Backbone NMR resonance assignments for the C terminal domain of the Streptococcus mutans adhesin P1.

Adhesin P1 (aka AgI/II) plays a pivotal role in mediating Streptococcus mutans attachment in the oral cavity, as well as in regulating biofilm development and maturation. P1's naturally occurring truncation product, Antigen II (AgII), adopts both soluble, monomeric and insoluble, amyloidogenic forms within the bacterial life cycle. Monomers are involved in important quaternary interactions that promote cell adhesion and the functional amyloid form promotes detachment of mature biofilms. The heterologous, 51-kD C123 construct comprises most of AgII and was previously characterized by X-ray crystallography. C123 contains three structurally homologous domains, C1, C2, and C3. NMR samples made using the original C123 construct, or its C3 domain, yielded moderately resolved NMR spectra. Using Alphafold, we re-analyzed the P1 sequence to better identify domain boundaries for C123, and in particular the C3 domain. We then generated a more tractable construct for NMR studies of the monomeric form, including quaternary interactions with other proteins. The addition of seven amino acids at the C-terminus greatly improved the spectral dispersion for C3 relative to the prior construct. Here we report the backbone NMR resonance assignments for the new construct and characterize some of its quaternary interactions. These data are in good agreement with the structure predicted by Alphafold, which contains additional ß-sheet secondary structure compared to the C3 domain in the C123 crystal structure for a construct lacking the seven C-terminal amino acids. Its quaternary interactions with known protein partners are in good agreement with prior competitive binding assays. This construct can be used for further NMR studies, including protein-protein interaction studies and assessing the impact of environmental conditions on C3 structure and dynamics within C123 as it transitions from monomer to amyloid form.

Structural and biochemical insights into CRISPR RNA processing by the Cas5c ribonuclease SMU1763 from Streptococcus mutans.

The cariogenic pathogen Streptococcus mutans contains two CRISPR systems (type I-C and type II-A) with the Cas5c protein (SmuCas5c) involved in processing of long CRISPR RNA transcripts (pre-crRNA) containing repeats and spacers to mature crRNA guides. In this study, we determined the crystal structure of SmuCas5c at a resolution of 1.72 Å, which revealed the presence of an N-terminal modified RNA recognition motif and a C-terminal twisted ß-sheet domain with four bound sulphate molecules. Analysis of surface charge and residue conservation of the SmuCas5c structure suggested the location of an RNA-binding site in a shallow groove formed by the RNA recognition motif domain with several conserved positively charged residues (Arg39, Lys52, Arg109, Arg127, and Arg134). Purified SmuCas5c exhibited metal-independent ribonuclease activity against single-stranded pre-CRISPR RNAs containing a stem-loop structure with a seven-nucleotide stem and a pentaloop. We found SmuCas5c cleaves substrate RNA within the repeat sequence at a single cleavage site located at the 3'-base of the stem but shows significant tolerance to substrate sequence variations downstream of the cleavage site. Structure-based mutational analysis revealed that the conserved residues Tyr50, Lys120, and His121 comprise the SmuCas5c catalytic residues. In addition, site-directed mutagenesis of positively charged residues Lys52, Arg109, and Arg134 located near the catalytic triad had strong negative effects on the RNase activity of this protein, suggesting that these residues are involved in RNA binding. Taken together, our results reveal functional diversity of Cas5c ribonucleases and provide further insight into the molecular mechanisms of substrate selectivity and activity of these enzymes.

A concerted probiotic activity to inhibit periodontitis-associated bacteria.

