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Streptococcus mutans is an etiologic agent of human dental caries that forms dental plaque biofilms containing functional amyloids. Three amyloidogenic proteins, P1, WapA, and Smu_63c were previously identified. C123 and AgA are naturally occurring amyloid-forming fragments of P1 and WapA, respectively. We determined that four amyloidophilic dyes, ThT, CDy11, BD-oligo, and MK-H4, differentiate C123, AgA, and Smu_63c amyloid from monomers, but non-specific binding to bacterial cells in the absence of amyloid precludes their utility for identifying amyloid in biofilms. Congo red-induced birefringence is a more specific indicator of amyloid formation and differentiates biofilms formed by wild-type S. mutans from a triple ΔP1/WapA/Smu_63c mutant with reduced biofilm forming capabilities. Amyloid accumulation is a late event, appearing in older S. mutans biofilms after 60 hours of growth. Amyloid derived from pure preparations of all three proteins is visualized by electron microscopy as mat-like structures. Typical amyloid fibers become evident following protease digestion to eliminate non-specific aggregates and monomers. Amyloid mats, similar in appearance to those reported in S. mutans biofilm extracellular matrices, are reconstituted by co-incubation of monomers and amyloid fibers. X-ray fiber diffraction of amyloid mats and fibers from all three proteins demonstrate patterns reflective of a cross-β amyloid structure.

Potassium (K + ) is the most abundant cation in the fluids of dental biofilm. The biochemical and biophysical functions of K + and a variety of K + transport systems have been studied for most pathogenic bacteria but not for oral pathogens. In this study, we establish the modes of K + acquisition in Streptococcus mutans and the importance of K + homeostasis for its virulence attributes. The S. mutans genome harbors four putative K + transport systems that included two Trk-like transporters (designated Trk1 and Trk2), one glutamate/K + cotransporter (GlnQHMP), and a channel-like K + transport system (Kch). Mutants lacking Trk2 had significantly impaired growth, acidogenicity, aciduricity, and biofilm formation. [K + ] less than 5 mM eliminated biofilm formation in S. mutans . The functionality of the Trk2 system was confirmed by complementing an Escherichia coli TK2420 mutant strain, which resulted in significant K + accumulation, improved growth, and survival under stress. Taken together, these results suggest that Trk2 is the main facet of the K + -dependent cellular response of S. mutans to environment stresses. IMPORTANCE Biofilm formation and stress tolerance are important virulence properties of caries-causing Streptococcus mutans . To limit these properties of this bacterium, it is imperative to understand its survival mechanisms. Potassium is the most abundant cation in dental plaque, the natural environment of S. mutans . K + is known to function in stress tolerance, and bacteria have specialized mechanisms for its uptake. However, there are no reports to identify or characterize specific K + transporters in S. mutans . We identified the most important system for K + homeostasis and its role in the biofilm formation, stress tolerance, and growth. We also show the requirement of environmental K + for the activity of biofilm-forming enzymes, which explains why such high levels of K + would favor biofilm formation.

The P1 adhesin (aka Antigen I/II or PAc) of the cariogenic bacterium Streptococcus mutans is a cell surface-localized protein involved in sucrose-independent adhesion and colonization of the tooth surface. The immunoreactive and adhesive properties of S. mutans suggest an unusual functional quaternary ultrastructure comprised of intact P1 covalently attached to the cell wall and interacting with non-covalently associated proteolytic fragments thereof, particularly the ~57-kDa C-terminal fragment C123 previously identified as Antigen II. S. mutans is capable of amyloid formation when grown in a biofilm and P1 is among its amyloidogenic proteins. The C123 fragment of P1 readily forms amyloid fibers in vitro suggesting it may play a role in the formation of functional amyloid during biofilm development. Using wild-type and P1-deficient strains of S. mutans, we demonstrate that solid state NMR (ssNMR) spectroscopy can be used to (1) globally characterize cell walls isolated from a Gram-positive bacterium and (2) characterize the specific binding of heterologously expressed, isotopically-enriched C123 to cell wall-anchored P1. Our results lay the groundwork for future high-resolution characterization of the C123/P1 ultrastructure and subsequent steps in biofilm formation via ssNMR spectroscopy, and they support an emerging model of S. mutans colonization whereby quaternary P1-C123 interactions confer adhesive properties important to binding to immobilized human salivary agglutinin.

