Spatiotemporal monitoring of a periodontal multispecies biofilm model: demonstration of prebiotic treatment responses.

Biofilms are complex polymicrobial communities which are often associated with human infections such as the oral disease periodontitis. Studying these complex communities under controlled conditions requires in vitro biofilm model systems that mimic the natural environment as close as possible. This study established a multispecies periodontal model in the drip flow biofilm reactor in order to mimic the continuous flow of nutrients at the air-liquid interface in the oral cavity. The design is engineered to enable real-time characterization. A community of five bacteria, Streptococcus gordonii-GFPmut3*, Streptococcus oralis-GFPmut3*, Streptococcus sanguinis-pVMCherry, Fusobacterium nucleatum, and Porphyromonas gingivalis-SNAP26 is visualized using two distinct fluorescent proteins and the SNAP-tag. The biofilm in the reactor develops into a heterogeneous, spatially uniform, dense, and metabolically active biofilm with relative cell abundances similar to those in a healthy individual. Metabolic activity, structural features, and bacterial composition of the biofilm remain stable from 3 to 6 days. As a proof of concept for our periodontal model, the 3 days developed biofilm is exposed to a prebiotic treatment with L-arginine. Multifaceted effects of L-arginine on the oral biofilm were validated by this model setup. L-arginine showed to inhibit growth and incorporation of the pathogenic species and to reduce biofilm thickness and volume. Additionally, L-arginine is metabolized by Streptococcus gordonii-GFPmut3* and Streptococcus sanguinis-pVMCherry, producing high levels of ornithine and ammonium in the biofilm. In conclusion, our drip flow reactor setup is promising in studying spatiotemporal behavior of a multispecies periodontal community.ImportancePeriodontitis is a multifactorial chronic inflammatory disease in the oral cavity associated with the accumulation of microorganisms in a biofilm. Not the presence of the biofilm as such, but changes in the microbiota (i.e., dysbiosis) drive the development of periodontitis, resulting in the destruction of tooth-supporting tissues. In this respect, novel treatment approaches focus on maintaining the health-associated homeostasis of the resident oral microbiota. To get insight in dynamic biofilm responses, our research presents the establishment of a periodontal biofilm model including Streptococcus gordonii, Streptococcus oralis, Streptococcus sanguinis, Fusobacterium nucleatum, and Porphyromonas gingivalis. The added value of the model setup is the combination of simulating continuously changing natural mouth conditions with spatiotemporal biofilm profiling using non-destructive characterization tools. These applications are limited for periodontal biofilm research and would contribute in understanding treatment mechanisms, short- or long-term exposure effects, the adaptation potential of the biofilm and thus treatment strategies.

Quantification of Extracellular DNA Network Abundance and Architecture within Streptococcus gordonii Biofilms Reveals Modulatory Factors.

Extracellular DNA (eDNA) is an important component of biofilm matrix that serves to maintain biofilm structural integrity, promotes genetic exchange within the biofilm, and provides protection against antimicrobial compounds. Advances in microscopy techniques have provided evidence of the cobweb- or lattice-like structures of eDNA within biofilms from a range of environmental niches. However, methods to reliably assess the abundance and architecture of eDNA remain lacking. This study aimed to address this gap by development of a novel, high-throughput image acquisition and analysis platform for assessment of eDNA networks in situ within biofilms. Utilizing Streptococcus gordonii as the model, the capacity for this imaging system to reliably detect eDNA networks and monitor changes in abundance and architecture (e.g., strand length and branch number) was verified. Evidence was provided of a synergy between glucans and eDNA matrices, while it was revealed that surface-bound nuclease SsnA could modify these eDNA structures under conditions permissive for enzymatic activity. Moreover, cross talk between the competence and hexaheptapeptide permease systems was shown to regulate eDNA release by S. gordonii. This novel imaging system can be applied across the wider field of biofilm research, with potential to significantly advance interrogation of the mechanisms by which the eDNA network architecture develops, how it can influence biofilm properties, and how it may be targeted for therapeutic benefit. IMPORTANCE Extracellular DNA (eDNA) is critical for maintaining the structural integrity of many microbial biofilms, making it an attractive target for the management of biofilms. However, our knowledge and targeting of eDNA are currently hindered by a lack of tools for the quantitative assessment of eDNA networks within biofilms. Here, we demonstrate use of a novel image acquisition and analysis platform with the capacity to reliably monitor the abundance and architecture of eDNA networks. Application of this tool to Streptococcus gordonii biofilms has provided new insights into how eDNA networks are stabilized within the biofilm and the pathways that can regulate eDNA release. This highlights how exploitation of this novel imaging system across the wider field of biofilm research has potential to significantly advance interrogation of the mechanisms by which the eDNA network architecture develops, how it can influence biofilm properties, and how it may be targeted for therapeutic benefit.

