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 β-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.


Amyloids represent an evolutionarily conserved fibrillar, cross-β-sheet quaternary structure of certain proteins in which the β-sheets laterally assemble in a noncovalent polymer to form fibers (14). Amyloid aggregates typically form structures with diameters ranging from 6 to 12 nm and share common biophysical properties such as detergent and protease resistance, sensitivity to formic acid dissolution, uptake of amyloidophilic dyes, and birefringent properties when stained with Congo red (CR) (4, 5). Although amyloids are commonly associated with human diseases related to protein misfolding, functional amyloids are known to play important biological roles across all domains of life (6). In addition to polysaccharides and extracellular DNA (eDNA), amyloids are increasingly detected as constituents of extracellular biofilm matrices, where they can influence growth at air-water interfaces (7), serve as a functional sink for quorum sensing molecules (8), or act as a scaffold to help maintain three-dimensional (3D) architecture, allowing for the formation of channels and wrinkles that determine spatial distribution of species within multispecies biofilms (1, 3, 913). Other functional roles of amyloids include regulation of genes and cell fate, toxicity against other microorganisms and host cells, and immune regulation of host responses (12). Several bacterial functional amyloids have been described in both Gram-positive and Gram-negative organisms, including the curli proteins from Escherichia coli and Salmonella spp., Bacillus subtilis TasA, Pseudomonas aeruginosa FapBC, Staphylococcus aureus Bap and phenol-soluble modulins (PSM), Enterococcus faecalis Esp, and Streptococcus mutans P1 (also known as SpaP or antigen I/II), WapA, and SMU_63c (12, 1419). Multiple factors, including pH, salt concentration, and surface hydrophobicity, have been reported to influence the propensity of proteins to form amyloids within bacterial biofilms (1, 10, 15, 20, 21).
A resident of the dental biofilm, S. mutans is a keystone pathogen in dental caries. The cariogenic potential of S. mutans can be largely attributed to several characteristics that are hallmarks of its successful coevolution with humans. First, S. mutans genomes encode a number of glucosyltransferase enzymes that convert sucrose into large glucan polymers, which compose the bulk of the extracellular polymeric matrix and are critical for the establishment and maintenance of the dental biofilm (22). In addition, S. mutans thrives in carbohydrate-rich environments, as it can metabolize a wide range of carbohydrates, generating acids (acidogenicity) and surviving and continuing to undergo glycolysis even at pHs below 5.0 (aciduricity) (23, 24).
In addition to the sucrose-dependent colonization mechanism that is based on glucan polymer synthesis, S. mutans also possesses sucrose-independent colonization mechanisms whereby cell surface-associated adhesins such as P1 and WapA mediate attachment to the salivary pellicle that coats the tooth enamel (25, 26). Of note, P1 and WapA also mediate binding to collagen-rich surfaces (27). More recently, genes encoding two closely related surface-associated collagen-binding proteins (CBPs), cnm and cbm, were identified in approximately 20% of S. mutans strains (2830). Cnm was shown to mediate invasion of epithelial and oral cell lines and binding to collagenous and laminin-rich substrates present in oral (dentin, cementum, and roots) and extraoral environments (heart valves) (27, 3133). Cnm and Cbm were shown to be important virulence factors implicated in systemic infections such as infective endocarditis, cerebral microbleeds, and hemorrhagic stroke (3438). Moreover, clinical studies have linked oral infection with CBP+ S. mutans strains with increased caries risk and poor caries outcomes (23, 27, 39, 40).
While Cnm has a predicted molecular mass of 54 kDa, it migrates at approximately 120 kDa when separated by SDS-PAGE. Recently, we discovered that this aberrant migration was, in great part, a result of Cnm posttranslational modification through glycosylation. Specifically, we identified a novel protein glycosylation machinery pgf (protein glycosyltransferase) in S. mutans (41) that was cotranscribed with cnm and responsible for its glycosylation in the serotype f OMZ175 strain (42). Inactivation of pgf genes resulted in a Cnm protein that migrated at approximately 90 kDa and that is highly susceptible to proteolysis, indicating a critical role for glycosylation in Cnm function and stability (41, 42). In silico analysis revealed that the threonine-rich repeat domain located in the Cnm C terminus is subjected to glycosylation, while the β-sheet-rich N terminus where the conserved collagen-binding domain (CBD) resides is not modified by the Pgf glycosylation machinery (42). In addition, the Pgf system was shown to also modify WapA, whose N-terminal truncation product antigen A (AgA) was shown to have amyloidogenic properties (41). Interestingly, homology models revealed a high similarity between the tertiary structures of the Cnm CBD with that of AgA (27). Based on the similarity between AgA and the Cnm CBD, we hypothesized that Cnm may also self-assemble into amyloid fibers. In the present study, we confirmed this prediction by using a panel of wild-type and mutant strains and different variant forms of purified Cnm (native glycosylated, recombinant nonglycosylated, and recombinant CBD). We show that Cnm is a major amyloidogenic protein in the Cnm+ S. mutans strain OMZ175, with its CBD being sufficient for amyloidogenesis but possibly requiring the threonine-rich repeat for maintenance of stability, especially in acidic environments. We also demonstrate that when in monomeric form, the main function of Cnm is collagen binding. However, when Cnm self-assembles into amyloids, its ability to competitively inhibit binding of S. mutans cells to collagenous substrates is lost. Hence, amyloid formation by Cnm might act as a negative regulatory mechanism to modulate collagen-binding activity within S. mutans biofilms.


