Involvement of sensor kinases in the stress tolerance response of Streptococcus mutans

The gram-positive bacterium Streptococcus mutans is the primary causative agent in the formation of dental caries in humans. The ability of S. mutans to adapt and to thrive in the hostile environment of the oral cavity suggests that this cariogenic pathogen is capable of sensing and responding to different environmental stimuli. This prompted us to investigate the role of two-component signal transduction systems (TCS), particularly the sensor kinases, in response to environmental stresses. Analysis of the annotated genome sequence of S. mutans indicates the presence of 13 putative TCS. Further bioinformatics analysis in our laboratory has identified an additional TCS in the genome of S. mutans. We verified the presence of the 14 sensor kinases by using PCR and Southern hybridization in 13 different S. mutans strains and found that not all of the sensor kinases are encoded by each strain. To determine the potential role of each TCS in the stress tolerance of S. mutans UA159, insertion mutations were introduced into the genes encoding the individual sensor kinases. We were successful in inactivating all of the sensor kinases, indicating that none of the TCS are essential for the viability of S. mutans. The mutant S. mutans strains were assessed for their ability to withstand various stresses, including osmotic, thermal, oxidative, and antibiotic stress, as well as the capacity to produce mutacin. We identified three sensor kinases, Smu486, Smu1128, and Smu1516, which play significant roles in stress tolerance of S. mutans strain UA159.     

Bacteriocin (Mutacin) Production by Streptococcus mutans Genome Sequence Reference Strain UA159: Elucidation of the Antimicrobial Repertoire by Genetic Dissection

Streptococcus mutans UA159, the genome sequence reference strain, exhibits nonlantibiotic mutacin activity. In this study, bioinformatic and mutational analyses were employed to demonstrate that the antimicrobial repertoire of strain UA159 includes mutacin IV (specified by the nlm locus) and a newly identified bacteriocin, mutacin V (encoded by SMU.1914c).     

SMU.746-SMU.747, a Putative Membrane Permease Complex,Is Involved in Aciduricity,Acidogenesis, and Biofilm Formation in Streptococcus mutans

Dental caries induced by Streptococcus mutans is one of the most prevalent chronic infectious diseases worldwide. The pathogenicity of S. mutans relies on the bacterium''s ability to colonize tooth surfaces and survive a strongly acidic environment. We performed an ISS1 transposon mutagenesis to screen for acid-sensitive mutants of S. mutans and identified an SMU.746-SMU.747 gene cluster that is needed for aciduricity. SMU.746 and SMU.747 appear to be organized in an operon and encode a putative membrane-associated permease. SMU.746- and SMU.747-deficient mutants showed a reduced ability to grow in acidified medium. However, the short-term or long-term acid survival capacity and F₁F₀ ATPase activity remained unaffected in the mutants. Furthermore, deletion of both genes did not change cell membrane permeability and the oxidative and heat stress responses. Growth was severely affected even with slight acidification of the defined medium (pH 6.5). The ability of the mutant strain to acidify the defined medium during growth in the presence of glucose and sucrose was significantly reduced, although the glycolysis rate was only slightly affected. Surprisingly, deletion of the SMU.746-SMU.747 genes triggered increased biofilm formation in low-pH medium. The observed effects were more striking in a chemically defined medium. We speculate that the SMU.746-SMU.747 complex is responsible for amino acid transport, and we discuss its possible role in colonization and survival in the oral environment.     

The transcriptional landscape of the deep-sea bacterium Photobacterium profundum in both a toxR mutant and its parental strain

Background

Mutans streptococci are a group of gram-positive bacteria including the primary cariogenic dental pathogen Streptococcus mutans and closely related species. Two component systems (TCSs) composed of a signal sensing histidine kinase (HK) and a response regulator (RR) play key roles in pathogenicity, but have not been comparatively studied for these oral bacterial pathogens.

