Synergistic effects of antimicrobial components of the human-derived composite amnion-chorion membrane on bacterial growth.

Introduction: The human-derived amnion-chorion membrane (ACM) has endogenous antimicrobial properties, which are important for preventing the colonization and survival of oral bacteria on exposed membranes. This project aimed to decipher the underlying mechanism by identifying the components of ACM that confer antibacterial properties. In addition, the antimicrobial efficacy of these identified components on oral bacteria was assessed. Methods: Four antimicrobial proteins, histone H2A/H2B, cathelicidin LL-37, lactoferrin, and lysozyme, were identified via mass spectrometry in ACM. These proteins were then assessed for their efficacy in killing Streptococcus gordonii Challis. Log-phased bacterial cells were cultured with the commercially available proteins that were identified in ACM, either individually or in combination, at different concentrations. After incubation for 8 or 24 hours, the bacteria were stained with a live/dead viability kit and analyzed via confocal microscopy. Results: The combination of these proteins effectively killed S. gordonii in a dose-dependent fashion after 8 or 24 hours of incubation. When each protein was tested individually, it killed S. gordonii at a much lower efficacy relative to the combinations. The synergistic effects of the antimicrobial protein combinations were also observed in both the viable cell count recovery and minimum inhibitory concentration assays. Discussion: By shedding light on the mechanisms in the ACM's antimicrobial property, this study may raise more awareness of the potential benefit of utilization of a membrane with endogenous antimicrobial properties in regeneration surgeries.

Phage-based biocontrol of Porphyromonas gingivalis through indirect targeting.

Bacteriophages offer an opportunity for chemical-free, precise control of problematic bacteria, but this approach can be limited when lytic phages are difficult to obtain for the target host. In such cases, phage-based targeting of cooperating or cross-feeding bacteria (e.g., Streptococcus gordonii) can be an effective approach to control the problematic bacteria (e.g., Porphyromonas gingivalis). Using a dual-species biofilm system, phage predation of S. gordonii (108 PFU·mL-1) decreased the abundance of pathogenic P. gingivalis by >99% compared with no-treatment controls, while also inhibiting the production of cytotoxic metabolic end products (butyric and propionic acids). Phage treatment upregulated genes associated with interspecies co-adhesion (5- to 8-fold) and quorum sensing (10-fold) in residual P. gingivalis, which is conducive to increased potential to bind to S. gordonii. Counterintuitively, lower-titer phage applications (104 PFU·mL-1) increased the production of extracellular polymeric substance (EPS) by 22% and biofilm biomass by 50%. This overproduction of EPS may contribute to the phenomenon where the biofilm separated into two distinct species layers, as observed by confocal laser scanning microscopy. Although more complex mixed-culture systems should be considered to delineate the merits and limitations of this novel biocontrol approach (which would likely require the use of phage cocktails), our results offer proof of concept that indirect phage-based targeting can expand the applicability of phage-based control of pathogenic bacteria for public health protection. IMPORTANCE: Lytic phages are valuable agents for targeted elimination of bacteria in diverse applications. Nevertheless, lytic phages are difficult to isolate for some target pathogens. We offer proof of concept that this limitation may be overcome via indirect phage targeting, which involves knocking out species that interact closely with and benefit the primary problematic target bacteria. Our target (P. gingivalis) only forms a periodontal pathogenic biofilm if the pioneer colonizer (S. gordonii) offers its surface for P. gingivalis to attach. Phage predation of the co-adhesive S. gordonii significantly reduced abundance of the target pathogen by >99%, decreased the total biofilm biomass by >44%, and suppressed its production of cytotoxic metabolic byproducts. Thus, this research extends the scope of phage-based biocontrol for public health protection.

Microbial interactions impact stress tolerance in a model oral community.

