Nkx2.3 transcription factor is a key regulator of mucous cell identity in salivary glands.

Saliva is vital to oral health, fulfilling multiple functions in the oral cavity. Three pairs of major salivary glands and hundreds of minor salivary glands contribute to saliva production. The secretory acinar cells within these glands include two distinct populations. Serous acinar cells secrete a watery saliva containing enzymes, while mucous acinar cells secrete a more viscous fluid containing highly glycosylated mucins. Despite their shared developmental origins, the parotid gland (PG) is comprised of only serous acinar cells, while the sublingual gland (SLG) contains predominantly mucous acinar cells. The instructive signals that govern the identity of serous versus mucous acinar cell phenotypes are not yet known. The homeobox transcription factor Nkx2.3 is uniquely expressed in the SLG. Disruption of the Nkx2.3 gene was reported to delay the maturation of SLG mucous acinar cells. To examine whether Nkx2.3 plays a role in directing the mucous cell phenotype, we analyzed SLG from Nkx2.3-/- mice using RNAseq, immunostaining and proteomic analysis of saliva. Our results indicate that Nkx2.3, most likely in concert with other transcription factors uniquely expressed in the SLG, is a key regulator of the molecular program that specifies the identity of mucous acinar cells.

Short-term and bystander effects of radiation on murine submandibular glands.

Many patients treated for head and neck cancers experience salivary gland hypofunction due to radiation damage. Understanding the mechanisms of cellular damage induced by radiation treatment is important in order to design methods of radioprotection. In addition, it is crucial to recognize the indirect effects of irradiation and the systemic responses that may alter saliva secretion. In this study, radiation was delivered to murine submandibular glands (SMGs) bilaterally, using a 137Cs gamma ray irradiator, or unilaterally, using a small-animal radiation research platform (SARRP). Analysis at 3, 24 and 48 h showed dynamic changes in mRNA and protein expression in SMGs irradiated bilaterally. Unilateral irradiation using the SARRP caused similar changes in the irradiated SMGs, as well as significant off-target, bystander effects in the non-irradiated contralateral SMGs.

Analysis of the Streptococcus mutans Proteome during Acid and Oxidative Stress Reveals Modules of Protein Coexpression and an Expanded Role for the TreR Transcriptional Regulator.

Streptococcus mutans promotes a tooth-damaging dysbiosis in the oral microbiota because it can form biofilms and survive acid stress better than most of its ecological competitors, which are typically health associated. Many of these commensals produce hydrogen peroxide; therefore, S. mutans must manage both oxidative stress and acid stress with coordinated and complex physiological responses. In this study, the proteome of S. mutans was examined during regulated growth in acid and oxidative stresses as well as in deletion mutants with impaired oxidative stress phenotypes, Δnox and ΔtreR. A total of 607 proteins exhibited significantly different abundances across the conditions tested, and correlation network analysis identified modules of coexpressed proteins that were responsive to the deletion of nox and/or treR as well as acid and oxidative stress. The data explained the reactive oxygen species (ROS)-sensitive and mutacin-deficient phenotypes exhibited by the ΔtreR strain. SMU.1069-1070, a poorly understood LytTR system, had an elevated abundance in the ΔtreR strain. S. mutans LytTR systems regulate mutacin production and competence, which may explain how TreR affects mutacin production. Furthermore, the protein cluster that produces mutanobactin, a lipopeptide important in ROS tolerance, displayed a reduced abundance in the ΔtreR strain. The role of Nox as a keystone in the oxidative stress response was also emphasized. Crucially, this data set provides oral health researchers with a proteome atlas that will enable a more complete understanding of the S. mutans stress responses that are required for pathogenesis, and will facilitate the development of new and improved therapeutic approaches for dental caries. IMPORTANCE Dental caries is the most common chronic infectious disease worldwide and disproportionately affects marginalized socioeconomic groups. Streptococcus mutans is considered a primary etiological agent of caries, with its pathogenicity being dependent on coordinated physiological stress responses that mitigate the damage caused by the oxidative and acid stress common within dental plaque. In this study, the proteome of S. mutans was examined during growth in acidic and oxidative stresses as well in nox and treR deletion mutants. A total of 607 proteins were differentially expressed across the strains/growth conditions, and modules of coexpressed proteins were identified, which enabled mapping the acid and oxidative stress responses across S. mutans metabolism. The presence of TreR was linked to mutacin production via LytTR system signaling and to oxidative stress via mutanobactin production. The data provided by this study will guide future research elucidating S. mutans pathogenesis and developing improved preventative and treatment modalities for dental caries.