In silico analyses suggests that the collagen-binding domain (CBD) of Cnm is aggregation-prone.

To evaluate the potential for Cnm amyloid formation, we used the bioinformatics tool AmyloPred2 to analyze the primary structure of Cnm from S. mutans OMZ175. The analysis predicted a high frequency of aggregation-prone amino acid residues in the N-terminal portion (within the CBD) (Fig. 1A and B), which coincides with the predicted β-sheet-rich region modeled using SWISS-MODEL (Fig. 1C). The threonine-rich repeat domain of Cnm could not be modeled, due to its high degree of structural disorder (defined as a lack of fixed three-dimensional structure), and therefore can be classified as an intrinsically disordered region (Fig. 2A) (11). It is possible, however, that this level of structural disorder is different when the protein is glycosylated. Such a disorder analysis using MobiDB requires a deposited UniProtKB sequence, and for this analysis, the Cnm sequence of strain TW871 (UniProtKB C4B6T3), which shares 98% and 100% identity with full-length Cnm and the Cnm CBD from S. mutans OMZ175, respectively, was used. To gain further insight into how the aggregation-prone N-terminal domain of Cnm may lead to amyloid formation, we performed an ab initio docking modeling of a Cnm dimer on GalaxyHomomer. The analysis predicted an interaction between the β-sheet regions of CBDs from different Cnm monomers (Fig. 2B) with a surface area of interaction of 1,095.1 Å2 and a docking score of 1,338.308. Portions of the β-sheet region of the interacting monomers also remained available for potential coupling with additional Cnm monomers, while the predicted unstructured acidic and glycosylated threonine-rich domains protruded outward without active participation in the predicted interaction (Fig. 2B).
FIG 1 The β-sheet-rich collagen-binding domain of S. mutans Cnm is predicted to be amyloidogenic. (A) Schematic representation of Cnm, including the secretion signal (SS), the A and B domains, and the LPXTG anchoring motif (AM). (B) Aggregation-prone peptides predicted by AmylPred2 are shown in red. The CBD is underlined, and the portion of the sequence that was modeled by SWISS-MODEL is highlighted in gray. (C) SWISS-MODEL homology model of the CBD of Cnm from S. mutans OMZ175 (based on model SMTL ID 2f6a.1).
FIG 2 The Cnm threonine-rich B domain as an intrinsically disordered region. (A) MobiDB estimation of structural disorder highlights a lack of fixed 3D structure of the B domain of the deposited Cnm sequence UniProtKB C4B6T3. (B) GalaxyHomomer modeling of full-length Cnm, by ab initio docking, combines ordered and disordered regions and illustrates likely interactions between β-sheet-rich CBDs of two different Cnm monomers (each in a different color, orange or green). The acidic and glycosylated threonine-rich domains protrude outward. Domains are indicated with brackets for the monomeric protein colored in orange (left).

Cnm and its variant forms take up the amyloidophilic dye Thioflavin-T.

After in silico analyses predicting that Cnm is prone to aggregation, we performed Thioflavin-T (ThT) fluorescence assays using purified Cnm in the presence or absence of the amyloid inhibitor tannic acid (43). In addition to full-length glycosylated Cnm purified from S. mutans OMZ175, we also tested a recombinant full-length Cnm (rCnm) and a truncated Cnm containing the CBD only (rCBD) purified from E. coli. Of note, both recombinant protein versions are not glycosylated, as E. coli lacks the Pgf machinery required for Cnm glycosylation. As shown in Fig. 3A, all three tested forms of Cnm incorporated the amyloidophilic ThT dye when amyloidogenesis was stimulated by stirring in pH 7.4 phosphate-buffered saline (PBS). Mechanical agitation of purified proteins is a common strategy to promote nucleation, enhancing amyloid fibril assembly (44). Moreover, ThT incorporation by all three Cnm versions was significantly inhibited by the amyloid inhibitor tannic acid (Fig. 3A). Collectively, these results suggest that both glycosylated and nonglycosylated Cnm can form amyloids and that the CBD is the main amyloidogenic unit of Cnm.
FIG 3 Thioflavin-T (ThT) fluorescence assay of native glycosylated S. mutans Cnm and recombinant nonglycosylated Cnm variants (rCnm and rCBD) purified from E. coli. (A) ThT uptake by purified Cnm and its variants. Purified rAgA and rGbpC were used as positive and negative controls, respectively. S, stirred; TA, stirred with tannic acid; U, unstirred. All values were normalized by molarity and relative to stirred Cnm. (B) Effect of pH on amyloidogenic properties of native Cnm and its derivatives. All values were normalized by molarity and are relative to Cnm at pH 7.4. Experiments were performed at least in triplicate, with statistical analyses performed using one-way ANOVA (for each group shown in panel A) or nonparametric t tests (for each pair shown in panel B). ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.
To determine whether pH can influence Cnm fibril assembly, we performed the same ThT assay under a neutral pH (7.4) and an acidic pH (5.5), the latter being similar to the pH of enamel dissolution provoked during dental plaque acidification by S. mutans (Fig. 3B). While the full-length forms of Cnm (Cnm and rCnm) did not show significant differences in dye uptake based on pH, ThT uptake by rCBD was significantly decreased at pH 5.5 compared to pH 7.4 (P < 0.05). This suggests that the unstructured threonine-rich domain may contribute to quaternary structure stability at lower pH.