Periodontitis can result in tooth loss and the associated chronic inflammation can provoke several severe systemic health risks. Adjunctive to mechanical treatment of periodontitis and as alternatives to antibiotics, the use of probiotic bacteria was suggested. In this study, the inhibitory effect of the probiotic Streptococcus salivarius subsp. salivarius strains M18 and K12, Streptococcus oralis subsp. dentisani 7746, and Lactobacillus reuteri ATCC PTA 5289 on anaerobic periodontal bacteria and Aggregatibacter actinomycetemcomitans was tested. Rarely included in other studies, we also quantified the inverse effect of pathogens on probiotic growth. Probiotics and periodontal pathogens were co-incubated anaerobically in a mixture of autoclaved saliva and brain heart infusion broth. The resulting genome numbers of the pathogens and of the probiotics were measured by quantitative real-time PCR. Mixtures of the streptococcal probiotics were also used to determine their synergistic, additive, or antagonistic effects. The overall best inhibitor of the periodontal pathogens was L. reuteri ATCC PTA 5289, but the effect is coenzyme B12-, anaerobiosis-, as well as glycerol-dependent, and further modulated by L. reuteri strain DSM 17938. Notably, in absence of glycerol, the pathogen-inhibitory effect could even turn into a growth spurt. Among the streptococci tested, S. salivarius M18 had the most constant inhibitory potential against all pathogens, followed by K12 and S. dentisani 7746, with the latter still having significant inhibitory effects on P. intermedia and A. actinomycetemcomitans. Overall, mixtures of the streptococcal probiotics did inhibit the growth of the pathogens equally or-in the case of A. actinomycetemcomitans- better than the individual strains. P. gingivalis and F. nucleatum were best inhibited by pure cultures of S. salivarius K12 or S. salivarius M18, respectively. Testing inverse effects, the growth of S. salivarius M18 was enhanced when incubated with the periodontal pathogens minus/plus other probiotics. In contrast, S. oralis subsp. dentisani 7746 was not much influenced by the pathogens. Instead, it was significantly inhibited by the presence of other streptococcal probiotics. In conclusion, despite some natural limits such as persistence, the full potential for probiotic treatment is by far not utilized yet. Especially, further exploring concerted activity by combining synergistic strains, together with the application of oral prebiotics and essential supplements and conditions, is mandatory.

Quorum Sensing in Streptococcus mutans Regulates Production of Tryglysin, a Novel RaS-RiPP Antimicrobial Compound.

The genus Streptococcus encompasses a large bacterial taxon that commonly colonizes mucosal surfaces of vertebrates and is capable of disease etiologies originating from diverse body sites, including the respiratory, digestive, and reproductive tracts. Identifying new modes of treating infections is of increasing importance, as antibiotic resistance has escalated. Streptococcus mutans is an important opportunistic pathogen that is an agent of dental caries and is capable of systemic diseases such as endocarditis. As such, understanding how it regulates virulence and competes in the oral niche is a priority in developing strategies to defend from these pathogens. We determined that S. mutans UA159 possesses a bona fide short hydrophobic peptide (SHP)/Rgg quorum-sensing system that regulates a specialized biosynthetic operon featuring a radical-SAM (S-adenosyl-l-methionine) (RaS) enzyme and produces a ribosomally synthesized and posttranslationally modified peptide (RiPP). The pairing of SHP/Rgg regulatory systems with RaS biosynthetic operons is conserved across streptococci, and a locus similar to that in S. mutans is found in Streptococcus ferus, an oral streptococcus isolated from wild rats. We identified the RaS-RiPP product from this operon and solved its structure using a combination of analytical methods; we term these RiPPs tryglysin A and B for the unusual Trp-Gly-Lys linkage. We report that tryglysins specifically inhibit the growth of other streptococci, but not other Gram-positive bacteria such as Enterococcus faecalis or Lactococcus lactis We predict that tryglysin is produced by S. mutans in its oral niche, thus inhibiting the growth of competing species, including several medically relevant streptococci.IMPORTANCE Bacteria interact and compete with a large community of organisms in their natural environment. Streptococcus mutans is one such organism, and it is an important member of the oral microbiota. We found that S. mutans uses a quorum-sensing system to regulate production of a novel posttranslationally modified peptide capable of inhibiting growth of several streptococcal species. We find inhibitory properties of a similar peptide produced by S. ferus and predict that these peptides play a role in interspecies competition in the oral niche.

Antifungal effects of Streptococcus mutans extract on Candida strains susceptible and resistant to fluconazole: An in vivo study.

Previous studies showed that the crude extract obtained from Streptococcus mutans inhibited the growth of Candida albicans reference strains. In this study, we evaluated whether the antifungal effects of S. mutans extract can be extended to clinical Candida isolates, including C. albicans and non-abicans strains with different susceptibilities to fluconazole. We verified that S. mutans extract increased the survival of Galleria mellonella larvae infected with C. albicans and C. glabrata and inhibited the fungal cells in hemolymph. These antifungal effects occurred for both fluconazole-susceptible and fluconazole-resistant strains. However, larvae infected by C. krusei were not affected by S. mutans extract. LAY SUMMARY: Streptococcus mutans crude extract shows antifungal effects on clinical Candida strains susceptible and resistant to fluconazole in Galleria mellonella model.