Streptococcus gordonii programs epithelial cells to resist ZEB2 induction by Porphyromonas gingivalis.

The polymicrobial microbiome of the oral cavity is a direct precursor of periodontal diseases, and changes in microhabitat or shifts in microbial composition may also be linked to oral squamous cell carcinoma. Dysbiotic oral epithelial responses provoked by individual organisms, and which underlie these diseases, are widely studied. However, organisms may influence community partner species through manipulation of epithelial cell responses, an aspect of the host microbiome interaction that is poorly understood. We report here that Porphyromonas gingivalis, a keystone periodontal pathogen, can up-regulate expression of ZEB2, a transcription factor which controls epithelial-mesenchymal transition and inflammatory responses. ZEB2 regulation by P. gingivalis was mediated through pathways involving ß-catenin and FOXO1. Among the community partners of P. gingivalis, Streptococcus gordonii was capable of antagonizing ZEB2 expression. Mechanistically, S. gordonii suppressed FOXO1 by activating the TAK1-NLK negative regulatory pathway, even in the presence of P. gingivalis Collectively, these results establish S. gordonii as homeostatic commensal, capable of mitigating the activity of a more pathogenic organism through modulation of host signaling.

Uncovering Roles of Streptococcus gordonii SrtA-Processed Proteins in the Biofilm Lifestyle.

Streptococcus gordonii is a commensal oral organism. Harmless in the oral cavity, S. gordonii is an opportunistic pathogen. S. gordonii adheres to body surfaces using surface adhesive proteins (adhesins), which are critical to subsequent formation of biofilm communities. As in most Gram-positive bacteria, S. gordonii surface proteins containing the C-terminal LPXTG motif cleavage sequence are processed by sortase A (SrtA) to become covalently attached to the cell wall. To characterize the functional diversity and redundancy in the family of SrtA-processed proteins, an S. gordonii DL1 markerless deletion mutant library was constructed of each of the 26 putative SrtA-processed proteins. Each library member was evaluated for growth in rich medium, biofilm formation on plastic, saliva and salivary fractions, cell surface hydrophobicity (CSH), hemagglutination, and integration into an ex vivo plaque biofilm community. Library members were compared to the non-SrtA-processed adhesins AbpA and AbpB. While no major growth differences in rich medium were observed, many S. gordonii LPXTG/A proteins impacted biofilm formation on one or more of the substrates. Several mutants showed significant differences in hemagglutination, hydrophobicity, or fitness in the ex vivo plaque model. From the identification of redundant and unique functions in these in vitro and ex vivo systems, functional stratification among the LPXTG/A proteins is apparent.IMPORTANCES. gordonii interactions with its environment depend on the complement of cell wall proteins. A subset of these cell wall proteins requires processing by the enzyme sortase A (SrtA). The identification of SrtA-processed proteins and their functional characterization will help the community to better understand how S. gordonii engages with its surroundings, including other microbes, integrates into the plaque community, adheres to the tooth surface, and hematogenously disseminates to cause blood-borne infections. This study identified 26 putative SrtA-processed proteins through creation of a markerless deletion mutant library. The library was subject to functional screens that were chosen to better understand key aspects of S. gordonii physiology and pathogenesis.

Biofilm Interactions of Candida albicans and Mitis Group Streptococci in a Titanium-Mucosal Interface Model.