The cariogenic bacterium Streptococcus mutans uses adhesin P1 to adhere to tooth surfaces, extracellular matrix components, and other bacteria. A composite model of P1 based on partial crystal structures revealed an unusual complex architecture in which the protein forms an elongated hybrid alpha/polyproline type II helical stalk by folding back on itself to display a globular head at the apex and a globular C-terminal region at the base. The structure of P1's N terminus and the nature of its critical interaction with the C-terminal region remained unknown, however. We have cocrystallized a stable complex of recombinant N- and C-terminal fragments and here describe a previously unidentified topological fold in which these widely discontinuous domains are intimately associated. The structure reveals that the N terminus forms a stabilizing scaffold by wrapping behind the base of P1's elongated stalk and physically "locking" it into place. The structure is stabilized through a highly favorable ΔG(solvation) on complex formation, along with extensive hydrogen bonding. We confirm the functional relevance of this intramolecular interaction using differential scanning calorimetry and circular dichroism to show that disruption of the proper spacing of residues 989-1001 impedes folding and diminishes stability of the full-length molecule, including the stalk. Our findings clarify previously unexplained functional and antigenic properties of P1.

Made available in DSpace on 2018-12-11T16:56:07Z (GMT). No. of bitstreams: 0 Previous issue date: 2014-01-01 National Institutes of Health Streptococcus mutans, a major etiological agent of human dental caries, lives primarily on the tooth surface in biofilms. Limited information is available concerning the extracellular DNA (eDNA) as a scaffolding matrix in S. mutans biofilms. This study demonstrates that S. mutans produces eDNA by multiple avenues, including lysis-independent membrane vesicles. Unlike eDNAs from cell lysis that were abundant and mainly concentrated around broken cells or cell debris with floating open ends, eDNAs produced via the lysis-independent pathway appeared scattered but in a structured network under scanning electron microscopy. Compared to eDNA production of planktonic cultures, eDNA production in 5- and 24-h biofilms was increased by >3- and >1.6-fold, respectively. The addition of DNase I to growth medium significantly reduced biofilm formation. In an in vitro adherence assay, added chromosomal DNA alone had a limited effect on S. mutans adherence to saliva-coated hydroxylapatite beads, but in conjunction with glucans synthesized using purified glucosyltransferase B, the adherence was significantly enhanced. Deletion of sortase A, the transpeptidase that covalently couples multiple surface-associated proteins to the cell wall peptidoglycan, significantly reduced eDNA in both planktonic and biofilm cultures. Sortase A deficiency did not have a significant effect on membrane vesicle production; however, the protein profile of the mutant membrane vesicles was significantly altered, including reduction of adhesin P1 and glucan-binding proteins B and C. Relative to the wild type, deficiency of protein secretion and membrane protein insertion machinery components, including Ffh, YidC1, and YidC2, also caused significant reductions in eDNA. © 2014, American Society for Microbiology. Center of Excellence in Oral and Craniofacial Biology, School of Dentistry, Louisiana State University Health Sciences Center, New Orleans, LA Center for Oral Biology, University of Rochester School of Medicine and Dentistry, Rochester, NY Department of Oral Biology, University of Florida School of Dentistry, Gainesville, FL Department of Comprehensive Dentistry and Biomaterials, School of Dentistry, Louisiana State University Health Sciences Center, New Orleans, LA Department of Orthodontics, University of Pennsylvania School of Dental Medicine, Philadelphia, PA Department of Microbiology, Immunology and Parasitology, School of Medicine, Louisiana State University Health Sciences Center, New Orleans, LA Department of Dental Materials and Prosthodontics, Araraquara Dental School, Universidade Estadual Paulista, UNESP, Sao Paulo Department of Microbiology and Immunology, Tulane University School of Medicine, New Orleans, LA Department of Dental Materials and Prosthodontics, Araraquara Dental School, Universidade Estadual Paulista, UNESP, Sao Paulo National Institutes of Health: DE08007 National Institutes of Health: DE19452 National Institutes of Health: DE21789 National Institutes of Health: DE22529 National Institutes of Health: EFRI-1137186