Transmission electron microscopy confirms that S. mutans Cnm and its variants form amyloids.

A defining feature of amyloid aggregates is protease resistance (4547). In S. mutans, protein amyloids form mat-like composite aggregates in which typical amyloid fibrillar structures are only revealed following protease digestion of protein monomers or protease-sensitive oligomeric intermediates (48). To obtain direct evidence that Cnm forms amyloids, stirred samples of full-length glycosylated Cnm, nonglycosylated Cnm (rCnm), and rCBD were treated with proteinase K and visualized by transmission electron microscopy (TEM) (Fig. 4). Fibrillar material indicative of amyloid aggregates was visualized for the recombinant forms of nonglycosylated Cnm (rCnm and rCBD). In contrast, a mat-like amyloid structure was visualized for glycosylated native Cnm (Fig. 4). Because S. mutans amyloid mats have been confirmed to exhibit the same characteristic X-ray fiber diffraction pattern as isolated amyloid fibers (48), and because glycosylation of Cnm has been shown to protect this protein from proteolytic degradation (41), the visualization of amyloid mats rather than fibers by TEM confirms the formation of amyloid by native glycosylated Cnm.
FIG 4 Transmission electron microscopy of stirred S. mutans Cnm, rCnm, rCBD, and rAgA (positive control) following treatment with proteinase K. Purified proteins were stirred to induce amyloid formation and then treated with proteinase K to remove residual protease-sensitive monomers. Fibrillar material was visualized in the nonglycosylated rCnm, rCBD, and rAgA preparations, whereas the native glycosylated Cnm sample displayed a mat-like structure, likely due to proteinase K resistance. Scale bars = 500 nm.

Cnm is a major amyloidogenic protein in OMZ175 biofilms.

Congo red is an amyloidophilic dye which provides amyloid-containing cell foci an aberrant coloration, typically yellow or orange-green, when viewed with polarized light through crossed filters (49). To determine whether Cnm amyloid fibrils can be detected in the S. mutans OMZ175 biofilm matrix, Congo red birefringence assays were performed on mature (5-day-old) biofilms using a panel of strains that include the parent OMZ175, the cnm deletion mutant (OMZ175Δcnm), and the srtA deletion mutant (OMZ175ΔsrtA). The srtA gene codes for the sortase A enzyme such that OMZ175ΔsrtA cannot cross-link LPXTG proteins, such as Cnm and the other characterized amyloidogenic proteins WapA and P1, to the cell envelope. Moreover, previous work has shown that biofilm-associated amyloid formation was diminished in an S. mutans ΔsrtA strain (16). As shown in Fig. 5A, strongly birefringent, tightly organized amyloid-containing aggregates were observed in mature biofilms formed by OMZ175, while less intense, more diffuse staining was observed in the Δcnm and ΔsrtA strains grown in media containing glucose (Fig. 5A). Low magnification visualization (data not shown) and quantification of amyloid aggregates per slide revealed that OMZ175 biofilms display significantly more birefringent foci than either mutant strain, with no significant differences between the two mutants (Fig. 5B).
FIG 5 Cnm is a major amyloidogenic protein of S. mutans OMZ175 biofilms. Five-day-old biofilm biomass of OMZ175, OMZ175Δcnm, and OMZ175ΔsrtA strains were stained with Congo red and visualized by polarizing light microscopy with crossed filters. (A) Representative micrographs of biofilm masses as seen under bright field and cross-polarized light filters. Organized birefringent (amyloid-containing) aggregates can only be observed for OMZ175 (black arrowhead), and disorganized low-birefringence aggregates are abundant in the mutant biofilms (white arrowheads). Scale bars = 100 μm. (B) Counts of birefringent foci per slide for each strain confirm that Cnm amyloids are prevalent within S. mutans OMZ175 biofilms compared to OMZ175Δcnm and OMZ175ΔsrtA strains (P = 0.0297). Experiments were performed at least in triplicate, with statistical analyses performed using one-way ANOVA. **, P < 0.01; ns, nonsignificant.

CBD monomers, but not amyloid fibers, competitively inhibit cell binding to collagenous substrates.

To gain insight into the biological relevance of the amyloidogenic property of Cnm, we developed a competitive binding assay in which purified rCBD monomers or fibrils were tested for their ability to interfere with the ability of the OMZ175 strain to adhere to collagen-coated microtiter plate wells or to premolar roots of which collagen is the main organic component (50, 51). We used rCBD for these assays because it can be produced in large quantities and encompasses the domain that mediates collagen binding and can form amyloid fibers. Figure 6A shows that rCBD monomers inhibited OMZ175 binding to immobilized collagen, whereas rCBD-derived amyloid fibrils did not (P < 0.0001). These results indicate that the rCBD monomers, but not the amyloid aggregates, bind to collagen-rich surfaces, thereby making it less available for binding by OMZ175 cells. In an ex vivo model, the competitive binding assay was performed using saliva-coated dental root sections, with significant similar results obtained (Fig. 6B). These results are consistent with in silico prediction of Cnm oligomerization via the β-sheets of the CBD domains (Fig. 2B), in that once Cnm-Cnm interactions occur, the CBD should no longer be available for interaction with collagen.
FIG 6 Amyloid aggregation of S. mutans Cnm diminishes its binding to collagenous substrates. (A) In vitro competitive inhibition assay in which collagen-coated microtiter plates were preincubated with increasing concentrations of rCBD monomers or corresponding concentrations of amyloid fibrils prior to incubation with OMZ175 cells. Values indicate binding relative to untreated wells that did not receive rCBD monomers or fibrils. rCBD monomers, but not amyloid fibrils, significantly inhibited OMZ175 adherence. (B) Ex vivo competitive inhibition assay using premolar root sections. Preincubation with 20 μg of rCBD monomers, but not with amyloid fibers, significantly diminished binding of OMZ175 to the root sections, resulting in lowered CFU recovery. Values are relative to untreated (no monomers or fibrils added). Experiments were performed at least in triplicate, with statistical analyses performed using one-way ANOVA. **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.