Understanding the molecular mechanisms governing microbial interactions is crucial for unraveling the complexities of microbial communities and their ecological impacts. Here, we employed a two-species model system comprising the oral bacteria Aggregatibacter actinomycetemcomitans and Streptococcus gordonii to investigate how synergistic and antagonistic interactions between microbes impact their resilience to environmental change and invasion by other microbes. We used an in vitro colony biofilm model and focused on two S. gordonii-produced extracellular molecules, L-lactate and H2O2, which are known to impact fitness of this dual-species community. While the ability of A. actinomycetemcomitans to cross-feed on S. gordonii-produced L-lactate enhanced its fitness during co-culture, this function showed little impact on the ability of co-cultures to resist environmental change. In fact, the ability of A. actinomycetemcomitans to catabolize L-lactate may be detrimental in the presence of tetracycline, highlighting the complexity of interactions under antimicrobial stress. Furthermore, H2O2, known for its antimicrobial properties, had negative impacts on both species in our model system. However, H2O2 production by S. gordonii enhanced A. actinomycetemcomitans tolerance to tetracycline, suggesting a protective role under antibiotic pressure. Finally, S. gordonii significantly inhibited the bacterium Serratia marcescens from invading in vitro biofilms, but this inhibition was lost during co-culture with A. actinomycetemcomitans and in a murine abscess model, where S. gordonii actually promoted S. marcescens invasion. These data indicate that microbial interactions can impact fitness of a bacterial community upon exposure to stresses, but these impacts are highly environment dependent. IMPORTANCE: Microbial interactions are critical modulators of the emergence of microbial communities and their functions. However, how these interactions impact the fitness of microbes in established communities upon exposure to environmental stresses is poorly understood. Here, we utilized a two-species community consisting of Aggregatibacter actinomycetemcomitans and Streptococcus gordonii to examine the impact of synergistic and antagonistic interactions on microbial resilience to environmental fluctuations and susceptibility to microbial invasion. We focused on the S. gordonii-produced extracellular molecules, L-lactate and H2O2, which have been shown to mediate interactions between these two microbes. We discovered that seemingly beneficial functions, such as A. actinomycetemcomitans cross-feeding on S. gordonii-produced L-Lactate, can paradoxically exacerbate vulnerabilities, such as susceptibility to antibiotics. Moreover, our data highlight the context-dependent nature of microbial interactions, emphasizing that a seemingly potent antimicrobial, such as H2O2, can have both synergistic and antagonistic effects on a microbial community dependent on the environment.

Effects of fluid shear stress on oral biofilm formation and composition and the transcriptional response of Streptococcus gordonii.

Biofilms are subjected to many environmental pressures that can influence community structure and physiology. In the oral cavity, and many other environments, biofilms are exposed to forces generated by fluid flow; however, our understanding of how oral biofilms respond to these forces remains limited. In this study, we developed a linear rocker model of fluid flow to study the impact of shear forces on Streptococcus gordonii and dental plaque-derived multispecies biofilms. We observed that as shear forces increased, S. gordonii biofilm biomass decreased. Reduced biomass was largely independent of overall bacterial growth. Transcriptome analysis of S. gordonii biofilms exposed to moderate levels of shear stress uncovered numerous genes with differential expression under shear. We also evaluated an ex vivo plaque biofilm exposed to fluid shear forces. Like S. gordonii, the plaque biofilm displayed decreased biomass as shear forces increased. Examination of plaque community composition revealed decreased diversity and compositional changes in the plaque biofilm exposed to shear. These studies help to elucidate the impact of fluid shear on oral bacteria and may be extended to other bacterial biofilm systems.

Does dental material type influence bacterial adhesion under the same polishing conditions? Direct observation using a fluorescent staining technique: An in vitro study.

Highly polished 3, 4, and 5 mol% yttria-stabilized zirconia and CAD/CAM composite resin samples were prepared, and the influence of surface roughness (Ra and Sa, 21 areas/group), wettability (contact angle and surface energy, 3 samples/group), and surface chemical composition (2 samples/group) on single-strain bacterial adhesion models (Porphyromonas gingivalis, Streptococcus oralis, Streptococcus sanguinis, Streptococcus gordonii, and Streptococcus mutans) were compared via fluorescent staining with graphical analysis (21 areas/group). Statistical analysis was performed using the Shapiro-Wilk test followed by one-way analysis of variance with Tukey's test or the Kruskal-Wallis test with Dunn's test (α=0.05) and linear regression. For dental zirconia with the same surface roughness, the yttria content did not significantly influence the initial bacterial adhesion. However, higher bacterial adhesion was detected for the composite resin owing to its high C, O, and Si contents. There was no correlation between surface energy and bacterial adhesion for any bacterial strain (p<0.005).

Measurement and analysis of microbial fluoride resistance in dental biofilm models.