Results

HKs and RRs of 8 newly sequenced mutans streptococci strains, including S. sobrinus DSM20742, S. ratti DSM20564 and six S. mutans strains, were identified and compared to the TCSs of S. mutans UA159 and NN2025, two previously genome sequenced S. mutans strains. Ortholog analysis revealed 18 TCS clusters (HK-RR pairs), 2 orphan HKs and 2 orphan RRs, of which 8 TCS clusters were common to all 10 strains, 6 were absent in one or more strains, and the other 4 were exclusive to individual strains. Further classification of the predicted HKs and RRs revealed interesting aspects of their putative functions. While TCS complements were comparable within the six S. mutans strains, S. sobrinus DSM20742 lacked TCSs possibly involved in acid tolerance and fructan catabolism, and S. ratti DSM20564 possessed 3 unique TCSs but lacked the quorum-sensing related TCS (ComDE). Selected computational predictions were verified by PCR experiments.

Conclusions

Differences in the TCS repertoires of mutans streptococci strains, especially those of S. sobrinus and S. ratti in comparison to S. mutans, imply differences in their response mechanisms for survival in the dynamic oral environment. This genomic level study of TCSs should help in understanding the pathogenicity of these mutans streptococci strains.     

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.     

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.     

Extracellular DNA and lipoteichoic acids interact with exopolysaccharides in the extracellular matrix of Streptococcus mutans biofilms

Streptococcus mutans-derived exopolysaccharides are virulence determinants in the matrix of biofilms that cause caries. Extracellular DNA (eDNA) and lipoteichoic acid (LTA) are found in cariogenic biofilms, but their functions are unclear. Therefore, strains of S. mutans carrying single deletions that would modulate matrix components were used: eDNA – ?lytS and ?lytT; LTA – ?dltA and ?dltD; and insoluble exopolysaccharide – ΔgtfB. Single-species (parental strain S. mutans UA159 or individual mutant strains) and mixed-species (UA159 or mutant strain, Actinomyces naeslundii and Streptococcus gordonii) biofilms were evaluated. Distinct amounts of matrix components were detected, depending on the inactivated gene. eDNA was found to be cooperative with exopolysaccharide in early phases, while LTA played a larger role in the later phases of biofilm development. The architecture of mutant strains biofilms was distinct (vs UA159), demonstrating that eDNA and LTA influence exopolysaccharide distribution and microcolony organization. Thus, eDNA and LTA may shape exopolysaccharide structure, affecting strategies for controlling pathogenic biofilms.     

A Conserved Streptococcal Membrane Protein,LsrS, Exhibits a Receptor-Like Function for Lantibiotics

Streptococcus mutans strain GS-5 produces a two-peptide lantibiotic, Smb, which displays inhibitory activity against a broad spectrum of bacteria, including other streptococci. For inhibition, lantibiotics must recognize specific receptor molecules present on the sensitive bacterial cells. However, so far no such receptor proteins have been identified for any lantibiotics. In this study, using a powerful transposon mutagenesis approach, we have identified in Streptococcus pyogenes a gene that exhibits a receptor-like function for Smb. The protein encoded by that gene, which we named LsrS, is a membrane protein belonging to the CAAX protease family. We also found that nisin, a monopeptide lantibiotic, requires LsrS for its optimum inhibitory activity. However, we found that LsrS is not required for inhibition by haloduracin and galolacticin, both of which are two-peptide lantibiotics closely related to Smb. LsrS appears to be a well-conserved protein that is present in many streptococci, including S. mutans. Inactivation of SMU.662, an LsrS homolog, in S. mutans strains UA159 and V403 rendered the cells refractory to Smb-mediated killing. Furthermore, overexpression of LsrS in S. mutans created cells more susceptible to Smb. Although LsrS and its homolog contain the CAAX protease domain, we demonstrate that inactivation of the putative active sites on the LsrS protein has no effect on its receptor-like function. This is the first report describing a highly conserved membrane protein that displays a receptor-like function for lantibiotics.     