Streptococci from the mitis group (represented mainly by Streptococcus mitis, Streptococcus oralis, Streptococcus sanguinis, and Streptococcus gordonii) form robust biofilms with Candida albicans in different experimental models. These microorganisms have been found in polymicrobial biofilms forming on titanium biomaterial surfaces in humans with peri-implant disease. The purpose of this work was to study mutualistic interactions in biofilms forming on titanium and their effect on the adjacent mucosa, using a relevant infection model. Single and mixed biofilms of C. albicans and each Streptococcus species were grown on titanium disks. Bacterial and fungal biovolume and biomass were quantified in these biofilms. Organotypic mucosal constructs were exposed to preformed titanium surface biofilms to test their effect on secretion of proinflammatory cytokines and cell damage. C. albicans promoted bacterial biofilms of all mitis Streptococcus species on titanium surfaces. This relationship was mutualistic since all bacterial species upregulated the efg1 hypha-associated gene in C. albicans Mixed biofilms caused increased tissue damage but did not increase proinflammatory cytokine responses compared to biofilms comprising Candida alone. Interestingly, spent culture medium from tissues exposed to titanium biofilms suppressed Candida growth on titanium surfaces.IMPORTANCE Our findings provide new insights into the cross-kingdom interaction between C. albicans and Streptococcus species representative of the mitis group. These microorganisms colonize titanium-based dental implant materials, but little is known about their ability to cause inflammation and damage of the adjacent mucosal tissues. Using an in vitro biomaterial-mucosal interface infection model, we showed that mixed biofilms of each species with C. albicans enhance tissue damage. One possible mechanism for this effect is the increased fungal hypha-associated virulence gene expression we observed in mixed biofilms with these species. Interestingly, we also found that the interaction of multispecies biofilms with organotypic mucosal surfaces led to the release of growth-suppressing mediators of Candida, which may represent a homeostatic defense mechanism of the oral mucosa against fungal overgrowth. Thus, our findings provide novel insights into biofilms on biomaterials that may play an important role in the pathogenesis of mucosal infections around titanium implants.

Amino Sugars Reshape Interactions between Streptococcus mutans and Streptococcus gordonii.

Amino sugars, particularly glucosamine (GlcN) and N-acetylglucosamine (GlcNAc), are abundant carbon and nitrogen sources supplied in host secretions and in the diet to the biofilms colonizing the human oral cavity. Evidence is emerging that these amino sugars provide ecological advantages to beneficial commensals over oral pathogens and pathobionts. Here, we performed transcriptome analysis on Streptococcus mutans and Streptococcus gordonii growing in single-species or dual-species cultures with glucose, GlcN, or GlcNAc as the primary carbohydrate source. Compared to glucose, GlcN caused drastic transcriptomic shifts in each species of bacteria when it was cultured alone. Likewise, cocultivation in the presence of GlcN yielded transcriptomic profiles that were dramatically different from the single-species results from GlcN-grown cells. In contrast, GlcNAc elicited only minor changes in the transcriptome of either organism in single- and dual-species cultures. Interestingly, genes involved in pyruvate metabolism were among the most significantly affected by GlcN in both species, and these changes were consistent with measurements of pyruvate in culture supernatants. Differing from what was found in a previous report, growth of S. mutans alone with GlcN inhibited the expression of multiple operons required for mutacin production. Cocultivation with S. gordonii consistently increased the expression of two manganese transporter operons (slo and mntH) and decreased expression of mutacin genes in S. mutans Conversely, S. gordonii appeared to be less affected by the presence of S. mutans but did show increases in genes for biosynthetic processes in the cocultures. In conclusion, amino sugars profoundly alter the interactions between pathogenic and commensal streptococci by reprogramming central metabolism.IMPORTANCE Carbohydrate metabolism is central to the development of dental caries. A variety of sugars available to dental microorganisms influence the development of caries by affecting the physiology, ecology, and pathogenic potential of tooth biofilms. Using two well-characterized oral bacteria, one pathogen (Streptococcus mutans) and one commensal (Streptococcus gordonii), in an RNA deep-sequencing analysis, we studied the impact of two abundant amino sugars on bacterial gene expression and interspecies interactions. The results indicated large-scale remodeling of gene expression induced by GlcN in particular, affecting bacterial energy generation, acid production, protein synthesis, and release of antimicrobial molecules. Our study provides novel insights into how amino sugars modify bacterial behavior, information that will be valuable in the design of new technologies to detect and prevent oral infectious diseases.

Porphyromonas gingivalis HmuY and Streptococcus gordonii GAPDH-Novel Heme Acquisition Strategy in the Oral Microbiome.

The oral cavity of healthy individuals is inhabited by commensals, with species of Streptococcus being the most abundant and prevalent in sites not affected by periodontal diseases. The development of chronic periodontitis is linked with the environmental shift in the oral microbiome, leading to the domination of periodontopathogens. Structure-function studies showed that Streptococcus gordonii employs a "moonlighting" protein glyceraldehyde-3-phosphate dehydrogenase (SgGAPDH) to bind heme, thus forming a heme reservoir for exchange with other proteins. Secreted or surface-associated SgGAPDH coordinates Fe(III)heme using His43. Hemophore-like heme-binding proteins of Porphyromonas gingivalis (HmuY), Prevotella intermedia (PinO) and Tannerella forsythia (Tfo) sequester heme complexed to SgGAPDH. Co-culturing of P. gingivalis with S. gordonii results in increased hmuY gene expression, indicating that HmuY might be required for efficient inter-bacterial interactions. In contrast to the DhmuY mutant strain, the wild type strain acquires heme and forms deeper biofilm structures on blood agar plates pre-grown with S. gordonii. Therefore, our novel paradigm of heme acquisition used by P. gingivalis appears to extend to co-infections with other oral bacteria and offers a mechanism for the ability of periodontopathogens to obtain sufficient heme in the host environment. Importantly, P. gingivalis is advantaged in terms of acquiring heme, which is vital for its growth survival and virulence.