The interactions between communities of microorganisms inhabiting the dental biofilm is a major determinant of oral health. These biofilms are periodically exposed to high concentrations of fluoride, which is present in almost all oral healthcare products. The microbes resist fluoride through the action of membrane export proteins. This chapter describes the culture, growth and harvest conditions of model three-species dental biofilm comprised of cariogenic pathogens Streptococcus mutans and Candida albicans and the commensal bacterium Streptococcus gordonii. In order to examine the role of fluoride export by S. mutans in model biofilms, procedures for generating a strain of S. mutans with a genetic knockout of the fluoride exporter are described. We present a case study examining the effects of this mutant strain on the biofilm mass, acid production and mineral dissolution under exposure to low levels of fluoride. These general approaches can be applied to study the effects of any gene of interest in physiologically realistic multispecies oral biofilms.

Fluoride export is required for the competitive fitness of pathogenic microorganisms in dental biofilm models.

Microorganisms resist fluoride toxicity using fluoride export proteins from one of several different molecular families. Cariogenic species Streptococcus mutans and Candida albicans extrude intracellular fluoride using a CLCF F-/H+ antiporter and FEX fluoride channel, respectively, whereas oral commensal eubacteria, such as Streptococcus gordonii, export fluoride using a Fluc fluoride channel. In this work, we examine how genetic knockout of fluoride export impacts pathogen fitness in single-species and three-species dental biofilm models. For biofilms generated using S. mutans with the genetic knockout of the CLCF transporter, exposure to low fluoride concentrations decreased S. mutans counts, synergistically reduced the populations of C. albicans, increased the relative proportion of oral commensal S. gordonii, and reduced properties associated with biofilm pathogenicity, including acid production and hydroxyapatite dissolution. Biofilms prepared with C. albicans with genetic knockout of the FEX channel also exhibited reduced fitness in the presence of fluoride but to a lesser degree. Imaging studies indicate that S. mutans is highly sensitive to fluoride, with the knockout strain undergoing complete lysis when exposed to low fluoride for a moderate amount of time. Biochemical purification of the S. mutans CLCF transporter and functional reconstitution establishes that the functional protein is a dimer encoded by a single gene. Together, these findings suggest that fluoride export by oral pathogens can be targeted by specific inhibitors to restore biofilm symbiosis in dental biofilms and that S. mutans is especially susceptible to fluoride toxicity. IMPORTANCE: Dental caries is a globally prevalent condition that occurs when pathogenic species, including Streptococcus mutans and Candida albicans, outcompete beneficial species, such as Streptococcus gordonii, in the dental biofilm. Fluoride is routinely used in oral hygiene to prevent dental caries. Fluoride also has antimicrobial properties, although most microbes possess fluoride exporters to resist its toxicity. This work shows that sensitization of cariogenic species S. mutans and C. albicans to fluoride by genetic knockout of fluoride exporters alters the microbial composition and pathogenic properties of dental biofilms. These results suggest that the development of drugs that inhibit fluoride exporters could potentiate the anticaries effect of fluoride in over-the-counter products like toothpaste and mouth rinses. This is a novel strategy to treat dental caries.

In vitro bacterial penetration and dissemination through dentinal tubules in roots filled with calcium silicate-based cements.

The aim of this study was to assess whether calcium silicate root fillings prevent bacterial penetration and to determine how bacteria penetrate roots. Extracted single-rooted, single-canal human teeth were decoronated, prepared and filled with ProRootMTA or Biodentine (n = 12 each). Positive and negative (n = 2 each) controls were not filled. A two-chamber model was used with Streptococcus gordonii. The lower compartment was evaluated for turbidity over 150 days. Roots were split and examined for bacteria via SEM. The chi-squared test was used for comparisons (α = 0.05). Experimental groups had bacteria in their coronal thirds. Tubules contained bacteria in 90.9% and 91.7% of areas examined in the Biodentine and ProRootMTA groups, respectively, with no significant difference (p = 0.914). Experimental and negative roots had no turbidity with no significant difference between Biodentine and ProRootMTA (p = 1.000). Positive controls had turbidity. Bacteria penetrate roots via dentine tubules of root-filled teeth. Biodentine was comparable to ProRoot MTA.

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.

Inflammatory macrophages exploited by oral streptococcus increase IL-1B release via NLRP6 inflammasome.