Improving the Efficiency of the Modified Hodge Test in KPC-Producing Klebsiella pneumoniae Isolates by Incorporating an EDTA Disk

Streptococcus mutans (S. mutans) is the main etiological agent of dental caries, and adheres to the tooth surface through the sortase A (SrtA)-mediated cell wall-anchored protein Pac. Inhibition of SrtA activity results in a marked reduction in the adhesion potential of S. mutans, and the frequency of dental caries. Morin is a natural plant extract that was previously reported to inhibit Staphylococcus aureus SrtA activity. Here, we demonstrate that morin has an inhibitory effect against S. mutans UA159 SrtA, with an IC₅₀ of 27.2 ± 2.6 μM. Western blotting demonstrated that 30 μM morin induced the partial release of the Pac protein into the supernatant. The biofilm mass of S. mutans was reduced in the presence of 30 μM morin, which was not caused by a decrease in S. mutans viability. These results indicate that morin might be important as a new agent to prevent caries.     

Structural identities of four glycosylated lipids in the oral bacterium Streptococcus mutans UA159

The cariogenic bacterium Streptococcus mutans is an important dental pathogen that forms biofilms on tooth surfaces, which provide a protective niche for the bacterium where it secretes organic acids leading to the demineralization of tooth enamel. Lipids, especially glycolipids are likely to be key components of these biofilm matrices. The UA159 strain of S. mutans was among the earliest microorganisms to have its genome sequenced. While the lipids of other S. mutans strains have been identified and characterized, lipid analyses of UA159 have been limited to a few studies on its fatty acids. Here we report the structures of the four major glycolipids from stationary-phase S. mutans UA159 cells grown in standing cultures. These were shown to be monoglucosyldiacylglycerol (MGDAG), diglucosyldiacylglycerol (DGDAG), diglucosylmonoacylglycerol (DGMAG) and, glycerophosphoryldiglucosyldiacylglycerol (GPDGDAG). The structures were determined by high performance thin-layer chromatography, mass spectrometry and nuclear magnetic resonance spectroscopy. The glycolipids were identified by accurate, high resolution, and tandem mass spectrometry. The identities of the sugar units in the glycolipids were determined by a novel and highly efficient NMR method. All sugars were shown to have α-glycosidic linkages and DGMAG was shown to be acylated in the sn-1 position by NMR. This is the first observation of unsubstituted DGMAG in any organism and the first mass spectrometry data for GPDGDAG.     

3′-Phosphoadenosine-5′-Phosphate Phosphatase Activity Is Required for Superoxide Stress Tolerance in Streptococcus mutans

