BrpA is involved in regulation of cell envelope stress responses in Streptococcus mutans.

Previous studies have shown that BrpA plays a major role in acid and oxidative stress tolerance and biofilm formation by Streptococcus mutans. Mutant strains lacking BrpA also display increased autolysis and decreased viability, suggesting a role for BrpA in cell envelope integrity. In this study, we examined the impact of BrpA deficiency on cell envelope stresses induced by envelope-active antimicrobials. Compared to the wild-type strain UA159, the BrpA-deficient mutant (TW14D) was significantly more susceptible to antimicrobial agents, especially lipid II inhibitors. Several genes involved in peptidoglycan synthesis were identified by DNA microarray analysis as downregulated in TW14D. Luciferase reporter gene fusion assays also revealed that expression of brpA is regulated in response to environmental conditions and stresses induced by exposure to subinhibitory concentrations of cell envelope antimicrobials. In a Galleria mellonella (wax worm) model, BrpA deficiency was shown to diminish the virulence of S. mutans OMZ175, which, unlike S. mutans UA159, efficiently kills the worms. Collectively, these results suggest that BrpA plays a role in the regulation of cell envelope integrity and that deficiency of BrpA adversely affects the fitness and diminishes the virulence of OMZ175, a highly invasive strain of S. mutans.

Zinc import mediated by AdcABC is critical for colonization of the dental biofilm by Streptococcus mutans in an animal model.

Trace metals are essential to all domains of life but toxic when found at high concentrations. Although the importance of iron in host-pathogen interactions is firmly established, contemporary studies indicate that other trace metals, including manganese and zinc, are also critical to the infectious process. In this study, we sought to identify and characterize the zinc uptake system(s) of Streptococcus mutans, a keystone pathogen in dental caries and a causative agent of bacterial endocarditis. Different than other pathogenic bacteria, including several streptococci, that encode multiple zinc import systems, bioinformatic analysis indicated that the S. mutans core genome encodes a single, highly conserved, zinc importer commonly known as AdcABC. Inactivation of the genes coding for the metal-binding AdcA (ΔadcA) or both AdcC ATPase and AdcB permease (ΔadcCB) severely impaired the ability of S. mutans to grow under zinc-depleted conditions. Intracellular metal quantifications revealed that both mutants accumulated less zinc when grown in the presence of a subinhibitory concentration of a zinc-specific chelator. Notably, the ΔadcCB strain displayed a severe colonization defect in a rat oral infection model. Both Δadc strains were hypersensitive to high concentrations of manganese, showed reduced peroxide tolerance, and formed less biofilm in sucrose-containing media when cultivated in the presence of the lowest amount of zinc that support their growth, but not when zinc was supplied in excess. Collectively, this study identifies AdcABC as the major high affinity zinc importer of S. mutans and provides preliminary evidence that zinc is a growth-limiting factor within the dental biofilm.

Association of Candida albicans and Cbp+ Streptococcus mutans with early childhood caries recurrence.

Early childhood caries (ECC) recurrence occurs in approximately 40% of treated cases within one year. The association of Streptococcus mutans and Candida albicans with the onset of ECC is well known. Also, S. mutans strains harboring collagen-binding proteins (Cbps) avidly bind to collagen-rich dentin and are linked to increased caries risk. Here, we investigated the presence of Cbp+ S. mutans and C. albicans in saliva and dental plaque of children with varying caries statuses, and their salivary microbiome. In this cross-sectional study, 143 children who were caries-free (n = 73), treated for ECC with no signs of recurrence after 6 months (n = 45), or treated for ECC and experiencing recurrence within 6 months following treatment (n = 25) were enrolled. Co-infection with C. albicans and S. mutans, especially Cbp+ S. mutans, was strongly associated with caries recurrence. Subjects of the recurrence group infected with Cbp+ S. mutans showed a greater burden of Candida spp. and of Mutans streptococci in dentin than those infected with Cbp- strains. Salivary microbiome analysis revealed that Streptococcus parasanguinis was overrepresented in the caries recurrence group. Our findings indicate that Cbp+ S. mutans and C. albicans are intimately associated with caries recurrence, contributing to the establishment of recalcitrant biofilms.

Inactivation of VicK affects acid production and acid survival of Streptococcus mutans.