Unraveling the sequence of cytosolic reactions in the export of GspB adhesin from Streptococcus gordonii.

Many pathogenic bacteria, including Streptococcus gordonii, possess a pathway for the cellular export of a single serine-rich-repeat protein that mediates the adhesion of bacteria to host cells and the extracellular matrix. This adhesin protein is O-glycosylated by several cytosolic glycosyltransferases and requires three accessory Sec proteins (Asp1-3) for export, but how the adhesin protein is processed for export is not well understood. Here, we report that the S. gordonii adhesin GspB is sequentially O-glycosylated by three enzymes (GtfA/B, Nss, and Gly) that attach N-acetylglucosamine and glucose to Ser/Thr residues. We also found that modified GspB is transferred from the last glycosyltransferase to the Asp1/2/3 complex. Crystal structures revealed that both Asp1 and Asp3 are related to carbohydrate-binding proteins, suggesting that they interact with carbohydrates and bind glycosylated adhesin, a notion that was supported by further analyses. We further observed that Asp1 also has an affinity for phospholipids, which is attenuated by Asp2. In summary, our findings support a model in which the GspB adhesin is sequentially glycosylated by GtfA/B, Nss, and Gly and then transferred to the Asp1/2/3 complex in which Asp1 mediates the interaction of the Asp1/2/3 complex with the lipid bilayer for targeting of matured GspB to the export machinery.

Fluorescence Tools Adapted for Real-Time Monitoring of the Behaviors of Streptococcus Species.

Tagging of bacteria with fluorescent proteins has become an essential component of modern microbiology. Fluorescent proteins can be used to monitor gene expression and biofilm growth and to visualize host-pathogen interactions. Here, we developed a collection of fluorescent protein reporter plasmids for Streptococcus mutans UA159 and other oral streptococci. Using superfolder green fluorescent protein (sfGFP) as a reporter for transcriptional activity, we were able to characterize four strong constitutive promoters in S. mutans These promoter-sfgfp fusions worked both for single-copy chromosomal integration and on a multicopy plasmid, with the latter being segregationally stable in the absence of selective pressure under the conditions tested. We successfully labeled S. mutans UA159, Streptococcus gordonii DL1, and Streptococcus sp. strain A12 with sfGFP, DsRed-Express2 (red), and citrine (yellow). To test these plasmids under more challenging conditions, we performed mixed-species biofilm experiments and separated fluorescent populations using fluorescence-activated cell sorting (FACS). This allowed us to visualize two streptococci at a time and quantify the amounts of each species simultaneously. These fluorescent reporter plasmids add to the genetic toolbox available for the study of oral streptococci.IMPORTANCE Oral streptococci are the most abundant bacteria in the mouth and have a major influence on oral health and disease. In this study, we designed and optimized the expression of fluorescent proteins in Streptococcus mutans and other oral streptococci. We monitored the levels of expression and noise (the variability in fluorescence across the population). We then created several fluorescent protein delivery systems (green, yellow, and red) for use in oral streptococci. The data show that we can monitor bacterial growth and interactions in situ, differentiating between different bacteria growing in biofilms, the natural state of the organisms in the human mouth. These new tools will allow researchers to study these bacteria in novel ways to create more effective diagnostic and therapeutic tools for ubiquitous infectious diseases.

Amino Sugars Modify Antagonistic Interactions between Commensal Oral Streptococci and Streptococcus mutans.

