromone in the 100 to 1,000 nM range. It was further observed that comS(+) (but not comS mutant) cultures developed a high level of competence in the late log and transition phases of growth in this CDM without the introduction of any synthetic stimulatory peptide. This endogenous competence development was accompanied by extracellular release of one or more signals that complemented a comS mutation at levels equivalent to 1 μM synthetic ComS(11-17).     

Carbohydrate Fermentation by Streptococcus cremoris and Streptococcus lactis Growing in Agar Gels

When lactic streptococci were embedded in agar gels and incubated at 30°C, the end products of carbohydrate fermentation depended on the initial cell density, which determined the subsequent distribution and size of colonies in the gel. With high initial cell densities, microcolonies formed close together and lactose and glucose were converted almost entirely to lactate. However, inoculation with a small number of cells, which then grew to form widely spaced and comparatively large colonies, resulted in up to 30% diversion of end product, usually to formate, ethanol, and acetate. In these “low-colony-density” gel cultures, the initial rate of fermentation was exponential and only lactate was formed. However, this rate then became linear and fermentation became progressively more heterolactic. Streptococcus lactis ML₈ was the only strain among the 10 tested which remained homolactic. Incubation at temperatures either above or below the optimum for growth and metabolism decreased the diversion to end products other than lactate. The change from homo- to heterolactic fermentation appears to be caused by carbohydrate depletion in the vicinity of the colony, so that fermentation is then limited by the diffusion of substrate. Growth of cells on gel surfaces exposed to air resulted in up to 40% diversion of end product from lactate, mainly to CO₂, acetoin, 2,3-butanediol, and acetate. Six of the 12 Streptococcus cremoris strains tested remained homolactic under these aerobic conditions, whereas all 8 of the S. lactis strains tested, including ML₈, were heterolactic.     

Inhibition of competence development, horizontal gene transfer and virulence in Streptococcus pneumoniae by a modified competence stimulating peptide

Competence stimulating peptide (CSP) is a 17-amino acid peptide pheromone secreted by Streptococcus pneumoniae. Upon binding of CSP to its membrane-associated receptor kinase ComD, a cascade of signaling events is initiated, leading to activation of the competence regulon by the response regulator ComE. Genes encoding proteins that are involved in DNA uptake and transformation, as well as virulence, are upregulated. Previous studies have shown that disruption of key components in the competence regulon inhibits DNA transformation and attenuates virulence. Thus, synthetic analogues that competitively inhibit CSPs may serve as attractive drugs to control pneumococcal infection and to reduce horizontal gene transfer during infection. We performed amino acid substitutions on conserved amino acid residues of CSP1 in an effort to disable DNA transformation and to attenuate the virulence of S. pneumoniae. One of the mutated peptides, CSP1-E1A, inhibited development of competence in DNA transformation by outcompeting CSP1 in time and concentration-dependent manners. CSP1-E1A reduced the expression of pneumococcal virulence factors choline binding protein D (CbpD) and autolysin A (LytA) in vitro, and significantly reduced mouse mortality after lung infection. Furthermore, CSP1-E1A attenuated the acquisition of an antibiotic resistance gene and a capsule gene in vivo. Finally, we demonstrated that the strategy of using a peptide inhibitor is applicable to other CSP subtype, including CSP2. CSP1-E1A and CSP2-E1A were able to cross inhibit the induction of competence and DNA transformation in pneumococcal strains with incompatible ComD subtypes. These results demonstrate the applicability of generating competitive analogues of CSPs as drugs to control horizontal transfer of antibiotic resistance and virulence genes, and to attenuate virulence during infection by S. pneumoniae.     