The regulation of acid production in and the tolerance to low pH of the cariogenic bacterium Streptococcus mutans have garnered considerable attention since both of these properties contribute substantially to the virulence of this organism. Frequent or prolonged exposure to acid end products, mainly lactic acid, that are present following the consumption of dietary sugars erodes the dental enamel, thereby initiating dental caries. Here we report the involvement of the S. mutans VicK sensor kinase in both the acidogenicity and the aciduricity of this bacterium. When cultures were supplemented with glucose, the glycolytic rate of a VicK null mutant was significantly decreased compared to the glycolytic rate of the wild type (P < 0.05), suggesting that there was impaired acid production. Not surprisingly, the VicK deletion mutant produced less lactic acid, while an acid tolerance response assay revealed that loss of VicK significantly enhanced the survival of S. mutans (P < 0.05). Compared to the survival rates of the wild type, the survival rates of the VicK-deficient mutant were drastically increased when cultures were grown at pH 3.5 with or without preexposure to a signal pH (pH 5.5). Global transcriptional analysis using DNA microarrays and S. mutans wild-type UA159 and VicK deletion mutant strains grown at neutral and low pH values revealed that loss of VicK significantly affected expression of 89 transcripts more than twofold at pH 5.5 (P < 0.001). The affected transcripts included genes with putative functions in transport and maintenance of cell membrane integrity. While our results provide insight into the acid-inducible regulon of S. mutans, here we imply a novel role for VicK in regulating intracellular pH homeostasis in S. mutans.

Invasion of human coronary artery endothelial cells by Streptococcus mutans OMZ175.

INTRODUCTION: Dissemination of oral bacteria into the bloodstream has been associated with eating, oral hygiene, and dental procedures; including tooth extraction, endodontic treatment, and periodontal surgery. Recently, studies identified Streptococcus mutans, the primary etiological agent of dental caries, as the most prevalent bacterial species found in clinical samples from patients who underwent heart valve and atheromatous plaque surgery. METHODS: By using antibiotic protection assays, we tested the capacity of 14 strains of S. mutans to invade primary human coronary artery endothelial cells (HCAEC). RESULTS: Serotype e strain B14 and serotype f strain OMZ175 of S. mutans were able to efficiently invade HCAEC. Among the tested strains, serotype f S. mutans OMZ175 was the most invasive, whereas strains of serotype c S. mutans, the most prevalent serotype in dental plaque, were not invasive. Based on its high invasion rate, we further investigated the invasive properties of serotype f OMZ175. Using transmission electron microscopy and antibiotic protection assays we demonstrate that S. mutans OMZ175 is capable of attaching to the HCAEC surface, entering the cells and surviving in HCAEC for at least 29 h. DISCUSSION: Our findings highlight a potential role for S. mutans in the pathogenesis of certain cardiovascular diseases.

The Biology of Streptococcus mutans.

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.

Whole genome sequence and phenotypic characterization of a Cbm+ serotype e strain of Streptococcus mutans.

We report the whole genome sequence of the serotype e Cbm+ strain LAR01 of Streptococcus mutans, a dental pathogen frequently associated with extra-oral infections. The LAR01 genome is a single circular chromosome of 2.1 Mb with a GC content of 36.96%. The genome contains 15 phosphotransferase system gene clusters, seven cell wall-anchored (LPxTG) proteins, all genes required for the development of natural competence and genes coding for mutacins VI and K8. Interestingly, the cbm gene is genetically linked to a putative type VII secretion system that has been found in Mycobacteria and few other Gram-positive bacteria. When compared with the UA159 type strain, phenotypic characterization of LAR01 revealed increased biofilm formation in the presence of either glucose or sucrose but similar abilities to withstand acid and oxidative stresses. LAR01 was unable to inhibit the growth of Strpetococcus gordonii, which is consistent with the genomic data that indicate absence of mutacins that can kill mitis streptococci. On the other hand, LAR01 effectively inhibited growth of other S. mutans strains, suggesting that it may be specialized to outcompete strains from its own species. In vitro and in vivo studies using mutational and heterologous expression approaches revealed that Cbm is a virulence factor of S. mutans by mediating binding to extracellular matrix proteins and intracellular invasion. Collectively, the whole genome sequence analysis and phenotypic characterization of LAR01 provides new insights on the virulence properties of S. mutans and grants further opportunities to understand the genomic fluidity of this important human pathogen.