Aerobic microorganisms have evolved different strategies to withstand environmental oxidative stresses generated by various reactive oxygen species (ROS). For the facultative anaerobic human oral pathogen Streptococcus mutans, the mechanisms used to protect against ROS are not fully understood, since it does not possess catalase, an enzyme that degrades hydrogen peroxide. In order to elucidate the genes that are essential for superoxide stress response, methyl viologen (MV)-sensitive mutants of S. mutans were generated via ISS1 mutagenesis. Screening of approximately 2,500 mutants revealed six MV-sensitive mutants, each containing an insertion in one of five genes, including a highly conserved hypothetical gene, SMU.1297. Sequence analysis suggests that SMU.1297 encodes a hypothetical protein with a high degree of homology to the Bacillus subtilis YtqI protein, which possesses an oligoribonuclease activity that cleaves nano-RNAs and a phosphatase activity that degrades 3′-phosphoadenosine-5′-phosphate (pAp) and 3′-phosphoadenosine-5′-phosphosulfate (pApS) to produce AMP; the latter activity is similar to the activity of the Escherichia coli CysQ protein, which is required for sulfur assimilation. SMU.1297 was deleted using a markerless Cre-loxP-based strategy; the SMU.1297 deletion mutant was just as sensitive to MV as the ISS1 insertion mutant. Complementation of the deletion mutant with wild-type SMU.1297, in trans, restored the parental phenotype. Biochemical analyses with purified SMU.1297 protein demonstrated that it has pAp phosphatase activity similar to that of YtqI but apparently lacks an oligoribonuclease activity. The ability of SMU.1297 to dephosphorylate pApS in vivo was confirmed by complementation of an E. coli cysQ mutant with SMU.1297 in trans. Thus, our results suggest that SMU.1297 is involved in superoxide stress tolerance in S. mutans. Furthermore, the distribution of homologs of SMU.1297 in streptococci indicates that this protein is essential for superoxide stress tolerance in these organisms.Streptococcus mutans, a gram-positive bacterium with a low G+C content, is widely considered the primary etiological agent of dental caries, a common human infectious disease (16, 23). S. mutans is also an important agent of infective endocarditis, as a large number of cases of viridans streptococcus-induced endocarditis are caused by S. mutans (18). During colonization of the oral cavity, S. mutans encounters various environmental stresses, including nutritional limitation, temperature fluctuation, osmotic shock, low pH conditions, radiation, toxins, and variations in oxygen tension (21). Despite these harsh conditions, S. mutans has developed multiple mechanisms for successful survival in the human host by forming diverse and densely populated biofilms on the tooth surface (4). The extraordinary ability of S. mutans to adapt and flourish in the diverse and adverse environment of the oral cavity emphasizes the fundamental importance of the need for detailed analyses of the molecular mechanisms of stress tolerance response in this organism.S. mutans is a facultative anaerobic organism, but it can tolerate aerobic conditions for colonization and survival. Like other streptococci, it does not possess cytochromes and therefore cannot carry out energy-conserving oxidative phosphorylation (2). However, irrespective of the growth conditions, S. mutans derives the energy for growth through fermentation of glucose and other sugars (26). This can lead to unwanted consequences, especially when the organism is exposed to aerobic conditions in the oral cavity. If the molecular oxygen is not fully reduced by the four-electron reduction step to water, it can undergo one- or two-electron reductions to form reactive superoxide radicals, hydroxyl radicals, and hydrogen peroxide, collectively known as reactive oxygen species (ROS) (19). These radicals, when accumulated in large amounts, can trigger oxidation of lipid, protein, and nucleic acid inside the cell, ultimately leading to cellular death (19, 20).Aerobic bacteria have developed multiple strategies to adapt and protect against ROS insults (19). These strategies include (i) enzymes that scavenge ROS, such as superoxide dismutases (SOD), catalases, and peroxidases; (ii) protein repair systems, such as thioredoxin; (iii) DNA damage repair enzymes such as RecA; and (iv) proteins that regulate intracellular iron level to ameliorate the generation of ROS. Although streptococci contain SOD, NADH oxidase, glutathione reductase, and other proteins to counter ROS threats, they do not contain catalase, a key protective enzyme against oxidative radicals. Therefore, the defense strategy against damage by ROS is significantly different in streptococci than in other bacteria. For example, the growth of S. mutans in planktonic or biofilm mode can influence the respiratory rates as well as the activities of the protective enzymes, such as SOD and NADH oxidase (31).Apart from studies related to the physiology of oxidative stress in S. mutans, very little information is available on the oxidative-stress response and its regulation in this organism. Many key regulatory genes, including members of the OxyR and SoxR families, which are involved in sensing and responding to ROS attacks, are not encoded in the genome of S. mutans (2). Instead, S. mutans has a PerR homolog, which has been shown to be involved in hydrogen peroxide stress response in this organism (21). The luxS gene of S. mutans, which encodes an enzyme that synthesizes the intercellular signaling molecule AI-2, is also involved in the oxidative-stress response (52). However, the exact mechanism by which LuxS participates in the oxidative-stress response is currently unknown. Furthermore, a recent investigation suggests that a two-component signal transduction system, ScnRK, is necessary for counteracting ROS in S. mutans (11).The major focus of this study was to identify the genes that are involved in the defense against superoxide stress of S. mutans strain UA159. Toward this end, a library of mutants was generated by insertion mutagenesis, and the mutants were screened for their sensitivity to methyl viologen (MV), a superoxide-generating compound. This study enabled the identification of five loci that are potentially involved in superoxide tolerance. One of the identified loci is SMU.1297, which encodes a protein homologous to YtqI of Bacillus subtilis. The biochemical characterization of SMU.1297 and its role in superoxide stress tolerance response are presented.     