Gene expression analysis of inflammation-related genes in macrophages treated with α-(1 → 3, 1 → 6)-D-glucan extracted from Streptococcus mutans.

Streptococcus mutans is a gram-positive bacterium that causes tooth decay. The exopolyssacharides, mostly glucans synthesized by the bacterium are responsible for establishing pathogenic bio-films associated with dental caries disease. The regulatory immune and inflammatory reactions implicated by the synthesized glucans are still not clearly understood. In this study, a water-soluble exopolyssacharide (WSP) was extracted from culture of Str. mutans. The structural properties of WSP, [α-(1 â†’ 3, 1 â†’ 6)-D-glucan] were confirmed using Fourier-transform infrared spectroscopy and 13C-nuclear magnetic resonance spectroscopy. Furthermore, the effects of WSP on the global gene expression of the macrophage-like RAW 264.7 cells were analyzed using mRNA-seq analysis. Using Gene Ontology analysis, we compiled a total of 24,421 genes that were upregulated or downregulated by more than 5.0-fold and 0.3-fold, respectively. Most of the transcripts were grouped under immune response and inflammation-related gene categories. Among the 802 immunity-related genes analyzed, chemokine ligand 7 (Ccl7), interleukin-1ß (IL-1ß), interleukin-1α (IL-1α) and interleukin-6 (IL-6) were upregulated after WSP exposure. In addition, among a total of 344 genes related to inflammation, Ccl7, IL-1α and IL-6 were upregulated. These results suggest that [α-(1 â†’ 3, 1 â†’ 6)-D-glucan] from Str. mutans produces activates macrophages and may contribute to the immune and inflammatory response to periodontal disease.

Structure Activity Relationship Study of the XIP Quorum Sensing Pheromone in Streptococcus mutans Reveal Inhibitors of the Competence Regulon.

The dental cariogenic pathogen Streptococcus mutans coordinates competence for genetic transformation via two peptide pheromones, competence stimulating peptide (CSP) and comX-inducing peptide (XIP). CSP is sensed by the comCDE system and induces competence indirectly, whereas XIP is sensed by the comRS system and induces competence directly. In chemically defined media (CDM), after uptake by oligopeptide permease, XIP interacts with the cytosolic receptor ComR to form the XIP::ComR complex that activates the expression of comX, an alternative sigma factor that initiates the transcription of late-competence genes. In this study, we set out to determine the molecular mechanism of XIP::ComR interaction. To this end, we performed systematic replacement of the amino acid residues in the XIP pheromone and assessed the ability of the mutated analogs to modulate the competence regulon in CDM. We were able to identify structural features that are important to ComR binding and activation. Our structure-activity relationship insights led us to construct multiple XIP-based inhibitors of the comRS pathway. Furthermore, when comCDE and comRS were both stimulated with CSP and XIP, respectively, a lead XIP-based inhibitor was able to maintain the inhibitory activity. Last, phenotypic assays were used to highlight the potential of XIP-based inhibitors to attenuate pathogenicity in S. mutans and to validate the specificity of these compounds to the comRS pathway within the competence regulon. The XIP-based inhibitors developed in this study can be used as lead scaffolds for the design and development of potential therapeutics against S. mutans infections.

Modulation of Antimicrobial Peptide Conformation and Aggregation by Terminal Lipidation and Surfactants.

The function and properties of peptide-based materials depend not only on the amino acid sequence but also on the molecular conformations. In this paper, we chose a series of peptides Gm(XXKK)nX-NH2 (m = 0, 3; n = 2, 3; X = I, L, and V) as the model molecules and studied the conformation regulation through N-terminus lipidation and their formulation with surfactants. The structural and morphological transition of peptide self-assemblies have also been investigated via transmission electron microscopy, atomic force microscopy, circular dichroism spectroscopy, and small-angle neutron scattering. With the terminal alkylation, the molecular conformation changed from random coil to ß-sheet or α-helix. The antimicrobial activities of alkylated peptide were different. C16-G3(IIKK)3I-NH2 showed antimicrobial activity against Streptococcus mutans, while C16-(IIKK)2I-NH2 and C16-G3(IIKK)2I-NH2 did not kill the bacteria. The surfactant sodium dodecyl sulfonate could rapidly induce the self-assemblies of alkylated peptides (C16-(IIKK)2I-NH2, C16-G3(IIKK)2I-NH2, C16-G3(VVKK)2V-NH2) from nanofibers to micelles, along with the conformation changing from ß-sheet to α-helix. The cationic surfactant hexadecyl trimethyl ammonium bromide made the lipopeptide nanofibers thinner, and nonionic surfactant polyoxyethylene (23) lauryl ether (C12EO23) induced the nanofibers much more intensively. Both the activity and the conformation of the α-helical peptide could be modulated by lipidation. Then, the self-assembled morphologies of alkylated peptides could also be further regulated with surfactants through hydrophobic, electrostatic, and hydrogen-bonding interactions. These results provided useful strategies to regulate the molecular conformations in peptide-based material functionalization.