N-Acetylglucosamine (GlcNAc) and glucosamine (GlcN) enhance the competitiveness of the laboratory strain DL1 of Streptococcus gordonii against the caries pathogen Streptococcus mutans Here, we examine how amino sugars affect the interaction of five low-passage-number clinical isolates of abundant commensal streptococci with S. mutans by utilizing a dual-species biofilm model. Compared to that for glucose, growth on GlcN or GlcNAc significantly reduced the viability of S. mutans in cocultures with most commensals, shifting the proportions of species. Consistent with these results, production of H2O2 was increased in most commensals when growing on amino sugars, and inhibition of S. mutans by Streptococcus cristatus, Streptococcus oralis, or S. gordonii was enhanced by amino sugars on agar plates. All commensals except S. oralis had higher arginine deiminase activities when grown on GlcN and, in some cases, GlcNAc. In ex vivo biofilms formed using pooled cell-containing saliva (CCS), the proportions of S. mutans were drastically diminished when GlcNAc was the primary carbohydrate. Increased production of H2O2 could account in large part for the inhibitory effects of CCS biofilms. Surprisingly, amino sugars appeared to improve mutacin production by S. mutans on agar plates, suggesting that the commensals have mechanisms to actively subvert antagonism by S. mutans in cocultures. Collectively, these findings demonstrate that amino sugars can enhance the beneficial properties of low-passage-number commensal oral streptococci and highlight their potential for moderating the cariogenicity of oral biofilms.IMPORTANCE Dental caries is driven by dysbiosis of oral biofilms in which dominance by acid-producing and acid-tolerant bacteria results in loss of tooth mineral. Our previous work demonstrated the beneficial effects of amino sugars GlcNAc and GlcN in promoting the antagonistic properties of a health-associated oral bacterium, Streptococcus gordonii, in competition with the major caries pathogen Streptococcus mutans Here, we investigated 5 low-passage-number clinical isolates of the most common streptococcal species to establish how amino sugars may influence the ecology and virulence of oral biofilms. Using multiple in vitro models, including a human saliva-derived microcosm biofilm, experiments showed significant enhancement by at least one amino sugar in the ability of most of these bacteria to suppress the caries pathogen. Therefore, our findings demonstrated the mechanism of action by which amino sugars may affect human oral biofilms to promote health.

Serine-Rich Repeat Adhesins Mediate Shear-Enhanced Streptococcal Binding to Platelets.

The binding of bacteria to platelets is thought to be a central event in the pathogenesis of infective endocarditis. The serine-rich repeat (SRR) glycoproteins of viridans group streptococci have been shown to mediate platelet binding in vitro and to contribute to virulence in animal models. However, it is not known whether SRR adhesins can mediate streptococcal binding under the high fluidic shear stress conditions present on the endocardial surface. We found that three streptococcal SRR adhesins (GspB, Hsa, and SrpA) with differing structures and sialoglycan binding specificities nevertheless exhibited similar biomechanical properties. All three adhesins mediated shear-enhanced streptococcal binding to immobilized platelets through the platelet receptor GPIbα. Shear-enhanced adhesion was manifested in three ways. First, the number of circulating streptococci binding via SRR adhesins to immobilized platelet receptors peaked at 1 dyn/cm2 Second, bound streptococci switched from weak rolling to strong stationary adhesion as shear stress increased to 10 dyn/cm2 Third, while a few streptococci detached each time the flow was increased, the majority of streptococci bound to platelets remained firmly attached through 20 to 80 dyn/cm2 (shear levels typical of arteries and the endocardium). Thus, all three adhesins mediated shear-enhanced streptococcal binding to platelets under the flow conditions found in heart valves. The ability of the SRR adhesins to mediate shear-enhanced binding strongly suggests that they form catch bonds that are activated by tensile force and provides a mechanism for the selective targeting of bacteria to platelet receptors immobilized on the endocardial surface.

Role of Neuraminidase-Producing Bacteria in Exposing Cryptic Carbohydrate Receptors for Streptococcus gordonii Adherence.

Streptococcus gordonii is an early colonizer of the oral cavity. Although a variety of S. gordonii adherence mechanisms have been described, current dogma is that the major receptor for S. gordonii is sialic acid. However, as many bacterial species in the oral cavity produce neuraminidase that can cleave terminal sialic acid, it is unclear whether S. gordonii relies on sialic acid for adherence to oral surfaces or if this species has developed alternative binding strategies. Previous studies have examined adherence to immobilized glycoconjugates and identified binding to additional glycans, but no prior studies have defined the contribution of these different glycan structures in adherence to oral epithelial cells. We determined that the majority of S. gordonii strains tested did not rely on sialic acid for efficient adherence. In fact, adherence of some strains was significantly increased following neuraminidase treatment. Further investigation of representative strains that do not rely on sialic acid for adherence revealed binding not only to sialic acid via the serine-rich repeat protein GspB but also to ß-1,4-linked galactose. Adherence to this carbohydrate occurs via an unknown adhesin distinct from those utilized by Streptococcus oralis and Streptococcus pneumoniae Demonstrating the potential biological relevance of binding to this cryptic receptor, we established that S. oralis increases S. gordonii adherence in a neuraminidase-dependent manner. These data suggest that S. gordonii has evolved to simultaneously utilize both terminal and cryptic receptors in response to the production of neuraminidase by other species in the oral environment.