Chronic inflammatory periodontal disease develops in part from the infiltration of a large number of classically activated inflammatory macrophages that release inflammatory cytokines important for disease progression, including inflammasome-dependent interleukin (IL)-1ß. Streptococcus gordonii is a normally commensal oral microorganism; while not causative, recent evidence indicates that commensal oral microbes are required for the full development of periodontal disease. We have recently reported that inflammatory macrophages counterintuitively allow for the increased survival of phagocytosed S. gordonii over nonactivated or alternatively activated macrophages. This survival is dependent on increased reactive oxygen species production within the phagosome of the inflammatory macrophages, and resistance by the bacterium and can result in S. gordonii damaging the phagolysosomes. Here, we show that activated macrophages infected with live S. gordonii release more IL-1ß than non-activated macrophages infected with either live or dead S. gordonii, and that the survival of oral Streptococci are more dependent on macrophage activation than other Gram positive microbes, both classical pathogens and commensals. We also find that S. gordonii-dependent inflammatory macrophage inflammasome activation requires the cytoplasmic NLRP6. Overall, our results suggest S. gordonii is capable of evading immune destruction, increasing inflammatory mediators, and increasing inflammatory macrophage response, and that this ability is increased under conditions of inflammation. This work reveals additional mechanisms by which normally commensal oral streptococci-macrophage interactions can change, resulting in increased release of mature IL-1ß, potentially contributing to an environment that perpetuates inflammation.

Antagonistic interactions by a high H2 O2 -producing commensal streptococcus modulate caries development by Streptococcus mutans.

Dental caries (tooth-decay) is caused by biofilms harboring polymicrobial communities on teeth that leads to the onset of localized areas of enamel demineralization. Streptococcus mutans has been clinically associated with severe caries in childhood. Although commensal bacteria can combat S. mutans using self-generated antimicrobials such as hydrogen peroxide (H2 O2 ), constant sugar-rich diet consumption disrupts microbial homeostasis shifting toward cariogenic community. Recently, Streptococcus oralis subsp. tigurinus strain J22, an oral isolate, was identified as a uniquely potent H2 O2 producer. Here, we assess whether a high H2 O2 -producing commensal streptococcus can modulate the spatial organization and virulence of S. mutans within biofilms. Using an experimental biofilm model, we find that the presence of S. oralis J22 can effectively inhibit the clustering, accumulation, and spatial organization of S. mutans on ex vivo human tooth surface, resulting in significant reduction of enamel demineralization. Notably, the generation of H2 O2 via pyruvate oxidase (SpxB) from S. oralis J22 is not repressed by sugars (a common repressor in other mitis group streptococci), resulting in enhanced inhibition of S. mutans growth (vs. Streptococcus gordonii). We further investigate its impact on biofilm virulence using an in vivo rodent caries model under sugar-rich diet. Coinfection of S. mutans with S. oralis results in reduced caries development compared to either species infected alone, whereas coinfection with S. gordonii has negligible effects, suggesting that the presence of an efficient, high H2 O2 -producer can disrupt S. mutans virulence. This work demonstrates that oral isolates with unusual high H2 O2 production may be capable of modulating biofilm cariogenicity in vivo. The findings also highlight the importance of bacterial antagonistic interactions within polymicrobial communities in health and in disease-causing state.

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.

E-Cigarette Aerosol Exposure Favors the Growth and Colonization of Oral Streptococcus mutans Compared to Commensal Streptococci.

E-cigarettes (e-cigs) have drastically increased in popularity during the last decade, especially among teenagers. While recent studies have started to explore the effect of e-cigs in the oral cavity, little is known about their effects on the oral microbiota and how they could affect oral health and potentially lead to disease, including periodontitis and head and neck cancers. To explore the impact of e-cigs on oral bacteria, we selected members of the genus Streptococcus, which are abundant in the oral cavity. We exposed the commensals Streptococcus sanguinis and Streptococcus gordonii and the opportunistic pathogen Streptococcus mutans, best known for causing dental caries, to e-liquids and e-cig aerosols with and without nicotine and with and without menthol flavoring and measured changes in growth patterns and biofilm formation. Our results demonstrate that e-cig aerosols hindered the growth of S. sanguinis and S. gordonii, while they did not affect the growth of S. mutans. We also show that e-cig aerosols significantly increased biofilm formation by S. mutans but did not affect the biofilm formation of the two commensals. We found that S. mutans exhibits higher hydrophobicity and coaggregation abilities along with higher attachment to OKF6 cells than S. sanguinis and S. gordonii. Therefore, our data suggest that e-cig aerosols have the potential to dysregulate oral bacterial homeostasis by suppressing the growth of commensals while enhancing the biofilm formation of the opportunistic pathogen S. mutans. This study highlights the importance of understanding the consequences of e-cig aerosol exposure on selected commensals and pathogenic species. Future studies modeling more complex communities will provide more insight into how e-cig aerosols and vaping affect the oral microbiota. IMPORTANCE Our study shows that e-cigarette aerosol exposure of selected bacteria known to be residents of the oral cavity hinders the growth of two streptococcal commensals while enhancing biofilm formation, hydrophobicity, and attachment for the pathogen S. mutans. These results indicate that e-cigarette vaping could open a niche for opportunistic bacteria such as S. mutans to colonize the oral cavity and affect oral health.