Characterization of an intermolecular quaternary interaction between discrete segments of the Streptococcus mutans adhesin P1 by NMR spectroscopy.

Cell surface-localized P1 adhesin (aka Antigen I/II or PAc) of the cariogenic bacterium Streptococcus mutans mediates sucrose-independent adhesion to tooth surfaces. Previous studies showed that P1's C-terminal segment (C123, AgII) is also liberated as a separate polypeptide, contributes to cellular adhesion, interacts specifically with intact P1 on the cell surface, and forms amyloid fibrils. Identifying how C123 specifically interacts with P1 at the atomic level is essential for understanding related virulence properties of S. mutans. However, with sizes of ~ 51 and ~ 185 kDa, respectively, C123 and full-length P1 are too large to achieve high-resolution data for full structural analysis by NMR. Here, we report on biologically relevant interactions of the individual C3 domain with A3VP1, a polypeptide that represents the apical head of P1 as it is projected on the cell surface. Also evaluated are C3's interaction with C12 and the adhesion-inhibiting monoclonal antibody (MAb) 6-8C. NMR titration experiments with 15 N-enriched C3 demonstrate its specific binding to A3VP1. Based on resolved C3 assignments, two binding sites, proximal and distal, are identified. Complementary NMR titration of A3VP1 with a C3/C12 complex suggests that binding of A3VP1 occurs on the distal C3 binding site, while the proximal site is occupied by C12. The MAb 6-8C binding interface to C3 overlaps with that of A3VP1 at the distal site. Together, these results identify a specific C3-A3VP1 interaction that serves as a foundation for understanding the interaction of C123 with P1 on the bacterial surface and the related biological processes that stem from this interaction. DATABASE: BMRB submission code: 27935.

Synthetic Simplification of Carolacton Enables Chemical Genetic Studies in Streptococcus mutans.

Understanding the broader biological impact of carolacton, a macrolactone natural product, has been ongoing for the past decade. Multiple studies have shown connections to regulatory systems, acid tolerance mechanisms, biofilm formation, and recently folate dehydrogenase (FolD). Progress elucidating the cause of biofilm-specific activity in Streptococcus mutans has been limited due to low-throughput analyses of carolacton-treated cells. We disclose the discovery of a simplified carolacton-inspired analog that demonstrates inhibitory activity against S. mutans biofilm cells. This discovery permitted a proof of concept chemical genetic screen of S. mutans mutants identifying the carbon catabolite protein A signaling pathway as a putative target.

Structure-Activity Relationships of the Competence Stimulating Peptide in Streptococcus mutans Reveal Motifs Critical for Membrane Protease SepM Recognition and ComD Receptor Activation.

Streptococcus mutans ( S. mutans) is a Gram-positive human pathogen that is one of the major contributors to dental caries, a condition with an economic cost of over $100 billion per year in the United States. S. mutans secretes a 21-amino-acid peptide termed the competence stimulating peptide (21-CSP) to assess its population density in a process termed quorum sensing (QS) and to initiate a variety of phenotypes such as biofilm formation and bacteriocin production. 21-CSP is processed by a membrane bound protease SepM into active 18-CSP, which then binds to the ComD receptor. This study seeks to determine the molecular mechanism that ties 21-CSP:SepM recognition and 18-CSP:ComD receptor binding and to identify QS modulators with distinct activity profiles. To this end, we conducted systematic replacement of the amino acid residues in both 21-CSP and 18-CSP and assessed the ability of the mutated analogs to modulate QS. We identified residues that are important to SepM recognition and ComD receptor binding. Our results shed light on the S. mutans competence QS pathway at the molecular level. Moreover, our structural insights of the CSP signal can be used to design QS-based anti-infective therapeutics against S. mutans.