Biology of Oral Streptococci.

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 article, we discuss the different oral environments inhabited by streptococci and the species that occupy each niche. Special attention is given to the taxonomy of Streptococcus, because this genus is now divided into eight distinct groups, and oral species are found in six 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 an 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.

Collagen-binding proteins of Streptococcus mutans and related streptococci.

The ability of Streptococcus mutans to interact with collagen through the expression of collagen-binding proteins (CBPs) bestows this oral pathogen with an alternative to the sucrose-dependent mechanism of colonization classically attributed to caries development. Based on the abundance and distribution of collagen throughout the human body, stringent adherence to this molecule grants S. mutans with the opportunity to establish infection at different host sites. Surface proteins, such as SpaP, WapA, Cnm and Cbm, have been shown to bind collagen in vitro, and it has been suggested that these molecules play a role in colonization of oral and extra-oral tissues. However, robust collagen binding is not achieved by all strains of S. mutans, particularly those that lack Cnm or Cbm. These observations merit careful dissection of the contribution from these different CBPs towards tissue colonization and virulence. In this review, we will discuss the current understanding of mechanisms used by S. mutans and related streptococci to colonize collagenous tissues, and the possible contribution of CBPs to infections in different sites of the host.

Inactivation of the spxA1 or spxA2 gene of Streptococcus mutans decreases virulence in the rat caries model.

In oral biofilms, the major environmental challenges encountered by Streptococcus mutans are acid and oxidative stresses. Previously, we showed that the transcriptional regulators SpxA1 and SpxA2 are involved in general stress survival of S. mutans with SpxA1 playing a primary role in activation of antioxidant and detoxification strategies whereas SpxA2 serves as a back up activator of oxidative stress genes. We have also found that spxA1 mutant strains (∆spxA1 and ∆spxA1∆spxA2) are outcompeted by peroxigenic oral streptococci in vitro and have impaired abilities to colonize the teeth of rats fed a highly cariogenic diet. Here, we show that the Spx proteins can also exert regulatory roles in the expression of additional virulence attributes of S. mutans. Competence activation is significantly impaired in Δspx strains and the production of mutacin IV and V is virtually abolished in ΔspxA1 strains. Unexpectedly, the ∆spxA2 strain showed increased production of glucans from sucrose, without affecting the total amount of bacteria within biofilms when compared with the parent strain. By using the rat caries model, we showed that the capacity of the ΔspxA1 and ΔspxA2 strains to cause caries on smooth tooth surfaces is significantly impaired. The ∆spxA2 strain also formed fewer lesions on sulcal surfaces. This report reveals that global regulation via Spx contributes to the cariogenic potential of S. mutans and highlights that animal models are essential in the characterization of bacterial traits implicated in virulence.

Transcriptional profile of glucose-shocked and acid-adapted strains of Streptococcus mutans.

The aciduricity of Streptococcus mutans is an important virulence factor of the organism, required to both out-compete commensal oral microorganisms and cause dental caries. In this study, we monitored transcriptional changes that occurred as a continuous culture of either an acid-tolerant strain (UA159) or an acid-sensitive strain (fabM::Erm) moved from steady-state growth at neutral pH, experienced glucose-shock and acidification of the culture, and transitioned to steady-state growth at low pH. Hence, the timing of elements of the acid tolerance response (ATR) could be observed and categorized as acute vs. adaptive ATR mechanisms. Modulation of branched chain amino acid biosynthesis, DNA/protein repair mechanisms, reactive oxygen species metabolizers and phosphoenolpyruvate:phosphotransferase systems occurred in the initial acute phase, immediately following glucose-shock, while upregulation of F1 F0 -ATPase did not occur until the adaptive phase, after steady-state growth had been re-established. In addition to the archetypal ATR pathways mentioned above, glucose-shock led to differential expression of genes suggesting a re-routing of resources away from the synthesis of fatty acids and proteins, and towards synthesis of purines, pyrimidines and amino acids. These adjustments were largely transient, as upon establishment of steady-state growth at acidic pH, transcripts returned to basal expression levels. During growth at steady-state pH 7, fabM::Erm had a transcriptional profile analogous to that of UA159 during glucose-shock, indicating that even during growth in rich media at neutral pH, the cells were stressed. These results, coupled with a recently established collection of deletion strains, provide a starting point for elucidation of the acid tolerance response in S. mutans.