Microbial amyloids have been a focus of increased attention due to their wide array of biofilm-associated functions, which include organization of biofilm structure, modulation of biofilm hydrophobicity and mechanical properties, control of quorum sensing, regulation of genes, toxicity, and modulation of host response (1, 813, 52). Functional amyloids have been discovered in pathogenic and nonpathogenic bacteria, and their formation is generally associated with growth in biofilms (1, 10, 45). The discovery of multiple functional amyloids in S. mutans (WapA, P1, and SMU_63c) made us wonder if Cnm, a surface protein that is also rich in β-sheet structure, could be part of this group. Here, we suggest that Cnm function might be modulated by its aggregation status.
An amyloid is a noncovalent oligomer of extended intermolecularly hydrogen-bonded β-sheets that self-assemble to form fibers. Regardless of the origin, amyloids share several properties, which include a fibrillar structure when viewed by electron microscopy, enhanced birefringence on binding to Congo red, and a cross-β-sheet structure in which the β-strands run perpendicular to the fiber axis, resulting in a characteristic X-ray fiber diffraction pattern (53). Our in silico analyses suggested that the CBD portion of Cnm assembles into amyloid aggregates, whereas the glycosylated threonine-rich repeat likely does not contribute to the amyloid fibril assembly. During oligomerization, the β-sheet structures of the monomeric units interact, with the glycosylated threonine-rich domains protruding outward. This model is consistent with our in vivo and ex vivo collagen-binding competitive inhibition assays, in which we observed that purified rCBD monomers competed with intact cells of S. mutans for binding to collagen-rich surfaces, while rCBD-derived amyloid did not. In other words, the loss of the collagen-binding function of Cnm following amyloid aggregation suggests that the same β-sheet-rich region that mediates collagen binding also functions as the elongation point for fibril formation, thus rendering it unavailable for its primary adhesive function. Depending on the protein, formation of amyloids can result in a loss or a gain of function of the aggregating polypeptide (4). Hence, amyloid aggregation represents a potential mechanism to regulate protein function as needed in different environments (54, 55). The oligomerization of Cnm into amyloids does not seem to be dependent upon glycosylation by the Pgf machinery since native glycosylated and nonglycosylated recombinant proteins both activated ThT fluorescence and displayed visible amyloid fibers when visualized by microscopy. The contribution of glycosylation to protein amyloidogenesis is still a relatively unexplored field, although the effect of glycosylation of the amyloid precursor protein of Aβ in Alzheimer’s disease has gained attention due to its regulatory role in the protein’s proteolytic processing (56, 57). Bacterial amyloids derived from glycoproteins have now been reported as components of the structural extracellular matrix of biofilms rich in ammonia-oxidizing bacteria and nitrite-oxidizing bacteria grown in aerobic and granular sludge (58). Further characterization of glycoprotein-derived amyloids is warranted, as these may demonstrate common features in different domains of life.
In addition to its correlation with increased caries risk, S. mutans Cnm has also been shown to contribute to increased acid tolerance (59). Therefore, it was relevant to investigate if the newfound amyloidogenic properties of Cnm are modulated by acidic pH. Based on ThT assays of Cnm, rCnm, and rCBD stirred in neutral compared to acidic pH, we found that in contrast to glycosylated Cnm and rCnm, the rCBD variant forms fewer amyloid fibrils at pH 5.5 than at pH 7.4. We speculate that the threonine-rich domain might contribute to fiber integrity of full-length Cnm at acidic pH. This effect may be related to the presence of an external layer of acidic threonines to accept protons, thus protecting the β-sheet core from unfolding by charge repulsion (60). This observation strengthens the notion that, although the threonine-rich domain is not related to the collagen-binding activity of Cnm, it supports that activity by contributing to the stability of the Cnm monomer. For instance, a previous study from our group demonstrated that glycosylation of the threonine-rich domain by the Pgf machinery protects Cnm against the proteolytic activity of proteinase K (41). Also, the effect of acidity in promoting amyloidogenesis has been well characterized for Aβ oligomers related to Alzheimer’s disease as a key factor for pathogenicity (61, 62). In S. mutans, neutral pH was shown to favor amyloid formation by WapA and P1, while the secreted amyloidogenic protein Smu_63c, which appears to be a negative regulator of biofilm cell density and genetic competence, is triggered to assemble into amyloid by acidic pH (15). Of note, formation of S. aureus Bap amyloids is also favored at acidic pH (15, 63).