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.     

Phenotypic Heterogeneity of Genomically-Diverse Isolates of Streptococcus mutans

High coverage, whole genome shotgun (WGS) sequencing of 57 geographically- and genetically-diverse isolates of Streptococcus mutans from individuals of known dental caries status was recently completed. Of the 57 sequenced strains, fifteen isolates, were selected based primarily on differences in gene content and phenotypic characteristics known to affect virulence and compared with the reference strain UA159. A high degree of variability in these properties was observed between strains, with a broad spectrum of sensitivities to low pH, oxidative stress (air and paraquat) and exposure to competence stimulating peptide (CSP). Significant differences in autolytic behavior and in biofilm development in glucose or sucrose were also observed. Natural genetic competence varied among isolates, and this was correlated to the presence or absence of competence genes, comCDE and comX, and to bacteriocins. In general strains that lacked the ability to become competent possessed fewer genes for bacteriocins and immunity proteins or contained polymorphic variants of these genes. WGS sequence analysis of the pan-genome revealed, for the first time, components of a Type VII secretion system in several S. mutans strains, as well as two putative ORFs that encode possible collagen binding proteins located upstream of the cnm gene, which is associated with host cell invasiveness. The virulence of these particular strains was assessed in a wax-worm model. This is the first study to combine a comprehensive analysis of key virulence-related phenotypes with extensive genomic analysis of a pathogen that evolved closely with humans. Our analysis highlights the phenotypic diversity of S. mutans isolates and indicates that the species has evolved a variety of adaptive strategies to persist in the human oral cavity and, when conditions are favorable, to initiate disease.     

The antimicrobial effects of deglycyrrhizinated licorice root extract on Streptococcus mutans UA159 in both planktonic and biofilm cultures

The objective of the study was to investigate the antimicrobial effects of deglycyrrhizinated licorice root extracts (DG-LRE) against Streptococcus mutans UA159 in both the planktonic and biofilm phases by determining the minimum inhibitory concentration and minimum bactericidal concentration, and by performing time-kill kinetic, growth, adhesion, and biofilm assays. The cell toxicity of DG-LRE on normal human gingival fibroblast (NHGF) cells was tested using a methyl thiazolyl tetrazolium assay. This study showed that DG-LRE had strong antimicrobial activity against S. mutans in the planktonic phase with little cytotoxic effect on NHGF cells. In addition, DG-LRE significantly inhibited biofilm formation by S. mutans UA159 at concentrations over 4 μg/ml for glucose or 16 μg/ml for sucrose, respectively, regardless of the presence of saliva-coating. To the best of our knowledge, this is the first report to provide evidence that DG-LRE demonstrates antimicrobial activity against S. mutans. These results suggest that DG-LRE can be used in developing oral hygiene products, such as gargling solution and dentifrice to prevent human dental caries.     

The influence of HtrA expression on the growth of Streptococcus mutans during acid stress

When proteins are damaged under stresses conditions, these proteins are either refolded or degraded by quality control system of molecular chaperones and protease. High-temperature requirement A (htrA) is of particular interest because it can perform the roles of both protease and a chaperone. HtrA plays an important role in maintaining the physiological homeostasis of bacteri against environmental stress such as elevated temperature, oxidative and osmotic stress. Inactivation of htrA genes can thus restrict the survival ability of bacteria. These observations suggested that htrA might be responsible for acid tolerance of Streptococcus mutans. In this study, we have generated an htrA mutant and an htrA-complemented strain of S. mutans K7 isolated from a Korean in order to investigate the role of htrA in growth under acidic conditions. In terms of growth under cidic conditions, the htrA mutant exhibited 20% to 23% lower growth than the control group. In ddition, glucosyltransferaseB nd glucosyltransferaseC expression levels significantly decreased. When the htrA expression level was restored by adding the htrA gene to the htrA mutant strain, the normal growth phenotype was restored under acid stress. Further, similar results were obtained for S. mutans UA159. Thus, htrA in S. mutans K7, as well as S. mutans UA159, can be concluded to play an important role during acid stress.