Cardiolipin occupancy profiles of YidC paralogs reveal the significance of respective TM2 helix residues in determining paralog-specific phenotypes.

YidC belongs to an evolutionarily conserved family of insertases, YidC/Oxa1/Alb3, in bacteria, mitochondria, and chloroplasts, respectively. Unlike Gram-negative bacteria, Gram-positives including Streptococcus mutans harbor two paralogs of YidC. The mechanism for paralog-specific phenotypes of bacterial YidC1 versus YidC2 has been partially attributed to the differences in their cytoplasmic domains. However, we previously identified a W138R gain-of-function mutation in the YidC1 transmembrane helix 2. YidC1W138R mostly phenocopied YidC2, yet the mechanism remained unknown. Primary sequence comparison of streptococcal YidCs led us to identify and mutate the YidC1W138 analog, YidC2S152 to W/A, which resulted in a loss of YidC2- and acquisition of YidC1-like phenotype. The predicted lipid-facing side chains of YidC1W138/YidC2S152 led us to propose a role for membrane phospholipids in specific-residue dependent phenotypes of S. mutans YidC paralogs. Cardiolipin (CL), a prevalent phospholipid in the S. mutans cytoplasmic membrane during acid stress, is encoded by a single gene, cls. We show a concerted mechanism for cardiolipin and YidC2 under acid stress based on similarly increased promoter activities and similar elimination phenotypes. Using coarse grain molecular dynamics simulations with the Martini2.2 Forcefield, YidC1 and YidC2 wild-type and mutant interactions with CL were assessed in silico. We observed substantially increased CL interaction in dimeric versus monomeric proteins, and variable CL occupancy in YidC1 and YidC2 mutant constructs that mimicked characteristics of the other wild-type paralog. Hence, paralog-specific amino acid- CL interactions contribute to YidC1 and YidC2-associated phenotypes that can be exchanged by point mutation at positions 138 or 152, respectively.

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.

Amyloid Aggregates Are Localized to the Nonadherent Detached Fraction of Aging Streptococcus mutans Biofilms.

The number of bacterial species recognized to utilize purposeful amyloid aggregation within biofilms continues to grow. The oral pathogen Streptococcus mutans produces several amyloidogenic proteins, including adhesins P1 (also known as AgI/II, PAc) and WapA, whose truncation products, namely, AgII and AgA, respectively, represent the amyloidogenic moieties. Amyloids demonstrate common biophysical properties, including recognition by Thioflavin T (ThT) and Congo red (CR) dyes that bind to the cross ß-sheet quaternary structure of amyloid aggregates. Previously, we observed amyloid formation to occur only after 60 h or more of S. mutans biofilm growth. Here, we extend those findings to investigate where amyloid is detected within 1- and 5-day-old biofilms, including within tightly adherent compared with those in nonadherent fractions. CR birefringence and ThT uptake demonstrated amyloid within nonadherent material removed from 5-day-old cultures but not within 1-day-old or adherent samples. These experiments were done in conjunction with confocal microscopy and immunofluorescence staining with AgII- and AgA-reactive antibodies, including monoclonal reagents shown to discriminate between monomeric protein and amyloid aggregates. These results also localized amyloid primarily to the nonadherent fraction of biofilms. Lastly, we show that the C-terminal region of P1 loses adhesive function following amyloidogenesis and is no longer able to competitively inhibit binding of S. mutans to its physiologic substrate, salivary agglutinin. Taken together, our results provide new evidence that amyloid aggregation negatively impacts the functional activity of a widely studied S. mutans adhesin and are consistent with a model in which amyloidogenesis of adhesive proteins facilitates the detachment of aging biofilms. IMPORTANCE Streptococcus mutans is a keystone pathogen and causative agent of human dental caries, commonly known as tooth decay, the most prevalent infectious disease in the world. Like many pathogens, S. mutans causes disease in biofilms, which for dental decay begins with bacterial attachment to the salivary pellicle coating the tooth surface. Some strains of S. mutans are also associated with bacterial endocarditis. Amyloid aggregation was initially thought to represent only a consequence of protein mal-folding, but now, many microorganisms are known to produce functional amyloids with biofilm environments. In this study, we learned that amyloid formation diminishes the activity of a known S. mutans adhesin and that amyloid is found within the nonadherent fraction of older biofilms. This finding suggests that the transition from adhesin monomer to amyloid facilitates biofilm detachment. Knowing where and when S. mutans produces amyloid will help in developing therapeutic strategies to control tooth decay and other biofilm-related diseases.

