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.

Sarcoidosis Associated with Enlarged Mediastinal Lymph Nodes with the Detection of Streptococcus gordonii and Cutibacterium acnes Using a Clone Library Method.

A 77-year-old Japanese woman with mediastinal lymphadenopathy and uveitis was diagnosed with sarcoidosis. The bacterial flora in biopsied samples from mediastinal lymph nodes was analyzed using a clone library method with Sanger sequencing of the 16S rRNA gene, and Streptococcus gordonii (52 of 71 clones) and Cutibacterium acnes (19 of 71 clones) were detected. No previous study has conducted a bacterial floral analysis using the Sanger method for the mediastinal lymph node in sarcoidosis, making this case report the first to document the presence of S. gordonii and C. acnes in the mediastinal lymph node of a patient with sarcoidosis.

Transcriptome and metabolome analyses of Streptococcus gordonii DL1 under acidic conditions.

OBJECTIVES: Streptococcus gordonii is associated with the formation of biofilms, especially those that comprise dental plaque. Notably, S. gordonii DL1 causes infective endocarditis (IE). Colonization of this bacterium requires a mechanism that can tolerate a drop in environmental pH by producing acid via its own sugar metabolism. The ability to survive acidic environmental conditions might allow the bacterium to establish vegetative colonization even in the endocardium due to inflammation-induced lowering of pH, increasing the risk of IE. At present, the mechanism by which S. gordonii DL1 survives under acidic conditions is not thoroughly elucidated. The present study was thus conducted to elucidate the mechanism(s) by which S. gordonii DL1 survives under acidic conditions. METHODS: We analyzed dynamic changes in gene transcription and intracellular metabolites in S. gordonii DL1 exposed to acidic conditions, using transcriptome and metabolome analyses. RESULTS: Transcriptome analysis revealed upregulation of genes involved in heat shock response and glycolysis, and down regulation of genes involved in phosphotransferase systems and biosynthesis of amino acids. The most upregulated genes were a beta-strand repeat protein of unknown function (SGO_RS06325), followed by copper-translocating P-type ATPase (SGO_RS09470) and malic enzyme (SGO_RS01850). The latter two of these contribute to cytoplasmic alkalinization. S. gordonii mutant strains lacking each of these genes showed significantly reduced survival under acidic conditions. Metabolome analysis revealed that cytoplasmic levels of several amino acids were reduced. CONCLUSIONS: S. gordonii survives the acidic conditions by recovering the acidic cytoplasm using the various activities, which are regulated at the transcriptional level.

Structural and dynamical changes of the Streptococcus gordonii metalloregulatory ScaR protein induced by Mn2+ ion binding.

Divalent metal ions are essential micronutrients for many intercellular reactions. Maintaining their homeostasis is necessary for the survival of bacteria. In Streptococcus gordonii, one of the primary colonizers of the tooth surface, the cellular concentration of manganese ions (Mn2+) is regulated by the manganese-sensing transcriptional factor ScaR which controls the expression of proteins involved in manganese homeostasis. To resolve the molecular mechanism through which the binding of Mn2+ ions increases the binding affinity of ScaR to DNA, a variety of computational (QM and MD) and experimental (ITC, DSC, EMSA, EPR, and CD) methods were applied. The computational results showed that Mn2+ binding induces a conformational change in ScaR that primarily affects the position of the DNA binding domains and, consequently, the DNA binding affinity of the protein. In addition, experimental results revealed a 1:4 binding stoichiometry between ScaR dimer and Mn2+ ions, while the computational results showed that the binding of Mn2+ ions in the primary binding sites is sufficient to induce the observed conformational change of ScaR.

Impact of Polymicrobial Infection on Fitness of Streptococcus gordonii In Vivo.

