As a major etiological agent of human dental caries, Streptococcus mutans resides primarily in biofilms that form on the tooth surfaces, also known as dental plaque. In addition to caries, S. mutans is responsible for cases of infective endocarditis with a subset of strains being indirectly implicated with the onset of additional extraoral pathologies. During the past 4 decades, functional studies of S. mutans have focused on understanding the molecular mechanisms the organism employs to form robust biofilms on tooth surfaces, to rapidly metabolize a wide variety of carbohydrates obtained from the host diet, and to survive numerous (and frequent) environmental challenges encountered in oral biofilms. In these areas of research, S. mutans has served as a model organism for ground-breaking new discoveries that have, at times, challenged long-standing dogmas based on bacterial paradigms such as Escherichia coli and Bacillus subtilis . In addition to sections dedicated to carbohydrate metabolism, biofilm formation, and stress responses, this article discusses newer developments in S. mutans biology research, namely, how S. mutans interspecies and cross-kingdom interactions dictate the development and pathogenic potential of oral biofilms and how next-generation sequencing technologies have led to a much better understanding of the physiology and diversity of S. mutans as a species.

f 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.

Summary Bacteria belonging to the genus Streptococcus are the first inhabitants of the oral cavity which can be acquired right after birth and thus play an important role in the assembly of the oral microbiota. In this chapter, we will discuss the different oral environments inhabited by streptococci and the species that occupy each niche. Special attention is given to the taxonomy of Streptococcus as this genus is now divided into 8 distinct groups where oral species are found in 6 of them. Oral streptococci produce an arsenal of adhesive molecules that allow them to efficiently colonize different tissues in the mouth. Also, they have a remarkable ability to metabolize carbohydrates via fermentation thereby generating acids as byproducts. Excessive acidification of the oral environment by aciduric species such as Streptococcus mutans is directly associated with the development of dental caries. However, less acid-tolerant species such as Streptococcus salivarius and Streptococcus gordonii produce large amounts of alkali displaying and important role in the acid-base physiology of the oral cavity. Another important characteristic of certain oral streptococci is their ability to generate hydrogen peroxide that can inhibit the growth of S. mutans. Thus, oral streptococci can also be beneficial to the host by producing molecules that are inhibitory to pathogenic species. Lastly, commensal and pathogenic streptococci residing in the oral cavity can eventually gain access to the bloodstream and cause systemic infections such as infective endocarditis.

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Streptococcus mutans antigen I/II (AgI/II) is a cell surface-localized protein adhesin that interacts with salivary components within the salivary pellicle. AgI/II contributes to virulence and has been studied as an immunological and structural target, but a fundamental understanding of its underlying architecture has been lacking. Here we report a high-resolution (1.8 Å) crystal structure of the A 3 VP 1 fragment of S. mutans AgI/II that demonstrates a unique fibrillar form (155 Å) through the interaction of two noncontiguous regions in the primary sequence. The A 3 repeat of the alanine-rich domain adopts an extended α-helix that intertwines with the P 1 repeat polyproline type II (PPII) helix to form a highly extended stalk-like structure heretofore unseen in prokaryotic or eukaryotic protein structures. Velocity sedimentation studies indicate that fulllength AgI/II that contains three A/P repeats extends over 50 nanometers in length. Isothermal titration calorimetry revealed that the high-affinity association between the A 3 and P 1 helices is enthalpically driven. Two distinct binding sites on AgI/II to the host receptor salivary agglutinin (SAG) were identified by surface plasmon resonance (SPR). The current crystal structure reveals that AgI/II family proteins are extended fibrillar structures with the number of alanine-and proline-rich repeats determining their length.bacterial adhesion | dental caries | Streptococcus | x-ray crystallography | fibrous proteins S treptococcus mutans is the causative agent of human dental caries (1) and its protein adhesin antigen I/II (AgI/II) is a known target of protective immunity (2). AgI/II family molecules are expressed by numerous oral streptococci (3) and homologs have also been identified in the invasive pathogens Streptococcus pyogenes and Streptococcus agalactiae (4) (Fig. S1). In addition to mediating adhesion to the tooth surface (5), AgI/II influences biofilm formation (6), promotes collagen-dependent bacterial invasion of dentin (7), and mediates adherence to human epithelial cells (8). Elimination of AgI/II results in decreased virulence (9), but despite three decades of study, a mechanistic understanding of the functional properties of the molecule has been stymied by a lack of understanding of its structure.Originally identified as AgI/II (10) (also called P1, PAc, or SpaP), members of this protein family contain between 1310 and 1653 amino acids (aa) beginning with an amino-terminal signal motif that directs secretion, followed by the A, V, and P regions (Fig. 1A). The A region typically consists of 3-4 alanine-rich repeats (82 residues each) with 23-30% alanine content. The P region has 3-4 proline-rich repeats (39 residues each) with ∼35% proline content. Nested between the A and P repeats is a segment commonly referred to as the V or variable region, which contains within it a stretch of ∼100 amino acids where most of the sequence variation among S. mutans AgI/II molecules is clustered (11). The crystal structure of the V region adopts a globular β-s...