Streptococcus gordonii DL1 evades polymorphonuclear leukocyte-mediated killing via resistance to lysozyme.

Streptococcus gordonii is an etiological bacterial agent of infective endocarditis. Although the pathogenesis mechanisms are not well understood, the interaction between streptococci and phagocytes is considered important for the development of infective endocarditis. Previous studies show that some S. gordonii strains, including DL1, survive in polymorphonuclear leukocytes (PMNs), whereas other strains such as SK12 are sensitive to PMN-dependent killing. In this study, we assessed the differences between the sensitivity of S. gordonii DL1 and S. gordonii SK12 to PMN-dependent killing. S. gordonii DL1 showed a higher survival when treated with PMNs than SK12. Both S. gordonii DL1 and S. gordonii SK12 showed high resistance to low pH condition. Compared to S. gordonii SK12, S. gordonii DL1 was sensitive to hydrogen peroxide. However, the resistance of S. gordonii DL1 to the tested bactericidal agents, especially lysozyme, was higher than that of SK12. Furthermore, we performed a bactericidal assay by treating a mixture of S. gordonii DL1 and SK12 with PMNs. S. gordonii DL1 did not enhance the survival of S. gordonii SK12 exposed to PMNs. These results indicated that S. gordonii DL1 is resistant to bactericidal agents that degrade bacteria in phagolysosomes. In addition, there was no secretory factor involved in the resistance to bactericidal agents. The findings of this study may help develop treatments for infective endocarditis caused by S. gordonii.

Short-chain fatty acids inhibit the biofilm formation of Streptococcus gordonii through negative regulation of competence-stimulating peptide signaling pathway.

Streptococcus gordonii, a Gram-positive commensal bacterium, is an opportunistic pathogen closely related to initiation and progression of various oral diseases, such as periodontitis and dental caries. Its biofilm formation is linked with the development of such diseases by enhanced resistance against antimicrobial treatment or host immunity. In the present study, we investigated the effect of short-chain fatty acids (SCFAs) on the biofilm formation of S. gordonii. SCFAs, including sodium acetate (NaA), sodium propionate (NaP), and sodium butyrate (NaB), showed an effective inhibitory activity on the biofilm formation of S. gordonii without reduction in bacterial growth. SCFAs suppressed S. gordonii biofilm formation at early time points whereas SCFAs did not affect its preformed biofilm. A quorum-sensing system mediated by competence-stimulating peptide (CSP) is known to regulate biofilm formation of streptococci. Interestingly, SCFAs substantially decreased mRNA expression of comD and comE, which are CSP-sensor and its response regulator responsible for CSP pathway, respectively. Although S. gordonii biofilm formation was enhanced by exogenous synthetic CSP treatment, such effect was not observed in the presence of SCFAs. Collectively, these results suggest that SCFAs have an anti-biofilm activity on S. gordonii through inhibiting comD and comE expression which results in negative regulation of CSP quorum-sensing system. SCFAs could be an effective anti-biofilm agent against S. gordonii for the prevention of oral diseases.

Regulation of olfactomedin 4 by Porphyromonas gingivalis in a community context.

At mucosal barriers, the virulence of microbial communities reflects the outcome of both dysbiotic and eubiotic interactions with the host, with commensal species mitigating or potentiating the action of pathogens. We examined epithelial responses to the oral pathogen Porphyromonas gingivalis as a monoinfection and in association with a community partner, Streptococcus gordonii. RNA-Seq of oral epithelial cells showed that the Notch signaling pathway, including the downstream effector olfactomedin 4 (OLFM4), was differentially regulated by P. gingivalis alone; however, regulation was overridden by S. gordonii. OLFM4 was required for epithelial cell migratory, proliferative and inflammatory responses to P. gingivalis. Activation of Notch signaling was induced through increased expression of the Notch1 receptor and the Jagged1 (Jag1) agonist. In addition, Jag1 was released in response to P. gingivalis, leading to paracrine activation. Following Jag1-Notch1 engagement, the Notch1 extracellular domain was cleaved by P. gingivalis gingipain proteases. Antagonism by S. gordonii involved inhibition of gingipain activity by secreted hydrogen peroxide. The results establish a novel mechanism by which P. gingivalis modulates epithelial cell function which is dependent on community context. These interrelationships have relevance for innate inflammatory responses and epithelial cell fate decisions in oral health and disease.