Streptococcus mutans SpxA2 relays the signal of cell envelope stress from LiaR to effectors that maintain cell wall and membrane homeostasis.

Streptococcus mutans is a major etiologic agent of dental caries, which is the most common chronic infectious disease worldwide. S. mutans is particularly adept at causing caries due to its exceptional capacity to form biofilms and its ability to survive acidic conditions that arrest acid production and growth in many more benign members of the oral microbiota. Two mechanisms utilized by S. mutans to tolerate acid are: modulation of the membrane fatty acid content and utilization of the F1 F0 -ATPase to pump protons out of the cytosol. In this study, the role of the spxA2 transcriptional regulator in these two pathways, and overall cell envelope homeostasis, was examined. Loss of spxA2 resulted in an increase in the proportion of saturated fatty acids in the S. mutans membrane and altered transcription of several genes involved in the production of these membrane fatty acids, including fabT and fabM. Furthermore, activity of the F1 F0 -ATPase was increased in the ∆spxA2 strain. Transcription of spxA2 was elevated in the presence of a variety of membrane stressors, and highly dependent on the liaR component of the LiaFSR system, which is known to sense cell envelope stress in many Gram-positive bacteria. Finally, deletion of ∆spxA2 led to altered susceptibility of S. mutans to membrane stressors. Overall, the results of this study indicate that spxA2 serves a crucial role in transmitting the signal of cell wall/membrane damage from the LiaFSR sensor to downstream effectors in the SpxA2 regulon which restore and maintain membrane and cell wall homeostasis.

Disruption of l-Rhamnose Biosynthesis Results in Severe Growth Defects in Streptococcus mutans.

The rhamnose-glucose cell wall polysaccharide (RGP) of Streptococcus mutans plays a significant role in cell division, virulence, and stress protection. Prior studies examined function of the RGP using strains carrying deletions in the machinery involved in RGP assembly. In this study, we explored loss of the substrate for RGP, l-rhamnose, via deletion of rmlD (encoding the protein responsible for the terminal step in l-rhamnose biosynthesis). We demonstrate that loss of rhamnose biosynthesis causes a phenotype similar to strains with disrupted RGP assembly (ΔrgpG and ΔrgpF strains). Deletion of rmlD not only caused a severe growth defect under nonstress growth conditions but also elevated susceptibility of the strain to acid and oxidative stress, common conditions found in the oral cavity. A genetic complement of the ΔrmlD strain completely restored wild-type levels of growth, whereas addition of exogenous rhamnose did not. The loss of rhamnose production also significantly disrupted biofilm formation, an important aspect of S. mutans growth in the oral cavity. Further, we demonstrate that loss of either rmlD or rgpG results in ablation of rhamnose content in the S. mutans cell wall. Taken together, these results highlight the importance of rhamnose production in both the fitness and the ability of S. mutans to overcome environmental stresses.IMPORTANCEStreptococcus mutans is a pathogenic bacterium that is the primary etiologic agent of dental caries, a disease that affects billions yearly. Rhamnose biosynthesis is conserved not only in streptococcal species but in other Gram-positive, as well as Gram-negative, organisms. This study highlights the importance of rhamnose biosynthesis in RGP production for protection of the organism against acid and oxidative stresses, the two major stressors that the organism encounters in the oral cavity. Loss of RGP also severely impacts biofilm formation, the first step in the onset of dental caries. The high conservation of the rhamnose synthesis enzymes, as well as their importance in S. mutans and other organisms, makes them favorable antibiotic targets for the treatment of disease.