TEM visualization of amyloid material induced by mechanical agitation of purified proteins, followed by treatment with proteinase K, revealed a mat-like structure for glycosylated Cnm but a fibrillar morphology for nonglycosylated Cnm and CBD. Mat-like aggregates have been proposed as a more biologically germane form of S. mutans amyloids that represents a supramolecular structure composed of amyloid fibers, monomers, and oligomer intermediates (48) where pure fibers, achieved by proteolytic digestion of S. mutans amyloid-containing mats, are likely only seen in laboratory settings. It has also been shown that the addition of monomeric protein to purified fibrils shifts the morphology of fibrils back to mats (48). Amyloid mats derived from purified S. mutans C123 (the amyloid-forming truncation derivative of P1), AgA (the amyloid-forming truncation product of WapA), and Smu_63c, as well as purified fibers produced by protease treatment of the mats, all demonstrated classical X-ray fiber diffraction patterns with distinct intensities at 4.8 Å in the meridional direction corresponding to the separation of strands in a β-sheet and distinct equatorial intensities at 10 Å corresponding to the distance between stacked β-sheets (48). Our current results revealed that proteinase K treatment of aggregated native glycosylated Cnm did not disrupt the amyloid mats, confirming our previous finding that glycosylation of Cnm confers resistance to proteinase K degradation of monomers (41). In contrast, protease-treated aggregates of nonglycosylated rCnm and rCBD were more fibrillar in nature. The visualization of amyloid mats and fiber morphologies, in conjunction with ThT uptake assays, provides direct evidence of amyloid formation by S. mutans Cnm.
To confirm Cnm amyloidogenic properties in vivo, Congo red-induced birefringence was evaluated for mature biofilms of the wild-type, Δcnm, and ΔsrtA strains. Of note, S. mutans harbors only one sortase-encoding gene, and inactivation of srtA results in a complete lack of LPXTG motif-containing proteins on the cell surface (64), including the amyloidogenic proteins P1, WapA, and Cnm. While not every sortase substrate is amyloidogenic (15), most amyloid-forming proteins identified in other bacteria are anchored to the cell surface (17, 63, 65). In S. mutans, elimination of srtA negatively impacted biofilm-associated amyloid formation, suggesting that cell surface protein anchoring contributes to this process (16). Organized amyloid-containing birefringent aggregates were visualized in 5-day-old S. mutans OMZ175 biofilms, and surprisingly, the OMZ175Δcnm mutant showed similarly diminished birefringent properties as the OMZ175ΔsrtA mutant compared to the parent OMZ175 strain. This indicates that Cnm is one of the most abundant amyloidogenic proteins of strain OMZ175 and is present in amyloid form within mature biofilms, likely as a component of the biofilm matrix. Notably, while spaP (encoding P1) is present in the OMZ175 genome, the expression of P1 appears to be downregulated in Cnm+ strains (66)—hence, the pronounced impact of cnm deletion on detection of biofilm-associated amyloids.
A shift in function between monomeric and fibrillar forms of an amyloidogenic protein was previously observed for S. aureus PSMs (19). Monomeric (soluble) and amyloid fibers (insoluble) of PSMα peptides were assessed for their ability to modulate the antibiotic tolerance to ciprofloxacin in S. aureus cultures. The presence of PSMα monomers led to a reduction in the number of persister cells tolerant to ciprofloxacin, while the presence of PSMα amyloid fibrils did not alter the persister cell phenotype under the same conditions. It was proposed that the PSMα fibrils might act as reservoirs of the active form of this virulence factor that could be later mobilized if needed during changes in environmental conditions, such as pH fluctuation, a factor known to influence fiber formation, and dissociation of certain amyloids (61, 67). Another functional shift was also described between monomeric and fibrillar forms of PSM whereby several PSM monomers (PSMα, PSMβ, and δ-toxin), known to contribute to biofilm detachment (68), displayed the opposite behavior of promoting biofilm integrity when sequestered into amyloids (18). Based on our collagen-binding competition assays, we observed that Cnm also undergoes a functional shift when in amyloid form. In biofilms of common laboratory strains of S. mutans that lack Cnm, amyloid material is more readily detected as the biofilm matures, usually after 60 h of growth (48). Whether this represents time and concentration-dependent amyloid nucleation leading to polymerization and elongation (69) or depends on other changing environmental conditions within the aging biofilm is not yet understood. The loss of Cnm collagen-binding activity as the protein transitions to amyloid form might suggest that it serves as a mechanism to regulate this property once adhesion and colonization have occurred and the biofilm is established. It is possible that Cnm amyloid fibrils present with other macromolecules such as eDNA and polysaccharides in mature biofilms might serve a scaffolding function to integrate biofilm cells and provide a substrate for attachment of other members of the biofilm community. Alternatively, accumulation of amyloid material in the matrix could result in consolidation and detachment of mature biofilms, as surface-anchored monomers would stop interacting with the collagenous substrates and interact instead with other monomers to form amyloid fibers within the biofilm. Studies are under way to evaluate the possible contribution of Cnm and other amyloids to the three-dimensional architecture and adhesion/detachment of bacteria during progression of S. mutans biofilm development. It will be important to establish whether the amyloid form of Cnm alters other functions already described for this virulence factor, including oral colonization, and epithelial and endothelial cell invasion (27, 34). Our results provide the foundation to address these questions in future studies. Our findings also may shed some light on an emerging paradigm of Gram-positive multifunctional proteins and their structural transitions from monomer to amyloid forms as an energy-effective strategy for biofilm modulation (17, 63, 70). Such new aspects of Cnm organization status (monomers and amyloid fibers) will inform future studies not only in S. mutans but also in a myriad of other organisms implicated in biofilm-related disease processes.