Modifying the Lantibiotic Mutacin 1140 for Increased Yield, Activity, and Stability.

Mutacin 1140 belongs to the epidermin family of type AI lantibiotics. This family has a broad spectrum of activity against Gram-positive bacteria. The binding of mutacin 1140 to lipid II leads to the inhibition of cell wall synthesis. Pharmacokinetic experiments with type AI lantibiotics are generally discouraging for clinical applications due to the short half-life of these compounds. The unprotected dehydrated and protease-susceptible residues outside the lanthionine rings may play a role in the short half-life in physiological settings. Previous mutagenesis work on mutacin 1140 has been limited to the lanthionine-forming residues, the C-terminally decarboxylated residue, and single amino acid substitutions at residues Phe1, Trp4, Dha5, and Arg13. To study the importance of the dehydrated (Dha5 and Dhb14) and protease-susceptible (Lys2 and Arg13) residues within mutacin 1140 for stability and bioactivity, each of these residues was evaluated for its impact on production and inhibitory activity. More than 15 analogs were purified, enabling direct comparison of the activities against a select panel of Gram-positive bacteria. The efficiency of the posttranslational modification (PTM) machinery of mutacin 1140 is highly restricted on its substrate. Analogs in the various intermediate stages of PTMs were observed as minor products following single point mutations at the 2nd, 5th, 13th, and 14th positions. The combination of alanine substitutions at the Dha5 and Dhb14 positions abolished mutacin 1140 production, while the production was restored by substitution of a Gly residue at one of these positions. Analogs with improved activity, productivity, and proteolytic stability were identified.IMPORTANCE Our findings show that the efficiency of mutacin 1140 PTMs is highly dependent on the core peptide sequence. Analogs in various intermediate stages of PTMs can be transported by the bacterium, which indicates that PTMs and transport are finely tuned for the native mutacin 1140 core peptide. Only certain combinations of amino acid substitutions at the Dha5 and Dhb14 dehydrated residue positions were tolerated. Observation of glutamylated core peptide analogs shows that dehydrations occur in a glutamate-dependent manner. Interestingly, mutations at positions outside rings A and B, the lipid II binding domain, would interfere with lipid II binding. Purified mutacin 1140 analogs have various activities and selectivities against different genera of bacteria, supporting the effort to generate analogs with higher specificity against pathogenic bacteria. The discovery of analogs with improved inhibitory activity against pathogenic bacteria, increased stability in the presence of protease, and higher product yields may promote the clinical development of this unique antimicrobial compound.

MapZ Forms a Stable Ring Structure That Acts As a Nanotrack for FtsZ Treadmilling in Streptococcus mutans.

Bacterial binary cell division requires accurate placement of division machinery. FtsZ, a vital component of the division machinery, can assemble into filaments and self-organize into a ring structure (Z ring) at the appropriate site for cell division. MapZ, a recently identified FtsZ regulator in Streptococcaceae, has been found to localize at the midcell where it helps to properly position the FtsZ ring. However, its mechanism is still unclear. Here, by using total internal reflection fluorescence microscopy, super-resolution imaging, and single molecule tracking, we investigated the mechanism by which MapZ controls the position of the FtsZ ring. Our results show that FtsZ exhibits a dynamic treadmilling motion in S. mutans. Importantly, depletion of MapZ leads to the unconstrained movement of treadmilling FtsZ filaments and a shorter lifetime of the constricting FtsZ ring, which is frequently misplaced. Furthermore, by revealing that MapZ forms an immobile ring-like nanostructure at the division site, our study suggests that MapZ forms a stable ring that acts as a nanotrack to guide and restrict treadmilling FtsZ filaments in S. mutans.

Specific chemiluminescent protocol for dual-site recognition of Streptococcus mutans utilizing strong affinity between teicoplanin and Gram-positive bacteria.