Streptococcus mutans requires mature rhamnose-glucose polysaccharides for proper pathophysiology, morphogenesis and cellular division.

The cell wall of Gram-positive bacteria has been shown to mediate environmental stress tolerance, antibiotic susceptibility, host immune evasion and overall virulence. The majority of these traits have been demonstrated for the well-studied system of wall teichoic acid (WTA) synthesis, a common cell wall polysaccharide among Gram-positive organisms. Streptococcus mutans, a Gram-positive odontopathogen that contributes to the enamel-destructive disease dental caries, lacks the capabilities to generate WTA. Instead, the cell wall of S. mutans is highly decorated with rhamnose-glucose polysaccharides (RGP), for which functional roles are poorly defined. Here, we demonstrate that the RGP has a distinct role in protecting S. mutans from a variety of stress conditions pertinent to pathogenic capability. Mutant strains with disrupted RGP synthesis failed to properly localize cell division complexes, suffered from aberrant septum formation and exhibited enhanced cellular autolysis. Surprisingly, mutant strains of S. mutans with impairment in RGP side chain modification grew into elongated chains and also failed to properly localize the presumed cell wall hydrolase, GbpB. Our results indicate that fully mature RGP has distinct protective and morphogenic roles for S. mutans, and these structures are functionally homologous to the WTA of other Gram-positive bacteria.

Inactivation of Streptococcus mutans genes lytST and dltAD impairs its pathogenicity in vivo.

Background: Streptococcus mutans orchestrates the development of a biofilm that causes dental caries in the presence of dietary sucrose, and, in the bloodstream, S. mutans can cause systemic infections. The development of a cariogenic biofilm is dependent on the formation of an extracellular matrix rich in exopolysaccharides, which contains extracellular DNA (eDNA) and lipoteichoic acids (LTAs). While the exopolysaccharides are virulence markers, the involvement of genes linked to eDNA and LTAs metabolism in the pathogenicity of S. mutans remains unclear. Objective and Design: In this study, a parental strain S. mutans UA159 and derivative strains carrying single gene deletions were used to investigate the role of eDNA (ΔlytS and ΔlytT), LTA (ΔdltA and ΔdltD), and insoluble exopolysaccharides (ΔgtfB) in virulence in a rodent model of dental caries (rats) and a systemic infection model (Galleria mellonella larvae). Results: Fewer carious lesions were observed on smooth and sulcal surfaces of enamel and dentin of the rats infected with ∆lytS, ∆dltD, and ΔgtfB (vs. the parental strain). Moreover, strains carrying gene deletions prevented the killing of larvae (vs. the parental strain). Conclusions: Altogether, these findings indicate that inactivation of lytST and dltAD impaired S. mutans cariogenicity and virulence in vivo.

Deficiency of BrpA in Streptococcus mutans reduces virulence in rat caries model.

Our recent studies have shown that BrpA in Streptococcus mutans plays a critical role in cell envelope biogenesis, stress responses, and biofilm formation. In this study, a 10-species consortium was used to assess how BrpA deficiency influences the establishment, persistence, and competitiveness of S. mutans during growth in a community under conditions typical of the oral cavity. Results showed that, like the wild-type, the brpA mutant was able to colonize and establish on the surfaces tested. Relative to the wild-type, however, the brpA mutant had a reduced ability to persist and grow in the 10-species consortium (P < .001). A rat caries model was also used to examine the effect of BrpA, as well as Psr, a BrpA paralog, on S. mutans cariogenicity. The results showed no major differences in infectivity between the wild-type and the brpA and psr mutants. Unlike the wild-type, however, infection with the brpA mutant, but not the psr mutant, showed no significant differences in both total numbers of carious lesions and caries severity, compared with the control group that received bacterial growth medium (P > .05). Metagenomic and quantitative polymerase chain reaction analysis showed that S. mutans infection caused major alterations in the composition of the rats' plaque microbiota and that significantly less S. mutans was identified in the rats infected with the brpA mutant compared with those infected with the wild-type and the psr mutant. These results further suggest that BrpA plays a critical role in S. mutans pathophysiology and that BrpA has potential as a therapeutic target in the modulation of S. mutans virulence.