The Cytoplasmic Domains of Streptococcus mutans Membrane Protein Insertases YidC1 and YidC2 Confer Unique Structural and Functional Attributes to Each Paralog.

Integral and membrane-anchored proteins are pivotal to survival and virulence of the dental pathogen, Streptococcus mutans. The bacterial chaperone/insertase, YidC, contributes to membrane protein translocation. Unlike Escherichia coli, most Gram-positive bacteria contain two YidC paralogs. Herein, we evaluated structural features that functionally delineate S. mutans YidC1 and YidC2. Bacterial YidCs contain five transmembrane domains (TMD), two cytoplasmic loops, and a cytoplasmic tail. Because S. mutans YidC1 (SmYidC1) and YidC2 (SmYidC2) cytoplasmic domains (CD) are less well conserved than are TMD, we engineered ectopic expression of the 14 possible YidC1-YidC2 CD domain swap combinations. Growth and stress tolerance of each was compared to control strains ectopically expressing unmodified yidC1 or yidC2. Acid and osmotic stress sensitivity are associated with yidC2 deletion. Sensitivity to excess zinc was further identified as a ΔyidC1 phenotype. Overall, YidC1 tolerated CD substitutions better than YidC2. Preferences toward particular CD combinations suggested potential intramolecular interactions. In silico analysis predicted salt-bridges between C1 and C2 loops of YidC1, and C1 loop and C-terminal tail of YidC2, respectively. Mutation of contributing residues recapitulated ΔyidC1- and ΔyidC2-associated phenotypes. Taken together, this work revealed the importance of cytoplasmic domains in distinct functional attributes of YidC1 and YidC2, and identified key residues involved in interdomain interactions.

Amyloid Aggregation of Streptococcus mutans Cnm Influences Its Collagen-Binding Activity.

The cnm gene, coding for the glycosylated collagen- and laminin-binding surface adhesin Cnm, is found in the genomes of approximately 20% of Streptococcus mutans clinical isolates and is associated with systemic infections and increased caries risk. Other surface-associated collagen-binding proteins of S. mutans, such as P1 and WapA, have been demonstrated to form an amyloid quaternary structure with functional implications within biofilms. In silico analysis predicted that the ß-sheet-rich N-terminal collagen-binding domain (CBD) of Cnm has a propensity for amyloid aggregation, whereas the threonine-rich C-terminal domain was predicted to be disorganized. In this study, thioflavin-T fluorescence and electron microscopy were used to show that Cnm forms amyloids in either its native glycosylated or recombinant nonglycosylated form and that the CBD of Cnm is the main amyloidogenic unit of Cnm. We then performed a series of in vitro, ex vivo, and in vivo assays to characterize the amylogenic properties of Cnm. In addition, Congo red birefringence indicated that Cnm is a major amyloidogenic protein of S. mutans biofilms. Competitive binding assays using collagen-coated microtiter plates and dental roots, a substrate rich in collagen, revealed that Cnm monomers inhibit S. mutans binding to collagenous substrates, whereas Cnm amyloid aggregates lose this property. Thus, while Cnm contributes to recognition and initial binding of S. mutans to collagen-rich surfaces, amyloid formation by Cnm might act as a negative regulatory mechanism to modulate collagen-binding activity within S. mutans biofilms and warrants further investigation. IMPORTANCE Streptococcus mutans is a keystone pathogen that promotes caries by acidifying the dental biofilm milieu. The collagen- and laminin-binding glycoprotein Cnm is a virulence factor of S. mutans. Expression of Cnm by S. mutans is hypothesized to contribute to niche expansion, allowing colonization of multiple sites in the body, including collagen-rich surfaces such as dentin and heart valves. Here, we suggest that Cnm function might be modulated by its aggregation status. As a monomer, its primary function is to promote attachment to collagenous substrates via its collagen-binding domain (CBD). However, in later stages of biofilm maturation, the same CBD of Cnm could self-assemble into amyloid fibrils, losing the ability to bind to collagen and likely becoming a component of the biofilm matrix. Our findings shed light on the role of functional amyloids in S. mutans pathobiology and ecology.