Concerted functions of Streptococcus gordonii surface proteins PadA and Hsa mediate activation of human platelets and interactions with extracellular matrix.

A range of Streptococcus bacteria are able to interact with blood platelets to form a thrombus (clot). Streptococcus gordonii is ubiquitous within the human oral cavity and amongst the common pathogens isolated from subjects with infective endocarditis. Two cell surface proteins, Hsa and Platelet adherence protein A (PadA), in S. gordonii mediate adherence and activation of platelets. In this study, we demonstrate that PadA binds activated platelets and that an NGR (Asparagine-Glycine-Arginine) motif within a 657 amino acid residue N-terminal fragment of PadA is responsible for this, together with two other integrin-like recognition motifs RGT and AGD. PadA also acts in concert with Hsa to mediate binding of S. gordonii to cellular fibronectin and vitronectin, and to promote formation of biofilms. Evidence is presented that PadA and Hsa are each reliant on the other's active presentation on the bacterial cell surface, suggesting cooperativity in functions impacting both colonization and pathogenesis.

BAR-encapsulated nanoparticles for the inhibition and disruption of Porphyromonas gingivalis-Streptococcus gordonii biofilms.

BACKGROUND: Porphyromonas gingivalis adherence to oral streptococci is a key point in the pathogenesis of periodontal diseases (Honda in Cell Host Microbe 10:423-425, 2011). Previous work in our groups has shown that a region of the streptococcal antigen denoted BAR (SspB Adherence Region) inhibits P. gingivalis/S. gordonii interaction and biofilm formation both in vitro and in a mouse model of periodontitis (Daep et al. in Infect Immun 74:5756-5762, 2006; Daep et al. in Infect immun 76:3273-3280, 2008; Daep et al. in Infect Immun 79:67-74, 2011). However, high localized concentration and prolonged exposure are needed for BAR to be an effective therapeutic in the oral cavity. METHODS: To address these challenges, we fabricated poly(lactic-co-glycolic acid) (PLGA) and methoxy-polyethylene glycol PLGA (mPEG-PLGA) nanoparticles (NPs) that encapsulate BAR peptide, and assessed the potency of BAR-encapsulated NPs to inhibit and disrupt in vitro two-species biofilms. In addition, the kinetics of BAR-encapsulated NPs were compared after different durations of exposure in a two-species biofilm model, against previously evaluated BAR-modified NPs and free BAR. RESULTS: BAR-encapsulated PLGA and mPEG-PLGA NPs potently inhibited biofilm formation (IC50 = 0.7 µM) and also disrupted established biofilms (IC50 = 1.3 µM) in a dose-dependent manner. In addition, BAR released during the first 2 h of administration potently inhibits biofilm formation, while a longer duration of 3 h is required to disrupt pre-existing biofilms. CONCLUSIONS: These results suggest that BAR-encapsulated NPs provide a potent platform to inhibit (prevent) and disrupt (treat) P. gingivalis/S. gordonii biofilms, relative to free BAR.

Starch Combined with Sucrose Provokes Greater Root Dentine Demineralization than Sucrose Alone.

Since there is no consensus about whether starch increases the cariogenic potential of sucrose, we used a validated 3-species biofilm model to evaluate if starch combined with sucrose provokes higher root dentine demineralization than sucrose alone. Biofilms (n = 18) composed by Streptococcus mutans (the most cariogenic bacteria), Actinomces naeslundii (which has amylolytic activity), and Streptococcus gordonii (which binds salivary amylase) were formed on root dentine slabs under exposure 8 ×/day to one of the following treatments: 0.9% NaCl, 1% starch, 10% sucrose, or a combination of 1% starch and 10% sucrose. Before each treatment, biofilms were pretreated with human whole saliva for 1 min. The pH of the culture medium was measured daily as an indicator of biofilm acidogenicity. After 96 h of growth, the biofilms were collected, and the biomass, bacteria viability, and polysaccharides were analyzed. Dentine demineralization was assessed by surface hardness loss (% SHL). Biofilm bioarchitecture was analyzed using confocal laser scanning microscopy. Treatment with a starch and sucrose combination provoked higher (p = 0.01) dentine demineralization than sucrose alone (% SHL = 53.2 ± 7.0 vs. 43.2 ± 8.7). This was supported by lower pH values (p = 0.007) of the culture medium after daily exposure to the starch and sucrose combination compared with sucrose (4.89 ± 0.29 vs. 5.19 ± 0.32). Microbiological and biochemical findings did not differ between biofilms treated with the combination of starch and sucrose and sucrose alone (p > 0.05). Our findings give support to the hypothesis that a starch and sucrose combination is more cariogenic for root dentine than sucrose alone.