Bacterial strains and growth conditions.

The strains used in this study are listed in Table 1. Escherichia coli strains were routinely grown in Luria-Bertani (LB) broth medium at 37°C. When required, 100 μg ml−1 ampicillin was added to LB broth or to agar plates. S. mutans strains were routinely cultured in brain heart infusion (BHI) medium at 37°C in a humidified 5% CO2 atmosphere. When required, 1 mg ml−1 kanamycin was added to BHI broth or to agar plates.
TABLE 1 Bacterial strains used in this study
SpeciesStrainGenotypeReference or source
Streptococcus mutansOMZ175Wild type, cnm+B. Guggenheim
OMZ175ΔsrtAΔsrtA, kanrThis study
OMZ175ΔcnmΔcnm, kanr34
OMZ175ΔvicKΔvicK, kanr78
Escherichia coliBL21F- ompT hsdSB (rB-mB) gal dcm (λDE3)New England Biolabs
BL21/pET-16b:rCBDBL21 with pET-16b carrying the CBD of Cnm (amino acids 32–319), ampr41
BL21/pET-16b:rCnmBL21 with pET-16b carrying full-length Cnm (amino acids 32–545), ampr77

Genetic manipulation of S. mutans.

A sortase A (srtA) null-mutant was created in OMZ175 by using the PCR-ligation strategy (71) to replace the srtA gene with a nonpolar kanamycin marker (72). The 700-bp region flanking the 5′ end of the srtA gene was amplified using the primers srtA.1F (5′-TGAGTCGCGATAATGATG-3′) and srtA.1RPstI (5′-GAAACCTGACTGCAGTTGGTATTC-3′), whereas the 700-bp flanking the 3′ end of srtA was amplified using srtA.2FPstI (5′-GAATACCAACTGCAGTCAGGTTTC-3′) and srtA.2R (5′-GCAGCGGTTCAACTAACTTCTC-3′). The underlined bases correspond to the PstI restriction site that was included for cloning purposes. After amplification, the two PCR fragments were digested with PstI and ligated to a nonpolar kanamycin resistance gene cassette that was obtained as a PstI fragment. The ligation mixture was used to transform S. mutans OMZ175, followed by plating onto BHI medium containing kanamycin (1 mg ml−1). The insertional inactivation of srtA was confirmed by PCR using the primers srtA.1F and srtA.2R followed by Sanger sequencing of this PCR product, demonstrating the presence of the kanamycin marker and confirming the srtA deletion.

In silico analysis.

The AmylPred2 software (73), which employs a consensus of several methods to predict amyloid formation propensity, was used to analyze the primary amino acid sequence of Cnm from S. mutans strain OMZ175. Homology modeling of the Cnm monomer was performed with SWISS-MODEL (74) based on model SMTL ID 2f6a.1 (Cnm sequence identity, 52.04%), and a homo-oligomerization model was obtained by ab initio docking on GalaxyHomomer (75). Protein structure disorder estimations were calculated using MobiDB based on the similar deposited Cnm sequence UniProtKB C4B6T3 (76).

Expression and purification of recombinant Cnm and CBD.

Both recombinant CBD and full-length Cnm (rCBD and rCnm, respectively) were expressed in E. coli (41, 77). The E. coli strain harboring the pET16b‐rCnm plasmid was grown in LB broth containing ampicillin to an optical density at 600 nm of ≈0.5 (37°C and 150 rpm). The expression of the His‐tagged protein was induced by the addition of 0.5 mM isopropyl‐β‐d‐thiogalactopyranoside (IPTG) for 20 h at 24°C. The strain harboring the pET16b-rCBD plasmid was also grown to an optical density at 600 nm of ≈0.5 (37°C and 150 rpm) and was induced with 0.5 mM IPTG for 4 h at 37°C. Cells were lysed, and recombinant proteins were purified under native conditions using the Ni‐NTA fast start kit (Qiagen) following the manufacturer’s instructions. Halt protease inhibitor single-use cocktail (Thermo Fischer Scientific) was added to the lysis buffer as the only modification to the protocol.

Expression and purification of native Cnm.

Native glycosylated Cnm was purified using a custom affinity chromatography column coupled to an anti-rCBD antibody as previously described by our lab (42, 77). Briefly, the OMZ175ΔvicK strain, known to overexpresses Cnm (78), was grown overnight and then lysed by bead beating in the presence of Halt protease inhibitor single-use cocktail (Thermo Fischer Scientific) and ethylenediaminetetraacetic acid (EDTA). The soluble protein fraction was bound to a custom N-hydroxysuccinimide (NHS)–anti-rCBD column with overnight rocking at 4°C. The column was then washed with 15 ml of 1× phosphate-buffered saline (PBS) at pH 7.2, and Cnm was eluted using 0.1 M glycine buffer (pH 2.5) for 5 min. Elutions were immediately neutralized with 1/10 volume of a basic 1 M Tris buffer (pH 8.0). Purified native Cnm was later concentrated and dialyzed against a 100 mM ammonium bicarbonate solution.

Protein quantification and SDS-PAGE.