Protein Interactomes of Streptococcus mutans YidC1 and YidC2 Membrane Protein Insertases Suggest SRP Pathway-Independent- and -Dependent Functions, Respectively.

Virulence properties of cariogenic Streptococcus mutans depend on integral membrane proteins. Bacterial cotranslational protein trafficking involves the signal recognition particle (SRP) pathway components Ffh and FtsY, the SecYEG translocon, and YidC chaperone/insertases. Unlike Escherichia coli, S. mutans survives loss of the SRP pathway and has two yidC paralogs. This study characterized YidC1 and YidC2 interactomes to clarify respective functions alone and in concert with the SRP and/or Sec translocon. Western blots of formaldehyde cross-linked or untreated S. mutans lysates were reacted with anti-Ffh, anti-FtsY, anti-YidC1, or anti-YidC2 antibodies followed by mass spectrometry (MS) analysis of gel-shifted bands. Cross-linked lysates of wild-type and ΔyidC2 strains were reacted with anti-YidC2-coupled Dynabeads, and cocaptured proteins were identified by MS. Last, YidC1 and YidC2 C-terminal tail-captured proteins were subjected to two-dimensional (2D) difference gel electrophoresis and MS analysis. Direct interactions of putative YidC1 and YidC2 binding partners were confirmed by bacterial two-hybrid assay. Our results suggest YidC2 works preferentially with the SRP pathway, while YidC1 is preferred for SRP-independent Sec translocon-mediated translocation. YidC1 and YidC2 autonomous pathways were also apparent. Two-hybrid assay identified interactions between holotranslocon components SecYEG/YajC and YidC1. Both YidC1 and YidC2 interacted with Ffh, FtsY, and chaperones DnaK and RopA. Putative membrane-localized substrates HlyX, LemA, and SMU_591c interacted with both YidC1 and YidC2. Identification of several Rgp proteins in the YidC1 interactome suggested its involvement in bacitracin resistance, which was decreased in ΔyidC1 and SRP-deficient mutants. Collectively, YidC1 and YidC2 interactome analyses has further distinguished these paralogs in the Gram-positive bacterium S. mutansIMPORTANCEStreptococcus mutans is a prevalent oral pathogen and major causative agent of tooth decay. Many proteins that enable this bacterium to thrive in its environmental niche and cause disease are embedded in its cytoplasmic membrane. The machinery that transports proteins into bacterial membranes differs between Gram-negative and Gram-positive organisms, an important difference being the presence of multiple YidC paralogs in Gram-positive bacteria. Characterization of a protein's interactome can help define its physiological role. Herein, we characterized the interactomes of S. mutans YidC1 and YidC2. Results demonstrated substantial overlap between their interactomes but also revealed several differences in their direct protein binding partners. Membrane transport machinery components were identified in the context of a large network of proteins involved in replication, transcription, translation, and cell division/cell shape. This information contributes to our understanding of protein transport in Gram-positive bacteria in general and informs our understanding of S. mutans pathogenesis.

The Impacts of Sortase A and the 4'-Phosphopantetheinyl Transferase Homolog Sfp on Streptococcus mutans Extracellular Membrane Vesicle Biogenesis.

Extracellular membrane vesicles (EMVs) are produced by many Gram-positive organisms, but information regarding vesiculogenesis is incomplete. We used single gene deletions to evaluate the impacts on Streptococcus mutans EMV biogenesis of Sortase A (SrtA), which affects S. mutans EMV composition, and Sfp, a 4'-phosphopantetheinyl transferase that affects Bacillus subtilis EMV stability. ΔsrtA EMVs were notably larger than Δsfp and wild-type (WT) EMVs. EMV proteins identified from all three strains are known to be involved in cell wall biogenesis and cell architecture, bacterial adhesion, biofilm cell density and matrix development, and microbial competition. Notably, the AtlA autolysin was not processed to its mature active form in the ΔsrtA mutant. Proteomic and lipidomic analyses of all three strains revealed multiple dissimilarities between vesicular and corresponding cytoplasmic membranes (CMs). A higher proportion of EMV proteins are predicted substrates of the general secretion pathway (GSP). Accordingly, the GSP component SecA was identified as a prominent EMV-associated protein. In contrast, CMs contained more multi-pass transmembrane (TM) protein substrates of co-translational transport machineries than EMVs. EMVs from the WT, but not the mutant strains, were enriched in cardiolipin compared to CMs, and all EMVs were over-represented in polyketide flavonoids. EMVs and CMs were rich in long-chain saturated, monounsaturated, and polyunsaturated fatty acids, except for Δsfp EMVs that contained exclusively polyunsaturated fatty acids. Lipoproteins were less prevalent in EMVs of all three strains compared to their CMs. This study provides insight into biophysical characteristics of S. mutans EMVs and indicates discrete partitioning of protein and lipid components between EMVs and corresponding CMs of WT, ΔsrtA, and Δsfp strains.