Streptococcus gordonii LuxS/autoinducer-2 quorum-sensing system modulates the dual-species biofilm formation with Streptococcus mutans.

Dental plaques are mixed-species biofilms that are related to the development of dental caries. Streptococcus mutans (S. mutans) is an important cariogenic bacterium that forms mixed-species biofilms with Streptococcus gordonii (S. gordonii), an early colonizer of the tooth surface. The LuxS/autoinducer-2(AI-2) quorum sensing system is involved in the regulation of mixed-species biofilms, and AI-2 is proposed as a universal signal for the interaction between bacterial species. In this work, a S. gordonii luxS deficient strain was constructed to investigate the effect of the S. gordonii luxS gene on dual-species biofilm formed by S. mutans and S. gordonii. In addition, AI-2 was synthesized in vitro by incubating recombinant LuxS and Pfs enzymes of S. gordonii together. The effect of AI-2 on S. mutans single-species biofilm formation and cariogenic virulence gene expression were also assessed. The results showed that luxS disruption in S. gordonii altered dual-species biofilm formation, architecture, and composition, as well as the susceptibility to chlorhexidine. And the in vitro synthesized AI-2 had a concentration-dependent effect on S. mutans biofilm formation and virulence gene expression. These findings indicate that LuxS/AI-2 quorum-sensing system of S. gordonii plays a role in regulating the dual-species biofilm formation with S. mutans.

l-Arginine Modifies the Exopolysaccharide Matrix and Thwarts Streptococcus mutans Outgrowth within Mixed-Species Oral Biofilms.

UNLABELLED: l-Arginine, a ubiquitous amino acid in human saliva, serves as a substrate for alkali production by arginolytic bacteria. Recently, exogenous l-arginine has been shown to enhance the alkalinogenic potential of oral biofilm and destabilize its microbial community, which might help control dental caries. However, l-arginine exposure may inflict additional changes in the biofilm milieu when bacteria are growing under cariogenic conditions. Here, we investigated how exogenous l-arginine modulates biofilm development using a mixed-species model containing both cariogenic (Streptococcus mutans) and arginolytic (Streptococcus gordonii) bacteria in the presence of sucrose. We observed that 1.5% (wt/vol) l-arginine (also a clinically effective concentration) exposure suppressed the outgrowth of S. mutans, favored S. gordonii dominance, and maintained Actinomyces naeslundii growth within biofilms (versus vehicle control). In parallel, topical l-arginine treatments substantially reduced the amounts of insoluble exopolysaccharides (EPS) by >3-fold, which significantly altered the three-dimensional (3D) architecture of the biofilm. Intriguingly, l-arginine repressed S. mutans genes associated with insoluble EPS (gtfB) and bacteriocin (SMU.150) production, while spxB expression (H2O2 production) by S. gordonii increased sharply during biofilm development, which resulted in higher H2O2 levels in arginine-treated biofilms. These modifications resulted in a markedly defective EPS matrix and areas devoid of any bacterial clusters (microcolonies) on the apatitic surface, while the in situ pH values at the biofilm-apatite interface were nearly one unit higher in arginine-treated biofilms (versus the vehicle control). Our data reveal new biological properties of l-arginine that impact biofilm matrix assembly and the dynamic microbial interactions associated with pathogenic biofilm development, indicating the multiaction potency of this promising biofilm disruptor. IMPORTANCE: Dental caries is one of the most prevalent and costly infectious diseases worldwide, caused by a biofilm formed on tooth surfaces. Novel strategies that compromise the ability of virulent species to assemble and maintain pathogenic biofilms could be an effective alternative to conventional antimicrobials that indiscriminately kill other oral species, including commensal bacteria. l-Arginine at 1.5% has been shown to be clinically effective in modulating cariogenic biofilms via alkali production by arginolytic bacteria. Using a mixed-species ecological model, we show new mechanisms by which l-arginine disrupts the process of biofilm matrix assembly and the dynamic microbial interactions that are associated with cariogenic biofilm development, without impacting the bacterial viability. These results may aid in the development of enhanced methods to control biofilms using l-arginine.