Protein concentrations were determined with the Micro BCA protein assay kit (Thermo Fischer Scientific), using a bovine serum albumin (BSA) standard curve as a reference for linear regression.
Denaturing SDS 10% (wt/vol) polyacrylamide gels were performed to check for protein purity and integrity. Precision Plus Protein dual color standards (Bio-Rad), ranging from 10 to 250 kDa were used as molecular weight markers. All samples (2.5 μg) were boiled for 5 min in Laemmli sample buffer before each run (79). Electrophoresis was performed for approximately 90 min in an ice bath at 25 mA constant current, and gels were stained using a Coomassie blue silver staining protocol (80).

In vitro induction of Cnm amyloid formation.

The following six protein variants were used in the in vitro assays: Cnm (native, glycosylated), rCnm, rCBD, rAgA (amyloidogenic; positive control), and recombinant glucan-binding protein C (rGpbC) (nonamyloidogenic; negative control). The purification of control proteins was described previously (15). Each protein was subjected to three different conditions—stirring, stirring in the presence of the amyloid inhibitor tannic acid, and no stirring (static). Mechanical agitation such as stirring promotes amyloid nucleation and is commonly used in studies investigating amyloidogenic properties of proteins (81). A total of 200 μl of each sample, which included PBS (1×, pH 7.4), tannic acid (100 μM, when present), and protein at 250 μg · ml−1, was placed in a 1.5-ml microcentrifuge tube with a 3 by 10-mm stir bar (except for the unstirred conditions). All samples were incubated for 5 days at 4°C. Stirred samples, with or without tannic acid, were placed on magnetic stirrers at maximum intensity. Amyloid material obtained by this method was further used in the thioflavin-T fluorescence assays, transmission electron microscopy imaging, and competitive binding assays.

Thioflavin-T fluorescence assays.

To test whether Cnm and its variant forms have amyloidogenic properties, we determined whether they take up the amyloidophilic thioflavin-T (ThT) as described elsewhere (15). A total of 22 μl of a 20 μM ThT solution in PBS was added to stirred, stirred in the presence of tannic acid, or unstirred samples prepared as described above, followed by incubation in the dark for 30 min at room temperature. Next, samples were transferred to fully dark 96-well plates, and fluorescence was read in a Synergy H1 hybrid multimode reader (BioTek) with excitation at 440 nm and reading at 485 nm. A PBS plus ThT blank was subtracted from each sample reading. To determine whether pH influences amyloid formation, ThT assays were also performed as described above, but the pH was adjusted to pH 7.4 or 5.5. To account for potential pH interference of ThT incorporation, 20 μl of 20× PBS at pH 7.4 was added to the samples before the fluorescence readings. PBS plus ThT blanks for each pH were subtracted from each sample reading. For all ThT assays, results are displayed as the relative fluorescence compared to stirred Cnm (at pH 7.4), in proportion to the molarity of each sample.

Transmission electron microscopy (TEM) of amyloid material following treatment with proteinase K.

Stirred samples of Cnm and its variants were treated with proteinase K (ProK) at a 10:1 (amyloid:ProK) molar ratio to degrade residual protein monomers as previously described (48). Following incubation for 3 h at 37°C, the reaction was stopped by addition of 2 mM phenylmethylsulfonyl fluoride (PMSF). After 5 min of incubation at room temperature, samples were centrifuged at 100,000 × g at 10°C for 30 min, and the pellets were washed twice with distilled water and then resuspended in 100 μl of distilled water. Samples were visualized on SDS-PAGE to confirm the absence of monomers (data not shown). Finally, samples were dried overnight, weighed on an analytical balance, and resuspended in distilled water to a concentration of 1 mg · ml−1. A 30-μl drop of each sample was placed under a 100-mesh Formvar-carbon-coated grid (FCF100-Cu-UB; Electron Microscopy Sciences, Hatfield, PA) with 1% uranyl acetate. Imaging was done on a Hitachi H-7600 TEM (Hitachi High Technologies America, Schaumburg, IL) with digital images acquired at 80 keV using an AMT digital camera.

Congo red birefringence assay of S. mutans biofilms.

Evaluation of Congo red-induced birefringence of amyloid material within S. mutans biofilms was performed as described previously (15, 16). Briefly, overnight cultures of strains OMZ175, OMZ175Δcnm, and OMZ175ΔsrtA grown in BHI broth were diluted 1:100 in biofilm medium plus 1% glucose (BMG) (82). Then, 200 μl of the diluted cultures was added to 96-well microtiter plates and incubated for 3 days at 37°C in a 5% CO2 atmosphere for biofilm formation. Spent medium was then removed by aspiration, and fresh BMG was replaced daily until the biofilms were 5 days old. The wells were gently washed with 1× sterile PBS, and biofilms were scraped off and centrifuged at 16,000 × g for 10 min. Pellets were resuspended in 10 μl of a Congo red solution and incubated in microtubes for 1 h at room temperature in the dark. CR solution was prepared by dissolving 2 g NaCl in 20 ml deionized H2O and by suspending 0.5 g CR in 80 ml 100 % ethanol; these two solutions were combined, filtered, and stored at room temperature as a stock solution. Samples were transferred to glass slides and visualized for birefringence as wet mounts using a Zeiss Scope A1 with a computer-controlled ProgRes C5 Jenoptik inverted camera equipped with cross-polarized filters. The number of birefringent foci were counted for each slide.

In vitro and ex vivo collagen-binding competition assays.