Enhanced purification coupled with biophysical analyses shows cross-ß structure as a core building block for Streptococcus mutans functional amyloids.

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-ß amyloid structure.

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.

Membrane proteomic analysis reveals overlapping and independent functions of Streptococcus mutans Ffh, YidC1, and YidC2.

A comparative proteomic analysis was utilized to evaluate similarities and differences in membrane samples derived from the cariogenic bacterium Streptococcus mutans, including the wild-type strain and four mutants devoid of protein translocation machinery components, specifically ∆ffh, ∆yidC1, ∆yidC2, or ∆ffh/yidC1. The purpose of this work was to determine the extent to which the encoded proteins operate individually or in concert with one another and to identify the potential substrates of the respective pathways. Ffh is the principal protein component of the signal recognition particle (SRP), while yidC1 and yidC2 are dual paralogs encoding members of the YidC/Oxa/Alb family of membrane-localized chaperone insertases. Our results suggest that the co-translational SRP pathway works in concert with either YidC1 or YidC2 specifically, or with no preference for paralog, in the insertion of most membrane-localized substrates. A few instances were identified in which the SRP pathway alone, or one of the YidCs alone, appeared to be most relevant. These data shed light on underlying reasons for differing phenotypic consequences of ffh, yidC1 or yidC2 deletion. Our data further suggest that many membrane proteins present in a ∆yidC2 background may be non-functional, that ∆yidC1 is better able to adapt physiologically to the loss of this paralog, that shared phenotypic properties of ∆ffh and ∆yidC2 mutants can stem from impacts on different proteins, and that independent binding to ribosomal proteins is not a primary functional activity of YidC2. Lastly, genomic mutations accumulate in a ∆yidC2 background coincident with phenotypic reversion, including an apparent W138R suppressor mutation within yidC1.

Streptococcus mutans yidC1 and yidC2 Impact Cell Envelope Biogenesis, the Biofilm Matrix, and Biofilm Biophysical Properties.

Proper envelope biogenesis of Streptococcus mutans, a biofilm-forming and dental caries-causing oral pathogen, requires two paralogs (yidC1 and yidC2) of the universally conserved YidC/Oxa1/Alb3 family of membrane integral chaperones and insertases. The deletion of either paralog attenuates virulence in vivo, but the mechanisms of disruption remain unclear. Here, we determined whether the deletion of yidC affects cell surface properties, extracellular glucan production, and/or the structural organization of the exopolysaccharide (EPS) matrix and biophysical properties of S. mutans biofilm. Compared to the wild type, the ΔyidC2 mutant lacked staining with fluorescent vancomycin at the division septum, while the ΔyidC1 mutant resembled the wild type. Additionally, the deletion of either yidC1 or yidC2 resulted in less insoluble glucan synthesis but produced more soluble glucans, especially at early and mid-exponential-growth phases. Alteration of glucan synthesis by both mutants yielded biofilms with less dry weight and insoluble EPS. In particular, the deletion of yidC2 resulted in a significant reduction in biofilm biomass and pronounced defects in the spatial organization of the EPS matrix, thus modifying the three-dimensional (3D) biofilm architecture. The defective biofilm harbored smaller bacterial clusters with high cell density and less surrounding EPS than those of the wild type, which was stiffer in compression yet more susceptible to removal by shear. Together, our results indicate that the elimination of either yidC paralog results in changes to the cell envelope and glucan production that ultimately disrupts biofilm development and EPS matrix structure/composition, thereby altering the physical properties of the biofilms and facilitating their removal. YidC proteins, therefore, represent potential therapeutic targets for cariogenic biofilm control.IMPORTANCE YidC proteins are membrane-localized chaperone insertases that are universally conserved in all bacteria and are traditionally studied in the context of membrane protein insertion and assembly. Both YidC paralogs of the cariogenic pathogen Streptococcus mutans are required for proper envelope biogenesis and full virulence, indicating that these proteins may also contribute to optimal biofilm formation in streptococci. Here, we show that the deletion of either yidC results in changes to the structure and physical properties of the EPS matrix produced by S. mutans, ultimately impairing optimal biofilm development, diminishing its mechanical stability, and facilitating its removal. Importantly, the universal conservation of bacterial yidC orthologs, combined with our findings, provide a rationale for YidC as a possible drug target for antibiofilm therapies.