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.

What are We Learning and What Can We Learn from the Human Oral Microbiome Project?

Extraordinary technological advances have greatly accelerated our ability to identify bacteria, at the species level, present in clinical samples taken from the human mouth. In addition, technologies are evolving such that the oral samples can be analyzed for their protein and metabolic products. As a result, pictures are the advent of personalized dental medicine is becoming closer to reality.

A Modified Chromogenic Assay for Determination of the Ratio of Free Intracellular NAD+/NADH in Streptococcus mutans.

Nicotinamide adenine dinucleotide is a coenzyme present in all kingdoms of life and exists in two forms: oxidized (NAD+) and reduced (NADH). NAD(H) is involved in a multitude of essential metabolic redox reactions, providing oxidizing or reducing equivalents. The ratio of free intracellular NAD+/NADH is fundamentally important in the maintenance of cellular redox homeostasis (Ying, 2008). Various chromogenic cycling assays have been used to determine the ratio of NAD+/NADH in both bacterial and mammalian cells for more than forty years (Bernofsky and Swan, 1973; Nisselbaum and Green, 1969). Here, we describe in detail an assay to determine the ratio of free intracellular NAD+ to NADH in Streptococcus mutans. This cycling assay is a modified version of the protocol first described by Bernofsky and Swan (Bernofsky and Swan, 1973), using the extraction buffer described by Frezza et al. (2011), followed by the reduced MTT precipitation described by Gibbon and Larher (Gibon and Larher, 1997). As depicted in Figure 1, alcohol dehydrogenase is used to drive a series of redox reactions utilizing exogenously added ethanol and NAD+ from sample extracts as initial substrates, phenazine ethosulfate (PES) as an electron carrier, and thiazolyl blue tetrazolium bromide (MTT) as a terminal electron acceptor. 6 M NaCl is used to stop the reaction. The reduced MTT (formazan dye) is purple in color and can be quantified by measuring absorbance at 570 nm. This protocol is divided into three steps: A. Preparation of cell pellets of S. mutans; B. Preparation of deproteinated cell extracts containing NADtotal or NADH; C. NAD+/NADH cycling assay. This method has proven robust in measuring the NAD+/NADH ratio in S. mutans under a variety of conditions, and should be applicable to other Gram-positive bacteria.

ß-Phosphoglucomutase contributes to aciduricity in Streptococcus mutans.

Streptococcus mutans encounters an array of sugar moieties within the oral cavity due to a varied human diet. One such sugar is ß-d-glucose 1-phosphate (ßDG1P), which must be converted to glucose 6-phosphate (G6P) before further metabolism to lactic acid. The conversion of ßDG1P to G6P is mediated by ß-phosphoglucomutase, which has not been previously observed in any oral streptococci, but has been extensively characterized and the gene designated pgmB in Lactococcus lactis. An orthologue was identified in S. mutans, SMU.1747c, and deletion of the gene resulted in the inability of the deletion strain to convert ßDG1P to G6P, indicating that SMU.1747c is a ß-phosphoglucomutase and should be designated pgmB. In this study, we sought to characterize how deletion of pgmB affected known virulence factors of S. mutans, specifically acid tolerance. The ΔpgmB strain showed a decreased ability to survive acid challenge. Additionally, the strain lacking ß-phosphoglucomutase had a diminished glycolytic profile compared with the parental strain. Deletion of pgmB had a negative impact on the virulence of S. mutans in the Galleria mellonella (greater wax worm) animal model. Our results indicate that pgmB plays a role at the juncture of carbohydrate metabolism and virulence.

The Streptococcus mutans aminotransferase encoded by ilvE is regulated by CodY and CcpA.