P1 is an adhesin on the surface of Streptococcus mutans.Destroying the high affinity interaction between the N and C termini of S. mutans P1 creates a non-adherent phenotype.The N terminus facilitates proper folding, function, and stability within recombinant P1.The relationship between folding, maturation, and cell surface assembly is critical to understanding the P1 mechanism of action. The adhesin P1 is localized on the surface of the oral pathogen Streptococcus mutans and facilitates an interaction with the glycoprotein complex salivary agglutinin that is comprised primarily of the scavenger receptor gp340. Recent crystal structures of P1 display an unusual structure in which the protein folds back upon itself to form an elongated hybrid helical stalk with a globular head at the apex and a globular C-terminal region at the base. The N terminus of P1 has not yet been characterized. In this report we describe the contribution of an interaction between the N-terminal and C-terminal portions of the protein that is required for proper function of P1 on the surface of S. mutans. Utilizing recombinant N-terminal and C-terminal fragments, we employed isothermal titration calorimetry and native gel electrophoresis to demonstrate that these fragments form a high affinity and stable complex in solution. Furthermore, circular dichroism and surface plasmon resonance measurements indicated that the N-terminal fragment contributes to the folding and increases the functionality of the C-terminal fragment in trans. Finally, we utilized circular dichroism, surface plasmon resonance, and differential scanning calorimetry to show that an N-terminal 106-amino acid segment within P1 contributes to the proper folding and function of the full-length recombinant molecule and increases the stability of its elongated hybrid helical stalk.

The adhesin known as Antigen I/II, P1 or PAc of the cariogenic dental pathogen Streptococcus mutans is a target of protective immunity and candidate vaccine antigen. Previously we demonstrated that immunization of mice with S. mutans complexed with anti-AgI/II monoclonal antibodies (MAbs) resulted in changes in the specificity, isotype and functionality of elicited anti-AgI/II antibodies in the serum of immunized mice compared to administration of bacteria alone. In the current study, an anti-AgI/II MAb reported in the literature to confer unexplained long term protection against S. mutans re-colonization following passive immunization in human clinical trials (MAb Guy’s 13), and expressed in tobacco plants (MAb Guy’s 13 plantibody), was evaluated for its potential immunomodulatory properties. Immunization of BALB/c mice with immune complexes of Guy’s 13 plantibody bound to S. mutans whole cells resulted in a similar change in specificity, isotype, and functionality of elicited anti-AgI/II antibodies as had been observed for other immunomodulatory MAbs. This new information, coupled with the recently solved crystal structure of the adhesin, now provides a rational explanation and plausible mechanism of action of passively administered Guy’s 13/Guy’s 13 plantibody in human clinical trials, and how long-term prevention of S. mutans carriage well past the application period of the therapeutic antibody could have been achieved.