Yeast-Yeast Interactions in Guava and Tomato Fruits.

The host specificity of yeast-yeast interactions was investigated for two yeast types, represented by six pairs of Pichia membranifaciens clade yeasts (Pichia membranifaciens or Issatchenkia occidentalis) with apiculate yeasts (Kloeckera apis, Kloeckera africana, or Saccharomycodes ludwigii), commonly found in fruits. Competitive interactions between the two types were detected in both ripe tomato and guava fruit pulp. The differences in growth rates and carrying capacities depended on fruit type (host) and culture conditions (monocultures versus bicultures). These differences were probably due to nutrient composition of each fruit. Pichia membranifaciens did not show host dependent responses, but the apiculate yeasts and Issatchenkia occidentalis did. Depending on yeast strain and culture conditions (i.e., monoculture or biculture), carbon, nitrogen, and vitamins were investigated as potential limiting growth factors in guava fruit. Both singular and multiple limiting nutrients were implicated.

Cnm is a major virulence factor of invasive Streptococcus mutans and part of a conserved three-gene locus.

Cnm, a collagen- and laminin-binding protein present in a subset of Streptococcus mutans strains, mediates binding to extracellular matrices (ECM), intracellular invasion and virulence in the Galleria mellonella model. Antibodies raised against Cnm were used to confirm expression and the cell surface localization of Cnm in the highly invasive OMZ175 strain. Sequence analysis identified two additional genes (cnaB and cbpA) encoding putative surface proteins immediately upstream of cnm. Inactivation of cnaB and cbpA in OMZ175, individually or in combination, did not decrease the ability of this highly invasive and virulent strain to bind to different ECM proteins, invade human coronary artery endothelial cells (HCAEC), or kill G. mellonella. Similarly, expression of cnaB and cbpA in the cnm(-) strain UA159 revealed that these genes did not enhance Cnm-related phenotypes. However, integration of cnm in the chromosome of UA159 significantly increased its ability to bind to collagen and laminin, invade HCAEC, and kill G. mellonella. Moreover, the presence of antibodies against Cnm nearly abolished the ability of OMZ175 to bind to collagen and laminin and invade HCAEC, and significantly protected G. mellonella against OMZ175 infection. We concluded that neither CnaB nor CbpA is necessary for the expression of Cnm-related traits. We also provided definitive evidence that Cnm is an important virulence factor and a suitable target for the development of novel preventive and therapeutic strategies to combat invasive S. mutans strains.

Transcriptome analysis of LuxS-deficient Streptococcus mutans grown in biofilms.

We previously reported that LuxS in Streptococcus mutans is involved in stress tolerance and biofilm formation. In this study, flowcells and confocal laser scanning microscopy were used to further examine the effects of LuxS-deficiency on biofilm formation. Similar to the wild-type strain (UA159), a strain deficient in LuxS (TW26D) bound efficiently to the flowcells and formed microcolonies 4 h after inoculation. Unlike UA159, which accumulated and formed compact, evenly distributed biofilms after 28 h, TW26D showed only loose, sporadic, thin biofilms. DNA microarray analysis revealed alterations in transcription of more than 60 genes in TW26D biofilms by at least 1.5-fold (P < 0.001). Among the upregulated genes were those for sugar-specific enzymes II of the phosphotransferase (PTS) system and the atp operon, which codes for the proton-pumping F-ATPase. Of the downregulated genes, several encode proteins with putative functions in DNA repair. Mutation of selected genes caused severe defects in the ability of the mutants to tolerate low pH and oxidative stress. These results provide additional proof that LuxS-deficiency causes global alterations in the expression of genes central to biofilm formation and virulence of S. mutans, including those involved in energy metabolism, DNA repair and stress tolerance.

Characterization of the Streptococcus sobrinus acid-stress response by interspecies microarrays and proteomics.