A novel dual-site recognition protocol was developed for chemiluminescent (CL) detection of Streptococcus mutans (S. mutans) based on a designed antibiotic-affinity strategy. Teicoplanin, a broad-spectrum antibiotic against Gram-positive bacteria, was adopted to functionalize magnetic particles and recognize S. mutans utilizing the strong affinity between this agent and D-Alanyl-D-Alanine peptide moieties in the bacterial cell wall. To achieve ideal specificity for S. mutans detection, rat immunoglobulin G2a (rat IgG2a) tagged with horseradish peroxidase (HRP) was used as the second recognition agent and signal tracer since Fab region of rat IgG2a could bind with streptococcal protein G highly expressed in the cell wall of S. mutans. Thus HRP-tagged sandwich complex of teicoplanin/S. mutans/rat IgG2a was formed on the magnetic particles, followed by a CL quantification of S. mutans based on a HRP-catalyzed luminol-H2O2-p-iodophenol CL reaction. This dual-site recognition protocol showed a linear range of 1.0 × 102-1.0 × 106 CFU mL-1 and a detection limit of 33 CFU mL-1 for S. mutans detection. The whole detection process could be completed within 70min. The recovery tests for food, environmental, pharmaceutical and biological samples showed acceptable recovery values between 83.0% and 110.0%, demonstrating its application potential for detection of bacteria in various sample matrixes.

Investigating the candidacy of the serotype specific rhamnan polysaccharide based glycoconjugates to prevent disease caused by the dental pathogen Streptococcus mutans.

Dental caries remains a major health issue and the Gram-positive bacterium Streptococcus mutans is considered as the major pathogen causing caries. More recently, S. mutans has been recognised as a cause of endocarditis, ulcerative colitis and fatty acid liver disease along with the likelihood of increased cerebral hemorrhage following a stroke if S. mutans is present systemically. We initiated this study to examine the vaccine candidacy of the serotype specific polysaccharides elaborated by S. mutans. We have confirmed the carbohydrate structures for the serotype specific rhamnan containing polysaccharides from serotypes c, f and k. We have prepared glycoconjugate vaccines using the rhamnan containing polymers from serotypes f and k and immunised mice and rabbits. We consistently obtained a robust immune response to the glycoconjugates with cross-reactivity consistent with the structural similarities of the polymers from the different serotypes. We developed an opsonophagocytic assay which illustrated the ability of the post-immune sera to facilitate opsonophagocytic killing of the homologous and heterologous serotypes at titers consistent with the structural homologies. We conclude that glycoconjugates of the rhamnan polymers of S. mutans are a potential vaccine candidate to target dental caries and other sequelae following the escape of S. mutans from the oral cavity.

One-step FPLC-size-exclusion chromatography procedure for purification of rDMBT1 6 kb with increased biological activity.

Deleted in Malignant Brain Tumor 1 (DMBT1, alias SAG or gp340) is a pattern recognition receptor involved in immune defense, cell polarization, differentiation and regeneration. To investigate the role of the protein in physiological and pathological processes, the protein has often been isolated from saliva or produced in vitro and purified by a multistep affinity purification procedure using bacteria, followed by FPLC. Here, we compared a simple, one-step FPLC-SEC protocol for purification of recombinant DMBT1 6 kb, with that of the standard bacteria affinity purification-based protocol. Our data suggest that our FPLC-SEC protocol yields DMBT1 in a more native conformation.

LT adjuvant modulates epitope specificity and improves the efficacy of murine antibodies elicited by sublingual vaccination with the N-terminal domain of Streptococcus mutans P1.

In this study, we evaluated the immunogenicity, protective efficacy and peptide-based immune signatures of antibodies raised in mice after sublingual immunization with a recombinant form of the P1 (aka AgI/II, PAc) adhesin (P139-512) of Streptococcus mutans, a major etiological agent of dental caries. Sublingual administration of P139-512 in combination with the mucosal adjuvant LTK4R (a derivative of heat-labile LT toxin) induced strong and long-lasting systemic and mucosal immune responses. Incorporation of the adjuvant resulted in an enhancement of the anti-adhesive and anti-colonization activity against S. mutans as evaluated both under in vitro and in vivo conditions. Incorporation of the adjuvant to the vaccine formulation also changed the epitope specificity of the induced antibodies as determined by immunological signatures of sera collected from vaccinated mice. Use of a peptide microarray library led to the identification of peptide targets recognized by antibodies in serum samples with enhanced anti-adhesive effects. Altogether, the results presented herein showed that the sublingual administration of a P1-based subunit vaccine represents a promising approach for the prevention of dental caries caused by S. mutans. In addition, the present study disclosed the role of adjuvants on the epitope specificity and functionality of antibodies raised by subunit vaccines.