Interactions between salivary agglutinin and the adhesin P1 of Streptococcus mutans contribute to bacterial aggregation and mediate sucrose-independent adherence to tooth surfaces. We have examined biofilm formation by S. mutans UA159, and derivative strains carrying mutations affecting the localization or expression of P1, in the presence of fluid-phase or adsorbed saliva or salivary agglutinin preparations. Whole saliva-and salivary agglutinin-induced aggregation of S. mutans was adversely affected by the loss of P1 and sortase (SrtA) but not by the loss of trigger factor (RopA). Fluid-phase salivary agglutinin and, to a lesser extent, immobilized agglutinin inhibited biofilm development by S. mutans in the absence of sucrose, and whole saliva was more effective at decreasing biofilm formation than salivary agglutinin. Inhibition of biofilm development by salivary agglutinin was differently influenced by particular mutations, with the P1-deficient strain displaying a greater inhibition of biofilm development than the SrtA-or RopA-deficient strains. As expected, biofilm-forming capacities of all strains in the presence of salivary preparations were markedly enhanced in the presence of sucrose, although biofilm formation by the mutants was less efficient than that by the parental strain. Aeration strongly inhibited biofilm development, and the presence of salivary components did not restore biofilm formation in aerated conditions. The results disclose a potent ability of salivary constituents to moderate biofilm formation by S. mutans through P1-dependent and P1-independent pathways.

The signal recognition particle (SRP)-translocation pathway is conserved in all three domains of life and delivers membrane and secretory proteins to the cytoplasmic membrane or endoplasmic reticulum. We determined the requirement in the cariogenic oral pathogen Streptocococcus mutans of the three universally conserved elements of the SRP pathway: Ffh͞SRP54, scRNA, and FtsY͞SR␣. Previously, we reported that insertional interruption of S. mutans ffh was not lethal, but resulted in acid sensitivity. To test whether S. mutans could survive extensive disruption of the SRP pathway, single and double deletions of genes encoding Ffh, scRNA, and FtsY were generated. Without environmental stressors, all mutant strains were viable, but unlike the wild-type, none could initiate growth at pH 5.0 or in 3.5% NaCl. Survival of challenge with 0.3 mM H2O2 was also diminished without ffh. Members of the YidC͞Oxa1͞Alb3 family are also ubiquitous, involved in the translocation and assembly of membrane proteins, and have been identified in prokaryotes͞mitochondria͞chloroplasts. Two genes encoding YidC homologs, YidC1 and YidC2, are present in streptococcal genomes with both expressed in S. mutans. Deletion of YidC1 demonstrated no obvious phenotype. Elimination of YidC2 resulted in a stress-sensitive phenotype similar to SRP pathway mutants. Mutants lacking both YidC2 and SRP components were severely impaired and barely able to grow, even in the absence of environmental stress. Here, we report the dispensability of the cotranslational SRP protein translocation system in a bacterium. In S. mutans, this pathway contributes to protection against rapid environmental challenge and may overlap functionally with YidC2.protein translocation ͉ streptococcus ͉ Ffh ͉ FtsY ͉ membrane biogenesis

Dental caries is a common infectious disease associated with acidogenic and aciduric bacteria, including Streptococcus mutans. Organisms that cause cavities form recalcitrant biofilms, generate acids from dietary sugars and tolerate acid end products. It has recently been recognized that micro-organisms can produce functional amyloids that are integral to biofilm development. We now show that the S. mutans cell-surface-localized adhesin P1 (antigen I/II, PAc) is an amyloid-forming protein. This conclusion is based on the defining properties of amyloids, including binding by the amyloidophilic dyes Congo red (CR) and Thioflavin T (ThT), visualization of amyloid fibres by transmission electron microscopy and the green birefringent properties of CR-stained protein aggregates when viewed under cross-polarized light. We provide evidence that amyloid is present in human dental plaque and is produced by both laboratory strains and clinical isolates of S. mutans. We provide further evidence that amyloid formation is not limited to P1, since bacterial colonies without this adhesin demonstrate residual green birefringence. However, S. mutans lacking sortase, the transpeptidase enzyme that mediates the covalent linkage of its substrates to the cell-wall peptidoglycan, including P1 and five other proteins, is not birefringent when stained with CR and does not form biofilms. Biofilm formation is inhibited when S. mutans is cultured in the presence of known inhibitors of amyloid fibrillization, including CR, Thioflavin S and epigallocatechin-3-gallate, which also inhibited ThT uptake by S. mutans extracellular proteins. Taken together, these results indicate that S. mutans is an amyloid-forming organism and suggest that amyloidogenesis contributes to biofilm formation by this oral microbe. INTRODUCTIONAmyloid represents a fibrous cross b-sheet quaternary structure comprised of ordered aggregates of peptides or proteins that demonstrate common biophysical properties (Nilsson, 2004). While amyloid formation has been extensively studied in the context of pathological states, for example Alzheimer's disease, the concept of functional amyloid has only recently emerged (Epstein & Chapman, 2008;Fowler et al., 2007;Gebbink et al., 2005;Maury, 2009a;. In bacteria, amyloid formation may be the rule rather than the exception . There are now numerous examples of surface-localized microbial proteins that aggregate and assemble into functional amyloid fibrils.Amyloid fibres have a tensile strength comparable to steel (Smith et al., 2006). The unique physical and morphological properties of amyloid and the fact that it can be formed in a regulated manner suggest that it probably represents a common quaternary structure that is widespread in biology. Therefore, it does not always represent a misfolded protein structure, but instead represents a lowenergy quaternary structure that can occur in the context of function or disease (Fowler et al., 2007). Amyloid is a non-covalent oligomer of extended intermolecular hydrogen-bonded b-sheets that s...