The aminotransferase IlvE was implicated in the acid tolerance response of Streptococcus mutans when a mutation in its gene resulted in an acid-sensitive phenotype (B. Santiago, M. MacGilvray, R. C. Faustoferri, and R. G. Quivey, Jr., J. Bacteriol. 194:2010-2019, 2012). The phenotype suggested that amino acid metabolism is important for acid adaptation, as turnover of branched-chain amino acids (bcAAs) could provide important signals to modulate expression of genes involved in the adaptive process. Previous studies have demonstrated that ilvE is regulated in response to the external pH, though the mechanism is not yet established. CodY and CcpA have been shown to regulate expression of branched-chain amino acid biosynthetic genes, suggesting that the ability to sense carbon flow and the nutritional state of the cell also plays a role in the regulation of ilvE. Electrophoretic mobility shift assays using the ilvE promoter and a purified recombinant CodY protein provided evidence of the physical interaction between CodY and ilvE. In order to elucidate the signals that contribute to ilvE regulation, cat reporter fusions were utilized. Transcriptional assays demonstrated that bcAAs are signaling molecules involved in the repression of ilvE through regulation of CodY. In a codY deletion background, ilvE transcription was elevated, indicating that CodY acts a repressor of ilvE transcription. Conversely, in a ccpA deletion background, ilvE transcription was reduced, showing that CcpA activated ilvE transcription. The effects of both regulators were directly relevant for transcription of ilvE under conditions of acid stress, demonstrating that both regulators play a role in acid adaptation.

Role of DNA base excision repair in the mutability and virulence of Streptococcus mutans.

The oral pathogen, Streptococcus mutans, possesses inducible DNA repair defences for protection against pH fluctuations and production of reactive oxygen metabolites such as hydrogen peroxide (H(2) O(2) ), which are present in the oral cavity. DNA base excision repair (BER) has a critical role in genome maintenance by preventing the accumulation of mutations associated with environmental factors and normal products of cellular metabolism. In this study, we examined the consequences of compromising the DNA glycosylases (Fpg and MutY) and endonucleases (Smx and Smn) of the BER pathway and their relative role in adaptation and virulence. Enzymatic characterization of the BER system showed that it protects the organism against the effects of the highly mutagenic lesion, 7,8-dihydro-8-oxo-2'-deoxyguanine (8-oxo-dG). S. mutans strains lacking a functional Fpg, MutY or Smn showed elevated spontaneous mutation frequencies; and, these mutator phenotypes correlated with the ability of the strains to survive killing by acid and oxidative agents. In addition, in the Galleria mellonella virulence model, strains of S. mutans deficient in Fpg, MutY and Smn showed increased virulence as compared with the parent strain. Our results suggest that, for S. mutans, mutator phenotypes, due to loss of BER enzymes, may confer an advantage to virulence of the organism.

The branched-chain amino acid aminotransferase encoded by ilvE is involved in acid tolerance in Streptococcus mutans.

The ability of Streptococcus mutans to produce and tolerate organic acids from carbohydrate metabolism represents a major virulence factor responsible for the formation of carious lesions. Pyruvate is a key metabolic intermediate that, when rerouted to other metabolic pathways such as amino acid biosynthesis, results in the alleviation of acid stress by reducing acid end products and aiding in maintenance of intracellular pH. Amino acid biosynthetic genes such as ilvC and ilvE were identified as being upregulated in a proteome analysis of Streptococcus mutans under acid stress conditions (A. C. Len, D. W. Harty, and N. A. Jacques, Microbiology 150:1353-1366, 2004). In Lactococcus lactis and Staphylococcus carnosus, the ilvE gene product is involved with biosynthesis and degradation of branched-chain amino acids, as well as in the production of branched-chain fatty acids (B. Ganesan and B. C. Weimer, Appl. Environ. Microbiol. 70:638-641, 2004; S. M. Madsen et al., Appl. Environ. Microbiol. 68:4007-4014, 2002; and M. Yvon, S. Thirouin, L. Rijnen, D. Fromentier, and J. C. Gripon, Appl. Environ. Microbiol. 63:414-419, 1997). Here we constructed and characterized an ilvE deletion mutant of S. mutans UA159. Growth experiments revealed that the ilvE mutant strain has a lag in growth when nutritionally limited for branched-chain amino acids. We further demonstrated that the loss of ilvE causes a decrease in acid tolerance. The ilvE strain exhibits a defect in F(1)-F(o) ATPase activity and has reduced catabolic activity for isoleucine and valine. Results from transcriptional studies showed that the ilvE promoter is upregulated during growth at low pH. Collectively, the results of this investigation show that amino acid metabolism is a component of the acid-adaptive repertoire of S. mutans.