Import and Metabolism of Glutathione by Streptococcus mutans

Glutathione (γ-GluCysGly, GSH) is not found in most gram-positive bacteria, but some appear to synthesize it and others, including Streptococcus mutans ATCC 33402, import it from their growth medium. Import of oxidized glutathione (GSSG) by S. mutans 33402 in 7H9 medium was shown to require glucose and to occur with an apparent K_m of 18 ± 5 μM. GSSG, GSH, S-methylglutathione, and homocysteine-glutathione mixed disulfide (hCySSG) were imported at comparable rates (measured by depletion of substrate in the medium), as was the disulfide of γ-GluCys. In contrast, the disulfide of CysGly was not taken up at a measurable rate, indicating that the γ-Glu residue is important for efficient transport. During incubation with GSSG, little GSSG was detected in cells but GSH and γ-GluCys accumulated during the first 30 min and then declined. No significant intracellular accumulation of Cys or sulfide was found. Transient intracellular accumulation of d/l-homocysteine, as well as GSH and γ-GluCys, was observed during import of hCySSG. Although substantial levels of GSH were found in cells when S. mutans was grown on media containing glutathione, such GSH accumulation had no effect on the growth rate. However, the presence of cellular GSH did protect against growth inhibition by the thiol-oxidizing agent diamide. Import of glutathione by S. mutans ATCC 25175, which like strain 33402 does not synthesize glutathione, occurred at a rate comparable to that of strain 33402, but three species which appear to synthesize glutathione (S. agalactiae ATCC 12927, S. pyogenes ATCC 8668, and Enterococcus faecalis ATCC 29212) imported glutathione at negligible or markedly lower rates.Bacteria import peptides composed of two to eight residues by means of a number of different multiprotein uptake systems or permeases (14). Of the bacterial permeases, those of Escherichia coli, Lactococcus lactis, and Salmonella typhimurium are the best studied (6, 7). In these organisms, there are individual permeases that have high affinity for dipeptides, tripeptides, dipeptides and tripeptides, or oligopeptides. Among the bacterial peptide permeases (14), there seems to be no discrimination of the specific amino acids of the transported peptides. However, switching the stereochemistry of C_α from l to d or modifying the C-terminal carboxylate or N-terminal amine of transported peptides significantly reduces the rate of transport. One transport system which does seem to recognize peptide residue side chains has been reported to exist in Enterococcus faecalis; this system transports only peptides that possess an N-terminal Asp or Glu (13).In 1978, we reported that glutathione (γ-GluCysGly, GSH) is not synthesized by most gram-positive bacteria (4), apparent exceptions being Streptococcus agalactiae and L. lactis (previously Streptococcus lactis). However, some of the gram-positive bacteria appeared to acquire GSH by import of another form of GSH from the growth medium. Uptake of glutathione by Streptococcus mutans was later studied by Thomas (16), who found that total cellular thiol content, and radioactivity from labeled GSH or oxidized GSH (GSSG), increased with the same kinetics. A careful study of L. lactis subsp. cremoris by Wiederholt and Steele (17) established that strain Z8 efficiently accumulates GSH when grown in medium supplemented with GSH but is unable to synthesize it, whereas strain C2 can neither import nor synthesize GSH. Species of Peptostreptococcus and Fusobacterium have been shown to markedly increase their production of H₂S, apparently derived by import of glutathione from the growth medium (2). Finally, cellular accumulation of radioactivity from radiolabeled GSH or GSSG added to the incubation medium has been demonstrated in Streptococcus pneumoniae, and a mutant in which the apparent transport of glutathione is blocked has been found (9).In a recent report (10), we provided evidence for accumulation of GSH through transport and synthesis of GSH by streptococci and enterococci, but the occurrence of these processes appeared to be species dependent and even, for some species, strain dependent. Such strain dependence appears most variable for L. lactis, where different strains can synthesize GSH, accumulate GSH by import, or do neither (4, 17). In the present research, we expand on our studies of streptococci in order to gain insight into the nature of the glutathione species transported, the fate of the glutathione once it enters the cell, and the function of glutathione in the cell.     

Transport and metabolism of citrate by Streptococcus mutans

Streptococcus mutans, a normal inhabitant of dental plaque, is considered a primary etiological agent of dental caries. Two virulence determinants of S. mutans are its acidogenicity and aciduricity (the ability to produce acid and the ability to survive and grow at low pH, respectively). Citric acid is ubiquitous in nature; it is a component of fruit juices, bones, and teeth. In lactic acid bacteria citrate transport has been linked to increased survival in acidic conditions. We identified putative citrate transport and metabolism genes in S. mutans, which led us to investigate citrate transport and metabolism. Our goals in this study were to determine the mechanisms of citrate transport and metabolism in S. mutans and to examine whether citrate modulates S. mutans aciduricity. Radiolabeled citrate was used during citrate transport to identify citrate metal ion cofactors, and thin-layer chromatography was used to identify metabolic end products of citrate metabolism. S. mutans was grown in medium MM4 with different citrate concentrations and pH values, and the effects on the growth rate and cell survival were monitored. Intracellular citrate inhibited the growth of the bacteria, especially at low pH. The most effective cofactor for citrate uptake by S. mutans was Fe(3+). The metabolic end product of citrate metabolism was aspartate, and a citrate transporter mutant was more citrate tolerant than the parent.     