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.

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.

Enhanced biofilm formation of Streptococcus gordonii with lipoprotein deficiency.

Streptococcus gordonii is a commensal Gram-positive bacterium that acts as an opportunistic pathogen that can cause apical periodontitis, endocarditis, and pneumonia. Biofilm formation of bacteria is important for the initiation and progression of such diseases. Although lipoproteins play key roles in physiological functions, the role of lipoproteins of S. gordonii in its biofilm formation has not been clearly understood. In this study, we investigated the role of lipoproteins of S. gordonii in the bacterial biofilm formation using its lipoprotein-deficient strain (Δlgt). The S. gordonii Δlgt exhibited increased biofilm formation on the human dentin slices or on the polystyrene surfaces compared to the wild-type strain, while its growth rate did not differ from that of the wild-type. In addition, the S. gordonii Δlgt strain exhibited the enhanced LuxS mRNA expression and AI-2 production, which is known to be a positive regulator of biofilm formation, compared to the wild-type. Concordantly, the augmented biofilm formation of S. gordonii Δlgt was attenuated by an AI-2 inhibitor, D-ribose. In addition, lipoproteins from purified S. gordonii inhibited the biofilm formation of S. gordonii wild-type and Δlgt. Taken together, these results suggest that lipoprotein-deficient S. gordonii form biofilms more effectively than the wild-type strain, which might be related to the AI-2 quorum-sensing system.

Bacteria Modify Candida albicans Hypha Formation, Microcolony Properties, and Survival within Macrophages.

Phagocytic cells are crucial components of the innate immune system preventing Candida albicans mucosal infections. Streptococcus gordonii and Pseudomonas aeruginosa often colonize mucosal sites, along with C. albicans, and yet interkingdom interactions that might alter the survival and escape of fungi from macrophages are not understood. Murine macrophages were coinfected with S. gordonii or P. aeruginosa, along with C. albicans to evaluate changes in fungal survival. S. gordonii increased C. albicans survival and filamentation within macrophage phagosomes, while P. aeruginosa reduced fungal survival and filamentation. Coinfection with S. gordonii resulted in greater escape of C. albicans from macrophages and increased size of fungal microcolonies formed on macrophage monolayers, while coinfection with P. aeruginosa reduced macrophage escape and produced smaller microcolonies. Microcolonies formed in the presence of P. aeruginosa cells outside macrophages also had significantly reduced size that was not found with P. aeruginosa phenazine deletion mutants. S. gordonii cells, as well as S. gordonii heat-fixed culture supernatants, increased C. albicans microcolony biomass but also resulted in microcolony detachment. A heat-resistant, trypsin-sensitive pheromone processed by S. gordonii Eep was needed for these effects. The majority of fungal microcolonies formed on human epithelial monolayers with S. gordonii supernatants developed as large floating structures with no detectable invasion of epithelium, along with reduced gene expression of C. albicansHYR1, EAP1, and HWP2 adhesins. However, a subset of C. albicans microcolonies was smaller and had greater epithelial invasiveness compared to microcolonies grown without S. gordonii Thus, bacteria can alter the killing and escape of C. albicans from macrophages and contribute to changes in C. albicans pathogenicity.IMPORTANCECandida albicans is the predominant fungus colonizing the oral cavity that can have both synergistic and antagonistic interactions with other bacteria. Interkingdom polymicrobial associations modify fungal pathogenicity and are believed to increase microbial resistance to innate immunity. However, it is not known how these interactions alter fungal survival during phagocytic killing. We demonstrated that secreted molecules of S. gordonii and P. aeruginosa alter C. albicans survival within the phagosome of macrophages and alter fungal pathogenic phenotypes, including filamentation and microcolony formation. Moreover, we provide evidence for a dual interaction between S. gordonii and C. albicans such that S. gordonii signaling peptides can promote C. albicans commensalism by decreasing microcolony attachment while increasing invasion in epithelial cells. Our results identify bacterial diffusible factors as an attractive target to modify virulence of C. albicans in polymicrobial infections.