Pathogenic microbial ecosystems are often polymicrobial, and interbacterial interactions drive emergent properties of these communities. In the oral cavity, Streptococcus gordonii is a foundational species in the development of plaque biofilms, which can contribute to periodontal disease and, after gaining access to the bloodstream, target remote sites such as heart valves. Here, we used a transposon sequencing (Tn-Seq) library of S. gordonii to identify genes that influence fitness in a murine abscess model, both as a monoinfection and as a coinfection with an oral partner species, Porphyromonas gingivalis. In the context of a monoinfection, conditionally essential genes were widely distributed among functional pathways. Coinfection with P. gingivalis almost completely changed the nature of in vivo gene essentiality. Community-dependent essential (CoDE) genes under the coinfection condition were primarily related to DNA replication, transcription, and translation, indicating that robust growth and replication are required to survive with P. gingivalis in vivo. Interestingly, a group of genes in an operon encoding streptococcal receptor polysaccharide (RPS) were associated with decreased fitness of S. gordonii in a coinfection with P. gingivalis. Individual deletion of two of these genes (SGO_2020 and SGO_2024) resulted in the loss of RPS production by S. gordonii and increased susceptibility to killing by neutrophils. P. gingivalis protected the RPS mutants by inhibiting neutrophil recruitment, degranulation, and neutrophil extracellular trap (NET) formation. These results provide insight into genes and functions that are important for S. gordonii survival in vivo and the nature of polymicrobial synergy with P. gingivalis. Furthermore, we show that RPS-mediated immune protection in S. gordonii is dispensable and detrimental in the presence of a synergistic partner species that can interfere with neutrophil killing mechanisms. IMPORTANCE Bacteria responsible for diseases originating at oral mucosal membranes assemble into polymicrobial communities. However, we know little regarding the fitness determinants of the organisms that initiate community formation. Here, we show that the extracellular polysaccharide of Streptococcus gordonii, while important for streptococcal survival as a monoinfection, is detrimental to survival in the context of a coinfection with Porphyromonas gingivalis. We found that the presence of P. gingivalis compensates for immune protective functions of extracellular polysaccharide, rendering production unnecessary. The results show that fitness determinants of bacteria in communities differ substantially from those of individual species in isolation. Furthermore, constituents of communities can undertake activities that relieve the burden of energetically costly biosynthetic reactions on partner species.

Streptococcus mutans dexA affects exopolysaccharides production and biofilm homeostasis.

OBJECTIVES: The study aimed to evaluate the role of Streptococcus mutans (S. mutans) dexA gene on biofilm structure and microecological distribution in multispecies biofilms. MATERIALS AND METHODS: A multispecies biofilm model consisting of S. mutans and its dexA mutants, Streptococcus gordonii (S. gordonii) and Streptococcus sanguinis (S. sanguinis) was constructed, and bacterial growth, biofilm architecture and microbiota composition were determined to study the effect of the S. mutans dexA on multispecies biofilms. RESULTS: Our results showed that either deletion or overexpression of S. mutans dexA had no effect on the planktonic growth of bacterium, while S. mutans dominated in the multispecies biofilms to form cariogenic biofilms. Furthermore, we revealed that the SmudexA+ group showed structural abnormality in the form of more fractures and blank areas. The morphology of the SmudexA group was sparser and more porous, with reduced and less agglomerated exopolysaccharides scaffold. Interestingly, the microbiota composition analysis provided new insights that the inhibition of S. gordonii and S. sanguinis was alleviated in the SmudexA group compared to the significantly suppressed condition in the other groups. CONCLUSION: In conclusion, deletion of S. mutans dexA gene re-modules biofilm structure and microbiota composition, thereby leading to decreased cariogenicity. Thus, the S. mutans dexA may be an important target for regulating the cariogenicity of dental plaque biofilms, expecting to be a probiotic for caries control.

Interspecies competition in oral biofilms mediated by Streptococcus gordonii extracellular deoxyribonuclease SsnA.

Extracellular DNA (eDNA) is a key component of many microbial biofilms including dental plaque. However, the roles of extracellular deoxyribonuclease (DNase) enzymes within biofilms are poorly understood. Streptococcus gordonii is a pioneer colonizer of dental plaque. Here, we identified and characterised SsnA, a cell wall-associated protein responsible for extracellular DNase activity of S. gordonii. The SsnA-mediated extracellular DNase activity of S. gordonii was suppressed following growth in sugars. SsnA was purified as a recombinant protein and shown to be inactive below pH 6.5. SsnA inhibited biofilm formation by Streptococcus mutans in a pH-dependent manner. Further, SsnA inhibited the growth of oral microcosm biofilms in human saliva. However, inhibition was ameliorated by the addition of sucrose. Together, these data indicate that S. gordonii SsnA plays a key role in interspecies competition within oral biofilms. Acidification of the medium through sugar catabolism could be a strategy for cariogenic species such as S. mutans to prevent SsnA-mediated exclusion from biofilms.

The Regulatory Effect of Coaggregation Between Fusobacterium nucleatum and Streptococcus gordonii on the Synergistic Virulence to Human Gingival Epithelial Cells.