Arginine-Ornithine Antiporter ArcD Controls Arginine Metabolism and Interspecies Biofilm Development of Streptococcus gordonii.

Arginine is utilized by the oral inhabitant Streptococcus gordonii as a substrate of the arginine deiminase system (ADS), eventually producing ATP and NH3, the latter of which is responsible for microbial resistance to pH stress. S. gordonii expresses a putative arginine-ornithine antiporter (ArcD) whose function has not been investigated despite relevance to the ADS and potential influence on inter-bacterial communication with periodontal pathogens that utilize amino acids as a main energy source. Here, we generated an S. gordonii ΔarcD mutant to explore the role of ArcD in physiological homeostasis and bacterial cross-feeding. First, we confirmed that S. gordonii ArcD plays crucial roles for mediating arginine uptake and promoting bacterial growth, particularly under arginine-limited conditions. Next, metabolomic profiling and transcriptional analysis of the ΔarcD mutant revealed that deletion of this gene caused intracellular accumulation of ornithine leading to malfunction of the ADS and suppression of de novo arginine biosynthesis. The mutant strain also showed increased susceptibility to low pH stress due to reduced production of ammonia. Finally, accumulation of Fusobacterium nucleatum was found to be significantly decreased in biofilm formed by the ΔarcD mutant as compared with the wild-type strain, although ornithine supplementation restored fusobacterium biovolume in dual-species biofilms with the ΔarcD mutant and also enhanced single species biofilm development by F. nucleatum. Our results are the first direct evidence showing that S. gordonii ArcD modulates not only alkali and energy production but also interspecies interaction with F. nucleatum, thus initiating a middle stage of periodontopathic biofilm formation, by metabolic cross-feeding.

Critical roles of arginine in growth and biofilm development by Streptococcus gordonii.

Streptococcus gordonii is an oral commensal and an early coloniser of dental plaque. In vitro, S. gordonii is conditionally auxotrophic for arginine in monoculture but biosynthesises arginine when coaggregated with Actinomyces oris. Here, we investigated the arginine-responsive regulatory network of S. gordonii and the basis for conditional arginine auxotrophy. ArcB, the catabolic ornithine carbamoyltransferase involved in arginine degradation, was also essential for arginine biosynthesis. However, arcB was poorly expressed following arginine depletion, indicating that arcB levels may limit S. gordonii arginine biosynthesis. Arginine metabolism gene expression was tightly co-ordinated by three ArgR/AhrC family regulators, encoded by argR, ahrC and arcR genes. Microarray analysis revealed that > 450 genes were regulated in response to rapid shifts in arginine concentration, including many genes involved in adhesion and biofilm formation. In a microfluidic salivary biofilm model, low concentrations of arginine promoted S. gordonii growth, whereas high concentrations (> 5 mM arginine) resulted in dramatic reductions in biofilm biomass and changes to biofilm architecture. Collectively, these data indicate that arginine metabolism is tightly regulated in S. gordonii and that arginine is critical for gene regulation, cellular growth and biofilm formation. Manipulating exogenous arginine concentrations may be an attractive approach for oral biofilm control.

Streptococcus gordonii comCDE (competence) operon modulates biofilm formation with Candida albicans.

Candida albicans is a pleiomorphic fungus that forms mixed species biofilms with Streptococcus gordonii, an early colonizer of oral cavity surfaces. Activation of quorum sensing (QS; intercellular signalling) promotes monospecies biofilm development by these micro-organisms, but the role of QS in mixed species communities is not understood. The comCDE genes in S. gordonii encode a sensor-regulator system (ComDE), which is activated by the comC gene product (CSP, competence stimulating peptide) and modulates expression of QS-regulated genes. Dual species biofilms of S. gordonii ΔcomCDE or ΔcomC mutants with C. albicans showed increased biomass compared to biofilms of S. gordonii DL1 wild-type with C. albicans. The ΔcomCDE mutant dual species biofilms in particular contained more extracellular DNA (eDNA), and could be dispersed with DNase I or protease treatment. Exogenous CSP complemented the S. gordonii ΔcomC transformation deficiency, as well as the ΔcomC-C. albicans biofilm phenotype. Purified CSP did not affect C. albicans hyphal filament formation but inhibited monospecies biofilm formation by C. albicans. The results suggest that the S. gordonii comCDE QS-system modulates the production of eDNA and the incorporation of C. albicans into dual species biofilms.