The Streptococcus mutans antigen I/II (AgI/II) is a cell surface-localized protein that adheres to salivary components and extracellular matrix molecules. Here we report the 2.5 Å resolution crystal structure of the complete C-terminal region of AgI/II. The C-terminal region is comprised of three major domains: C(1), C(2), and C(3). Each domain adopts a DE-variant IgG fold, with two β-sheets whose A and F strands are linked through an intramolecular isopeptide bond. The adherence of the C-terminal AgI/II fragments to the putative tooth surface receptor salivary agglutinin (SAG), as monitored by surface plasmon resonance, indicated that the minimal region of binding was contained within the first and second DE-variant-IgG domains (C(1) and C(2)) of the C terminus. The minimal C-terminal region that could inhibit S. mutans adherence to SAG was also confirmed to be within the C(1) and C(2) domains. Competition experiments demonstrated that the C- and N-terminal regions of AgI/II adhere to distinct sites on SAG. A cleft formed at the intersection between these C(1) and C(2) domains bound glucose molecules from the cryo-protectant solution, revealing a putative binding site for its highly glycosylated receptor SAG. Finally, electron microscopy images confirmed the elongated structure of AgI/II and enabled building a composite tertiary model that encompasses its two distinct binding regions.

Streptococcus mutans antigen I/II (AgI/II) protein was one of the first cell wall-anchored adhesins identified in Gram-positive bacteria. It mediates attachment of S. mutans to tooth surfaces and has been a focus for immunization studies against dental caries. The AgI/II family polypeptides recognize salivary glycoproteins, and are also involved in biofilm formation, platelet aggregation, tissue invasion and immune modulation. The genes encoding AgI/II family polypeptides are found among Streptococcus species indigenous to the human mouth, as well as in Streptococcus pyogenes, S. agalactiae and S. suis. Evidence of functionalities for different regions of the AgI/II proteins has emerged. A sequence motif within the C-terminal portion of Streptococcus gordonii SspB (AgI/II) is bound by Porphyromonas gingivalis, thus promoting oral colonization by this anaerobic pathogen. The significance of other epitopes is now clearer following resolution of regional crystal structures. A new picture emerges of the central V (variable) region, predicted to contain a carbohydrate-binding trench, being projected from the cell surface by a stalk formed by an unusual association between an N-terminal alpha-helix and a C-terminal polyproline helix. This presentation mode might be important in determining functional conformations of other Gram-positive surface proteins that have adhesin domains flanked by alpha-helical and proline-rich regions.

Researchers now recognize the utility of surface plasmon resonance technology to evaluate interactions of microbial pathogens with host components. The surface adhesin and candidate vaccine antigen P1 of Streptococcus mutans, the main causative agent of dental caries, interacts with a high molecular weight glycoprotein called salivary agglutinin, or gp340, in the salivary pellicle. We optimized a BIAcore assay to measure P1-mediated Ca(2+) dependent binding of S. mutans whole cells to this physiological ligand immobilized on a Pioneer F1 sensor chip. Regeneration conditions allowed cells to be eluted from the sensor chip permitting multiple reuse of the agglutinin-coated surface. An isogenic P1-deficient S. mutans mutant did not bind to immobilized agglutinin demonstrating specificity of the detected interaction. Glutaraldehyde-fixation of bacterial cells showed the assay measured a whole cell-ligand interaction and was not an artifact of solubilized or leached proteins. Adherence inhibition assays demonstrated varying degrees of disruption of the S. mutans-agglutinin interaction by anti-P1 monoclonal antibodies recognizing different epitopes, whereas a polyclonal reagent demonstrated more complete inhibition. This report describes an improved method to assess salivary agglutinin-mediated adherence of S. mutans in vitro under physiological-like conditions and to evaluate the effectiveness of antibodies of differing specificities to inhibit binding.