For in vitro competitive inhibition assays, wells of a 96-well plate were coated with 100 μl of 40 μg · ml−1 rat tail type I collagen in sterile PBS (Life Technologies, NY) at 4°C for 18 h. Then, wells were gently washed three times with PBS and blocked with 200 μl of PBS containing 5% bovine serum albumin (wt/vol) for 1.5 h at 37°C. The wells were washed again and then incubated for 1 h at 37°C with 50 μl of PBS (untreated) or with PBS containing rCBD monomers or rCBD amyloid fibers at the indicated concentrations (0.002 to 20 μg). Following washing, 100 μl of bacterial suspensions of S. mutans OMZ175 grown in BHI to an optical density at 600 nm of ≈0.35 was added to quadruplicate wells of each experimental condition and incubated for 3 h at 37°C in 5% CO2. Following washing, wells were air-dried and stained for 1 min with 200 μl of 0.05% crystal violet (wt/vol), washed with PBS containing 0.01% Tween 20 (vol/vol) to remove excess unbound crystal violet, and then treated with 200 μl of a 7% solution of acetic acid (vol/vol) to resuspend bound crystal violet. Absorbance was read at 575 nm on a Synergy H1 hybrid multimode reader (BioTek). As a background control, absorbance was measured in wells containing collagen only (no bacteria) and subtracted from test values. Readings were normalized based on CFU counts of the initial inocula plated on tryptic soy agar (TSA).
For ex vivo competitive inhibition assays, we followed the protocol previously published by our lab (31) with some modifications. First, 21 surgically extracted nonimpacted premolars, which are typically discarded after extraction, were obtained from the University of Florida’s dental clinic at the College of Dentistry (IRB201500591) and were cleaned of all soft tissue detritus and cut horizontally with a circular saw blade 17 mm from the crown, exposing a similar surface area of dentin on the root fragments. Prior to the assay, tooth fragments were rinsed clean by six rounds of 30 s of sonication in 4 ml of sterile water. Then, the tooth fragments were disinfected for 1 h in 70% ethanol and rehydrated overnight in 1× PBS. Surface disinfection was assessed by plating on TSA and incubation for 48 h at 37°C and 5% CO2 (vol/vol). Each tooth fragment was then submersed in 1 ml of sterile clarified human pooled saliva collected from healthy subjects (IRB201600877) for 30 min and incubated for 1 h at 37°C in 500 μl of 1× PBS containing 0.2, 2, or 20 μg of either rCBD monomer or rCBD purified amyloid fibers. Untreated tooth fragments served as a basis for comparison. Each sample group contained three replicates. Treated and untreated root sections were then submerged in 1 ml of a mid-log-phase culture (OD600, ≈0.5) of OMZ175 in BHI broth for 5 min. Loosely bound cells were removed by three subsequent washes of each root fragment in 14 ml of sterile 1× PBS at 60 rpm for 5 min. Strongly adherent cells were then removed by three rounds of sonication of 10 s each, in 1× PBS, with 30 s on ice between each round. Sonicants were plated on TSA, and the CFU were enumerated after 48 h of incubation at 37°C and 5% CO2 (vol/vol).

Statistical analyses.

One-way analysis of variance (ANOVA) with Tukey’s post hoc comparison test was performed for ThT fluorescence under different stirring conditions and tannic acid presence, as well as for counts of birefringent foci and competitive collagen-binding assays. A nonparametric t test was used to compare pairs of neutral and acidic pH ThT assays for each type of protein. For all assays, ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.


We thank Matthew Alzate, Ana Barran-Berdon, and Joshua Lovelace for technical assistance.
This work was supported by NIH/NIDCR R01 DE021789 to L.J.B and R01 DE022559 to J.A. and J.A.L.
We declare no conflicts of interest.


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Published In

cover image Applied and Environmental Microbiology
Applied and Environmental Microbiology
Volume 87Number 2114 October 2021
eLocator: e01149-21
Editor: Andrew J. McBain, University of Manchester
PubMed: 34406827


Received: 9 June 2021
Accepted: 15 August 2021
Accepted manuscript posted online: 18 August 2021
Published online: 14 October 2021


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  1. Cnm
  2. functional amyloid
  3. collagen-binding protein
  4. Streptococcus mutans
  5. biofilms
  6. oral microbiology
  7. surface proteins



Nicholas M. di Cologna
Department of Oral Biology, University of Florida, College of Dentistry, Gainesville, Florida, USA
Sandip Samaddar
Department of Oral Biology, University of Florida, College of Dentistry, Gainesville, Florida, USA
Carolina A. Valle
Department of Oral Biology, University of Florida, College of Dentistry, Gainesville, Florida, USA
Jonathan Vargas
Department of Oral Biology, University of Florida, College of Dentistry, Gainesville, Florida, USA
Alejandro Aviles-Reyes
Department of Oral Biology, University of Florida, College of Dentistry, Gainesville, Florida, USA
Joyce Morales
Department of Oral Biology, University of Florida, College of Dentistry, Gainesville, Florida, USA
Tridib Ganguly
Department of Oral Biology, University of Florida, College of Dentistry, Gainesville, Florida, USA
Roberta Pileggi
Department of Endodontics, University of Florida, College of Dentistry, Gainesville, Florida, USA
Department of Oral Biology, University of Florida, College of Dentistry, Gainesville, Florida, USA
Department of Oral Biology, University of Florida, College of Dentistry, Gainesville, Florida, USA
Department of Oral Biology, University of Florida, College of Dentistry, Gainesville, Florida, USA


Andrew J. McBain
University of Manchester

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