Characterization of the pgf operon involved in the posttranslational modification of Streptococcus mutans surface proteins.

Protein glycosylation has been described as the most abundant and complex post-translational modification occurring in nature. Recent studies have enhanced our view of how this modification occurs in bacteria highlighting the role of protein glycosylation in various processes such as biofilm formation, virulence and host-microbe interactions. We recently showed that the collagen- and laminin-binding adhesin Cnm of the dental pathogen Streptococcus mutans is post-translationally modified by the PgfS glycosyltransferase. Following this initial identification of Cnm as a glycoprotein, we have now identified additional genes (pgfM1, pgfE and pgfM2) that are also involved in the posttranslational modification of Cnm. Similar to the previously characterized ΔpgfS strain, inactivation of pgfM1, pgfE or pgfM2 directly impacts Cnm by altering its migration pattern, proteolytic stability and function. In addition, we identified the wall-associated protein A (WapA) as an additional substrate of Pgf-dependent modification. We conclude that the pgS-pgfM1-pgfE-pgfM2 operon encodes for a protein machinery that can modify, likely through the addition of glycans, both core and non-core gene products in S. mutans.

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.

Functional amyloids in Streptococcus mutans, their use as targets of biofilm inhibition and initial characterization of SMU_63c.

Amyloids have been identified as functional components of the extracellular matrix of bacterial biofilms. Streptococcus mutans is an established aetiologic agent of dental caries and a biofilm dweller. In addition to the previously identified amyloidogenic adhesin P1 (also known as AgI/II, PAc), we show that the naturally occurring antigen A derivative of S. mutans wall-associated protein A (WapA) and the secreted protein SMU_63c can also form amyloid fibrils. P1, WapA and SMU_63c were found to significantly influence biofilm development and architecture, and all three proteins were shown by immunogold electron microscopy to reside within the fibrillar extracellular matrix of the biofilms. We also showed that SMU_63c functions as a negative regulator of biofilm cell density and genetic competence. In addition, the naturally occurring C-terminal cleavage product of P1, C123 (also known as AgII), was shown to represent the amyloidogenic moiety of this protein. Thus, P1 and WapA both represent sortase substrates that are processed to amyloidogenic truncation derivatives. Our current results suggest a novel mechanism by which certain cell surface adhesins are processed and contribute to the amyloidogenic capability of S. mutans. We further demonstrate that the polyphenolic small molecules tannic acid and epigallocatechin-3-gallate, and the benzoquinone derivative AA-861, which all inhibit amyloid fibrillization of C123 and antigen A in vitro, also inhibit S. mutans biofilm formation via P1- and WapA-dependent mechanisms, indicating that these proteins serve as therapeutic targets of anti-amyloid compounds.

Specific binding of a naturally occurring amyloidogenic fragment of Streptococcus mutans adhesin P1 to intact P1 on the cell surface characterized by solid state NMR spectroscopy.

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.

Trk2 Potassium Transport System in Streptococcus mutans and Its Role in Potassium Homeostasis, Biofilm Formation, and Stress Tolerance.

UNLABELLED: 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.

Identification of a supramolecular functional architecture of Streptococcus mutans adhesin P1 on the bacterial cell surface.