Membrane proteins are rarely identified in two-dimensional electrophoretic (2-DE) proteomics maps. This is due to low abundancy, poor solubility, and inherent hydrophobicity leading to self-aggregation during the first dimension. In this study, membrane proteins from the Gram-positive bacterium Streptococcus mutans were solubilized using three different methods and evaluated by 2-DE. In the first method, the extraction was performed using sodium dodecyl sulfate (SDS) followed by solubilization with a chaotropic buffer and precipitation with methanol/chloroform. The second method was based on temperature-dependent phase partitioning using Triton X-114 followed by purification using the ReadyPrep 2-D clean-up kit from Bio-Rad. The third method involved extraction using the organic solvents trifluoroethanol (TFE) and chloroform, which produced three separate phases. The upper aqueous phase, enriched with TFE, gave the highest overall protein yield and best 2-DE resolution. Protein spot identification by nanoelectrospray quadrupole time of flight (QTOF)-tandem mass spectrometry (MS/MS) revealed known membrane and surface-associated proteins. This is the first report describing the successful solubilization and 2-D electrophoresis of membrane proteins from a Gram-positive bacterium.

The c protein (Ibc) of group B streptococci (GBS) is associated with at least four antigens (alpha, beta, gamma, delta). To assess the virulence potential of these antigens, 255 GBS isolates recovered from septic neonates, healthy neonates, and pregnant women were serotyped and surveyed for reactivity with sera to c protein and the four associated antigens. A radioimmunoassay using intact bacteria was used to detect the GBS antigens. In contrast to earlier reports, most (66%) of the type III strains expressed the c protein. Except for the gamma antigen, none of the other c protein-associated antigens showed an increased association with pathogenic strains independent of the polysaccharide antigens. The gamma antigen was expressed by 15 of 41 c protein-positive early-onset strains and by 4 of 38 c protein-positive late-onset strains (P = .007). This association was independent of the type-specific antigen, suggesting a potential role for the gamma antigen as a virulence factor in GBS strains causing early-onset sepsis.

During characterization of the surface antigens of serotype III group B streptococci (GBS), a protein with an apparent Mr~ 173 500 migrating on a SDS – polyacrylamide gel was found to have an N-terminal amino acid sequence identical to that of the plasmin receptor (Plr) of group A streptococci, a surface-localized glyceraldehyde-3-phosphate dehydrogenase (GAPDH). This work begins to characterize GBS GAPDH and to assess its functional activity on the cell surface. The 1.0-kb gapC gene of GBS was amplified by PCR. plr and gapC demonstrated 87% homology. An anti-Plr monoclonal antibody reacted with GBS whole cells, suggesting GBS GAPDH is surface localized. Multiple serotypes of GBS demonstrated functional GAPDH on their surfaces. The anti-Plr monoclonal antibody recognized GBS protein bands of approximately 41 and 173.5 kDa, by Western blot. Presumably, these represent monomeric and tetrameric forms of the GAPDH molecule. GBS GAPDH was demonstrated by Western blot analysis to interact with lys- and glu-plasminogens. Fluid-phase GBS GAPDH interacted, by means of ELISA, with immobilized lys-plasminogen, glu-plasminogen, actin, and fibrinogen. Enzymatically active GAPDH, capable of binding cytoskeletal and extracellular matrix proteins, is expressed on the surface of GBS.Key words: group B streptococci, glyceraldehyde-3-phosphate dehydrogenase.

A two-stage radioimmunoassay (RIA) has been developed that identifies type-specific antigens on the surfaces of group B streptococci. In addition to detecting the type-specific carbohydrate antigens Ia, Ib, II, and III, the RIA identified four unique antigens reactive with the Centers for Disease Control's c-protein typing antiserum. Analysis of hot-acid extracts by immunoelectrophoresis confirmed that two of these reactivities corresponded to the reported alpha and beta antigens of the c-protein marker. In addition, the identification of two heretofore unidentified antigens, gamma and delta, by the two-stage RIA is detailed in this report. The assay uses intact bacteria and does not require hot-acid extraction, a procedure thus enabling detection of acid-labile antigens in their native unmodified form. The semiquantitative RIA requires less typing reagent than does precipitin testing and is more objective, reproducible, and rapid. The assay described here could be applied to the detection of any cell surface antigen for which a monospecific antiserum is available.