Conformational dynamics of the essential sensor histidine kinase WalK.

Two-component systems (TCSs) are key elements in bacterial signal transduction in response to environmental stresses. TCSs generally consist of sensor histidine kinases (SKs) and their cognate response regulators (RRs). Many SKs exhibit autokinase, phosphoryltransferase and phosphatase activities, which regulate RR activity through a phosphorylation and dephosphorylation cycle. However, how SKs perform different enzymatic activities is poorly understood. Here, several crystal structures of the minimal catalytic region of WalK, an essential SK from Lactobacillus plantarum that shares 60% sequence identity with its homologue VicK from Streptococcus mutans, are presented. WalK adopts an asymmetrical closed structure in the presence of ATP or ADP, in which one of the CA domains is positioned close to the DHp domain, thus leading both the ß- and γ-phosphates of ATP/ADP to form hydrogen bonds to the ℇ- but not the δ-nitrogen of the phosphorylatable histidine in the DHp domain. In addition, the DHp domain in the ATP/ADP-bound state has a 25.7° asymmetrical helical bending coordinated with the repositioning of the CA domain; these processes are mutually exclusive and alternate in response to helicity changes that are possibly regulated by upstream signals. In the absence of ATP or ADP, however, WalK adopts a completely symmetric open structure with its DHp domain centred between two outward-reaching CA domains. In summary, these structures of WalK reveal the intrinsic dynamic properties of an SK structure as a molecular basis for multifunctionality.

Self-assembling anticaries mucosal vaccine containing ferritin cage nanostructure and glucan-binding region of S. mutans glucosyltransferase effectively prevents caries formation in rodents.

Anticaries protein vaccines that induce a mucosal immune response are not effective. Therefore, development of effective and convenient anticaries vaccines is a priority of dental research. Here we generated self-assembling nanoparticles by linking the glucan-binding region of Streptococcus mutans glucosyltransferase (GLU) to the N-terminal domain of ferritin to determine whether these novel nanoparticles enhanced the immunogenicity of an anticaries protein vaccine against GLU in rodents. We constructed the expression plasmid pET28a-GLU-FTH and purified the proteins from bacteria using size-exclusion chromatography. BALB/c mice were used to evaluate the ability of GLU-ferritin (GLU-FTH) nanoparticles to induce GLU-specific mucosal and systemic responses. The protective efficiency of GLU-FTH nanoparticles was compared with that of GLU alone or a mixture of GLU and poly(I:C) after administering an intranasal infusion to Wistar rats. The phagocytosis and maturation of dendritic cells (DCs) exposed in vitro to the nanoparticles were assessed using flow cytometry. The GLU-FTH nanoparticle vaccine elicited significantly higher levels of GLU-specific antibodies compared with GLU or a mixture of GLU and poly(I:C). Immunization with GLU-FTH achieved lower caries scores compared with those of the other vaccines. Administration of GLU-FTH nanoparticles enhanced phagocytosis by DCs and their maturation. Thus, self-assembling GLU-FTH is a highly effective anticaries mucosal vaccine that enhanced antibody production and inhibited S. mutans infection in rodents.

Sensing of Streptococcus mutans by microscopic imaging ellipsometry.

Microscopic imaging ellipsometry is an optical technique that uses an objective and sensing procedure to measure the ellipsometric parameters ? and ? in the form of microscopic maps. This technique is well known for being noninvasive and label-free. Therefore, it can be used to detect and characterize biological species without any impact. Microscopic imaging ellipsometry was used to measure the optical response of dried Streptococcus mutans cells on a glass substrate. The ellipsometric ? and ? maps were obtained with the Optrel Multiskop system for specular reflection in the visible range ( ? = 450 to 750 nm). The ? and ? images at 500, 600, and 700 nm were analyzed using three different theoretical models with single-bounce, two-bounce, and multibounce light paths to obtain the optical constants and height distribution. The obtained images of the optical constants show different aspects when comparing the single-bounce analysis with the two-bounce or multibounce analysis in detecting S. mutans samples. Furthermore, the height distributions estimated by two-bounce and multibounce analyses of S. mutans samples were in agreement with the thickness values measured by AFM, which implies that the two-bounce and multibounce analyses can provide information complementary to that obtained by a single-bounce light path.