Mutation of the NADH oxidase gene (nox) reveals an overlap of the oxygen- and acid-mediated stress responses in Streptococcus mutans.

NADH oxidase (Nox) is a flavin-containing enzyme used by Streptococcus mutans to reduce dissolved oxygen encountered during growth in the oral cavity. In this study, we characterized the role of the NADH oxidase in the oxidative and acid stress responses of S. mutans. A nox-defective mutant strain of S. mutans and its parental strain, the genomic type strain UA159, were exposed to various oxygen concentrations at pH values of 5 and 7 to better understand the adaptive mechanisms used by the organism to withstand environmental pressures. With the loss of nox, the activities of oxygen stress response enzymes such as superoxide dismutase and glutathione oxidoreductase were elevated compared to those in controls, resulting in a greater adaptation to oxygen stress. In contrast, the loss of nox led to a decreased ability to grow in a low-pH environment despite an increased resistance to severe acid challenge. Analysis of the membrane fatty acid composition revealed that for both the nox mutant and UA159 parent strain, growth in an oxygen-rich environment resulted in high proportions of unsaturated membrane fatty acids, independent of external pH. The data indicate that S. mutans membrane fatty acid composition is responsive to oxidative stress, as well as changes in environmental pH, as previously reported (E. M. Fozo and R. G. Quivey, Jr., Appl. Environ. Microbiol. 70:929-936, 2004). The heightened ability of the nox strain to survive acidic and oxidative environmental stress suggests a multifaceted response system that is partially dependent on oxygen metabolites.

Smx nuclease is the major, low-pH-inducible apurinic/apyrimidinic endonuclease in Streptococcus mutans.

The causative agent of dental caries in humans, Streptococcus mutans, outcompetes other bacterial species in the oral cavity and causes disease by surviving acidic conditions in dental plaque. We have previously reported that the low-pH survival strategy of S. mutans includes the ability to induce a DNA repair system that appears to involve an enzyme with exonuclease functions (K. Hahn, R. C. Faustoferri, and R. G. Quivey, Jr., Mol. Microbiol 31:1489-1498, 1999). Here, we report overexpression of the S. mutans apurinic/apyrimidinic (AP) endonuclease, Smx, in Escherichia coli; initial characterization of its enzymatic activity; and analysis of an smx mutant strain of S. mutans. Insertional inactivation of the smx gene eliminates the low-pH-inducible exonuclease activity previously reported. In addition, loss of Smx activity renders the mutant strain sensitive to hydrogen peroxide treatment but relatively unaffected by acid-mediated damage or near-UV irradiation. The smx strain of S. mutans was highly sensitive to the combination of iron and hydrogen peroxide, indicating the likely production of hydroxyl radical by Fenton chemistry with concomitant formation of AP sites that are normally processed by the wild-type allele. Smx activity was sufficiently expressed in E. coli to protect an xth mutant strain from the effects of hydrogen peroxide treatment. The data indicate that S. mutans expresses an inducible, class II-like AP endonuclease, encoded by the smx gene, that exhibits exonucleolytic activity and is regulated as part of the acid-adaptive response of the organism. Smx is likely the primary, if not the sole, AP endonuclease induced during growth at low pH values.

The F-ATPase operon promoter of Streptococcus mutans is transcriptionally regulated in response to external pH.

Streptococcus mutans F-ATPase, the major component of the acid-adaptive response of the organism, is transcriptionally upregulated at low pH. Fusions of the F-ATPase promoter to chloramphenicol acetyltransferase indicated that pH-dependent expression is still observed with a short promoter that contains a domain conserved between streptococcal ATPase operons.