P1 (antigen I/II) is a sucrose-independent adhesin of Streptococcus mutans whose functional architecture on the cell surface is not fully understood. S. mutans cells subjected to mechanical extraction were significantly diminished in adherence to immobilized salivary agglutinin but remained immunoreactive and were readily aggregated by fluid-phase salivary agglutinin. Bacterial adherence was restored by incubation of postextracted cells with P1 fragments that contain each of the two known adhesive domains. In contrast to untreated cells, glutaraldehyde-treated bacteria gained reactivity with anti-C-terminal monoclonal antibodies (mAbs), whereas epitopes recognized by mAbs against other portions of the molecule were masked. Surface plasmon resonance experiments demonstrated the ability of apical and C-terminal fragments of P1 to interact. Binding of several different anti-P1 mAbs to unfixed cells triggered release of a C-terminal fragment from the bacterial surface, suggesting a novel mechanism of action of certain adherence-inhibiting antibodies. We also used atomic force microscopy-based single molecule force spectroscopy with tips bearing various mAbs to elucidate the spatial organization and orientation of P1 on living bacteria. The similar rupture lengths detected using mAbs against the head and C-terminal regions, which are widely separated in the tertiary structure, suggest a higher order architecture in which these domains are in close proximity on the cell surface. Taken together, our results suggest a supramolecular organization in which additional P1 polypeptides, including the C-terminal segment originally identified as antigen II, associate with covalently attached P1 to form the functional adhesive layer.

Binding forces of Streptococcus mutans P1 adhesin.

Streptococcus mutans is a Gram-positive oral bacterium that is a primary etiological agent associated with human dental caries. In the oral cavity, S. mutans adheres to immobilized salivary agglutinin (SAG) contained within the salivary pellicle on the tooth surface. Binding to SAG is mediated by cell surface P1, a multifunctional adhesin that is also capable of interacting with extracellular matrix proteins. This may be of particular importance outside of the oral cavity as S. mutans has been associated with infective endocarditis and detected in atherosclerotic plaque. Despite the biomedical importance of P1, its binding mechanisms are not completely understood. In this work, we use atomic force microscopy-based single-molecule and single-cell force spectroscopy to quantify the nanoscale forces driving P1-mediated adhesion. Single-molecule experiments show that full-length P1, as well as fragments containing only the P1 globular head or C-terminal region, binds to SAG with relatively weak forces (∼50 pN). In contrast, single-cell analyses reveal that adhesion of a single S. mutans cell to SAG is mediated by strong (∼500 pN) and long-range (up to 6000 nm) forces. This is likely due to the binding of multiple P1 adhesins to self-associated gp340 glycoproteins. Such a cooperative, long-range character of the S. mutans-SAG interaction would therefore dramatically increase the strength and duration of cell adhesion. We also demonstrate, at single-molecule and single-cell levels, the interaction of P1 with fibronectin and collagen, as well as with hydrophobic, but not hydrophilic, substrates. The binding mechanism (strong forces, cooperativity, broad specificity) of P1 provides a molecular basis for its multifunctional adhesion properties. Our methodology represents a valuable approach to probe the binding forces of bacterial adhesins and offers a tractable methodology to assess anti-adhesion therapy.

An intramolecular lock facilitates folding and stabilizes the tertiary structure of Streptococcus mutans adhesin P1.

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.

Immunogenicity and in vitro and in vivo protective effects of antibodies targeting a recombinant form of the Streptococcus mutans P1 surface protein.

Streptococcus mutans is a major etiologic agent of dental caries, a prevalent worldwide infectious disease and a serious public health concern. The surface-localized S. mutans P1 adhesin contributes to tooth colonization and caries formation. P1 is a large (185-kDa) and complex multidomain protein considered a promising target antigen for anticaries vaccines. Previous observations showed that a recombinant P1 fragment (P1(39-512)), produced in Bacillus subtilis and encompassing a functional domain, induces antibodies that recognize the native protein and interfere with S. mutans adhesion in vitro. In the present study, we further investigated the immunological features of P1(39-512) in combination with the following different adjuvants after parenteral administration to mice: alum, a derivative of the heat-labile toxin (LT), and the phase 1 flagellin of S. Typhimurium LT2 (FliCi). Our results demonstrated that recombinant P1(39-512) preserves relevant conformational epitopes as well as salivary agglutinin (SAG)-binding activity. Coadministration of adjuvants enhanced anti-P1 serum antibody responses and affected both epitope specificity and immunoglobulin subclass switching. Importantly, P1(39-512)-specific antibodies raised in mice immunized with adjuvants showed significantly increased inhibition of S. mutans adhesion to SAG, with less of an effect on SAG-mediated bacterial aggregation, an innate defense mechanism. Oral colonization of mice by S. mutans was impaired in the presence of anti-P1(39-512) antibodies, particularly those raised in combination with adjuvants. In conclusion, our results confirm the utility of P1(39-512) as a potential candidate for the development of anticaries vaccines and as a tool for functional studies of S. mutans P1.