Streptococcus mutans and Streptococcus sobrinus are considered the primary organisms responsible for human dental caries. The ability to generate acids and to adapt to low pH conditions is directly associated with the cariogenic potential of these bacteria. To survive acidic conditions, both species have been shown to mount an acid-tolerance response (ATR). However, previous characterization of the S. sobrinus ATR identified critical differences in the mechanisms of acid adaptation between S. mutans and S. sobrinus. Here, interspecies microarray and proteomic approaches were used to identify novel, previously unrecognized genes and pathways that participate in the S. sobrinus acid-stress response. The results revealed that, among other things, metabolic alterations that enhance energy generation and upregulation of the malolactic fermentation enzyme activity constitute important acid-resistance properties in S. sobrinus. Some of these acid adaptive traits are shared by S. mutans and might be considered optimal targets for therapeutic treatments designed to control dental caries.

Fluoride and organic weak acids as respiration inhibitors for oral streptococci in acidified environments.

Oxygen metabolism (respiration) of Streptococcus mutans GS-5 involving NADH oxidases, mainly of the H(2)O-producing type, was found to be acid sensitive, as was NADH oxidase activity of cell extracts. Respiration of intact cells in acidified media was also highly sensitive to fluoride, with a 50% inhibitory concentration of about 0.02 mM at pH 4. In contrast, NADH oxidases in cell extracts were fluoride insensitive. Fluoride inhibition of respiration of intact cells was related to weak-acid effects leading to enhanced proton permeability of cells, cytoplasmic acidification and resultant acid inhibition of NADH oxidases and glycolysis. Organic weak acids, such as indomethacin and benzoate, were also effective inhibitors. H(2)O(2) production by intact cells of Streptococcus sanguis NCTC 10904, a peroxide producer, was similarly inhibited by fluoride or organic weak acids in acidified media. Thus, weak acids act as respiratory inhibitors for oral streptococci indirectly by acidifying the cytoplasm rather than acting as direct inhibitors of NADH oxidases.

The incidence of killer activity and extracellular proteases in tropical yeast communities.

The presence of killer and proteolytic yeasts was studied among 944 isolates representing 105 species from tropical yeast communities. We found 13 killer toxin producing species, with Pichia kluyveri being the most frequent. Other killer yeast isolates were Candida apis, Candida bombicola, Candida fructus, Candida krusei, Candida sorbosa, Hanseniaspora uvarum, Issatchenkia occidentalis, Kloeckera apis, Kluyveromyces marxianus, Pichia membranaefaciens, Pichia ohmeri-like, and Sporobolomyces roseus. The communities from which killer yeasts were isolated had strains sensitive to them, and there were interspecific and intraspecific differences in the spectra of their killer activities. Pichia kluyveri had the broadest spectra of activity against sensitive isolates, and it apparently produced different toxins. The coexistence of sensitive and killer yeasts using the same substrate suggests that there is spatial separation in microhabitats or temporal separation in different stages of successions. Basidiomycetous yeasts were more frequently proteolytic than ascomycetous yeasts. Extracellular proteases could be important for the yeasts to have access to more nitrogen nutrients and obtain a better balance with available carbon sources.

Effects of apigenin and tt-farnesol on glucosyltransferase activity, biofilm viability and caries development in rats.

Propolis, a resinous hive product secreted by Apis mellifera bees, has been shown to reduce the incidence of dental caries in rats. Several compounds, mainly polyphenolics, have been identified in propolis. Apigenin and tt-farnesol demonstrated biological activity against mutans streptococci. We determined here their effects, alone or in combination, on glucosyltransferase activity, biofilm viability, and development of caries in rats. Sprague-Dawley rats were infected with Streptococcus sobrinus 6715 and treated topically twice daily as follows: (1) tt-farnesol, (2) apigenin, (3) vehicle control, (4) fluoride, (5) apigenin +tt-farnesol, and (6) chlorhexidine. Apigenin (1.33 mM) inhibited the activity of glucosyltransferases in solution (90-95%) and on the surface of saliva-coated hydroxyapatite beads (35-58%); it was devoid of antibacterial activity. tt-Farnesol (1.33 mM) showed modest antibacterial activity against biofilms and its effects on glucosyltransferases were minimal. The incidence of smooth-surface caries was significantly reduced by apigenin +tt-farnesol (60%), fluoride (70%), and chlorhexidine (72%) treatments compared to control (P < 0.05).