Disulfide reduction and sulfhydryl uptake by Streptococcus mutans

Incubation of Streptococcus mutans cells with certain disulfide compounds resulted in accumulation of reduced sulfhydryl compounds in the extracellular medium or in both the medium and the cells. Oxidized lipoic acid and lipoamide competed for reduction. At high concentrations, these compounds were reduced at rates comparable to that of glucose metabolism, and all of the increase in sulfhydryls was in the medium. Cystamine did not compete with these compounds for reduction but was also reduced at high rates and low apparent affinity, and all of the cysteamine produced from cystamine accumulated in the medium. In contrast, glutathione disulfide (GSSG) and L-cystine were reduced slowly but with high apparent affinity, and 60 to 80% of the increase in sulfhydryls was intracellular. NADH-dependent lipoic acid or lipoamide reductase activity was present in the particulate (wall-plus-membrane) fraction, whereas NADPH-dependent GSSG reductase activity was present in the soluble (cytoplasmic) fraction. Two transport systems for disulfide and sulfhydryl compounds were distinguished. GSSG, L-cystine, and reduced glutathione competed for uptake. L-Cysteine was taken up by a separate system that also accepted L-penicillamine and D-cysteine as substrates. Uptake of glutathione or L-cysteine, or the uptake and reduction of GSSG or L-cystine, resulted in up to a 10-fold increase in cell sulfhydryl content that raised intracellular concentrations to between 30 and 40 mM. These reductase and transport systems enable S. mutans cells to create a reducing environment in both the extracellular medium and the cytoplasm.     

Inhibition of Streptococcus mutans biofilm formation by Streptococcus salivarius FruA

The oral microbial flora consists of many beneficial species of bacteria that are associated with a healthy condition and control the progression of oral disease. Cooperative interactions between oral streptococci and the pathogens play important roles in the development of dental biofilms in the oral cavity. To determine the roles of oral streptococci in multispecies biofilm development and the effects of the streptococci in biofilm formation, the active substances inhibiting Streptococcus mutans biofilm formation were purified from Streptococcus salivarius ATCC 9759 and HT9R culture supernatants using ion exchange and gel filtration chromatography. Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry analysis was performed, and the results were compared to databases. The S. salivarius HT9R genome sequence was determined and used to indentify candidate proteins for inhibition. The candidates inhibiting biofilms were identified as S. salivarius fructosyltransferase (FTF) and exo-beta-d-fructosidase (FruA). The activity of the inhibitors was elevated in the presence of sucrose, and the inhibitory effects were dependent on the sucrose concentration in the biofilm formation assay medium. Purified and commercial FruA from Aspergillus niger (31.6% identity and 59.6% similarity to the amino acid sequence of FruA from S. salivarius HT9R) completely inhibited S. mutans GS-5 biofilm formation on saliva-coated polystyrene and hydroxyapatite surfaces. Inhibition was induced by decreasing polysaccharide production, which is dependent on sucrose digestion rather than fructan digestion. The data indicate that S. salivarius produces large quantities of FruA and that FruA alone may play an important role in multispecies microbial interactions for sucrose-dependent biofilm formation in the oral cavity.     

Functional genomics approach to identifying genes required for biofilm development by Streptococcus mutans

Streptococcus mutans, the primary etiological agent of human dental caries, is an obligate biofilm-forming bacterium. The goals of this study were to identify the gene(s) required for biofilm formation by this organism and to elucidate the role(s) that some of the known global regulators of gene expression play in controlling biofilm formation. In S. mutans UA159, the brpA gene (for biofilm regulatory protein) was found to encode a novel protein of 406 amino acid residues. A strain carrying an insertionally inactivated copy of brpA formed longer chains than did the parental strain, aggregated in liquid culture, and was unable to form biofilms as shown by an in vitro biofilm assay. A putative homologue of the enzyme responsible for synthesis of autoinducer II (AI-2) of the bacterial quorum-sensing system was also identified in S. mutans UA159, but insertional inactivation of the gene (luxS(Sm)) did not alter colony or cell morphology or diminish the capacity of S. mutans to form biofilms. We also examined the role of the homologue of the Bacillus subtilis catabolite control protein CcpA in S. mutans in biofilm formation, and the results showed that loss of CcpA resulted in about a 60% decrease in the ability to form biofilms on an abiotic surface. From these data, we conclude that CcpA and BrpA may regulate genes that are required for stable biofilm formation by S. mutans.