In subgingival plaque biofilms, Fusobacterium nucleatum is closely related to the occurrence and development of periodontitis. Streptococcus gordonii, as an accessory pathogen, can coaggregate with periodontal pathogens, facilitating the subgingival colonization of periodontal pathogens. Studies have shown that F. nucleatum can coaggregate with S. gordonii and colonize the subgingival plaque. However, most studies have focused on monocultures or coinfection of species and the potential impact of coaggregation between the two species on periodontal interactions to human gingival epithelial cells (hGECs) remains poorly understood. The present study explored the effect of coaggregation between F. nucleatum and S. gordonii on subgingival synergistic virulence to hGECs. The results showed that coaggregation inhibited the adhesion and invasion of F. nucleatum to hGECs compared with that in the F. nucleatum monoculture and coinfection group. Coaggregation and coinfection with F. nucleatum both enhanced S. gordonii adhesion to hGECs, but neither of the two groups affected S. gordonii invasion to hGECs compared with S. gordonii monoculture. The gene expression levels of TLR2 and TLR4 in hGECs in the coaggregation group were higher than those in the monoculture groups but lower than those in the coinfection group. Compared with coinfection, the coaggregation inhibited apoptosis of hGECs and promoted the secretion of the proinflammatory cytokines TNF-α and IL-6 by hGECs, showed a synergistic inflammatory effect, while coaggregation inhibited the secretion of the anti-inflammatory cytokine TGF-ß1. Coaggregation enhanced the phosphorylation of p65, p38, and JNK proteins and therefore activated the NF-κB and MAPK signaling pathways. Pretreatment with a pathway antagonist/inhibitor decreased the phosphorylation levels of proteins and the secretion of TNF-α and IL-6. In conclusion, coaggregation inhibited the adhesion and invasion of F. nucleatum to hGECs. However, it enhanced the adhesion of S. gordonii to hGECs. Compared with coinfection, coaggregation inhibited the apoptosis of hGECs. The coaggregation coordinately promoted the secretion of TNF-α and IL-6 by hGECs through the TLR/NF-κB and TLR/MAPK signaling pathways while inhibiting the secretion of TGF-ß1, thus aggravating the inflammatory response of hGECs.

The Role of Glycoside Hydrolases in S. gordonii and C. albicans Interactions.

Candida albicans can coaggregate with Streptococcus gordonii and cocolonize in the oral cavity. Saliva provides a vital microenvironment for close interactions of oral microorganisms. However, the level of fermentable carbohydrates in saliva is not sufficient to support the growth of multiple species. Glycoside hydrolases (GHs) that hydrolyze glycoproteins are critical for S. gordonii growth in low-fermentable-carbohydrate environments such as saliva. However, whether GHs are involved in the cross-kingdom interactions between C. albicans and S. gordonii under such conditions remains unknown. In this study, C. albicans and S. gordonii were cocultured in heart infusion broth with a low level of fermentable carbohydrate. Planktonic growth, biofilm formation, cell aggregation, and GH activities of monocultures and cocultures were examined. The results revealed that the planktonic growth of cocultured S. gordonii in a low-carbohydrate environment was elevated, while that of cocultured C. albicans was reduced. The biomass of S. gordonii in dual-species biofilms was higher than that of monocultures, while that of cocultured C. albicans was decreased. GH activity was observed in S. gordonii, and elevated activity of GHs was detected in S. gordonii-C. albicans cocultures, with elevated expression of GH-related genes of S. gordonii. By screening a mutant library of C. albicans, we identified a tec1Δ/Δ mutant strain that showed reduced ability to promote the growth and GH activities of S. gordonii compared with the wild-type strain. Altogether, the findings of this study demonstrate the involvement of GHs in the cross-kingdom metabolic interactions between C. albicans and S. gordonii in an environment with low level of fermentable carbohydrates. IMPORTANCE Cross-kingdom interactions between Candida albicans and oral streptococci such as Streptococcus gordonii have been reported. However, their interactions in a low-fermentable-carbohydrate environment like saliva is not clear. The current study revealed glycoside hydrolase-related cross-kingdom communications between S. gordonii and C. albicans under the low-fermentable-carbohydrate condition. We demonstrate that C. albicans can promote the growth and metabolic activities of S. gordonii by elevating the activities of cell-wall-anchored glycoside hydrolases of S. gordonii. C. albicans gene TEC1 is critical for this cross-kingdom metabolic communication.

Hemagglutinating properties of a Streptococcus gordonii strain expressing sialic acid-binding adhesin homolog with low binding site similarity to that of strain DL1.

OBJECTIVES: The Hsa adhesin of Streptococcus gordonii strain DL1 was previously identified as a hemagglutinin that binds specifically to sialoglycoconjugates. We recently found that among oral streptococcal species, S. gordonii strains most frequently express Hsa homologs on the bacterial cell surface. However, the effect of amino acid sequence diversity of nonrepetitive region 2 (NR2), a putative binding site of Hsa, on antigenicity and hemagglutinating (HA) properties is unclear due to difficulties in DNA sequencing the NR2 coding region. The aim of this study was to elucidate the similarity of the low NR2 antigenicity Hsa homolog of strain NDU1118 to that of strain DL1 and the association of the homolog with HA properties of the strain. METHODS: The hsa homolog of NDU1118 was sequenced using a long-read next-generation sequencer, and the Hsa homolog was assessed by alignment analysis of the deduced amino acid sequences. The hsa mutant of NDU1118 was generated by insertion of the erythromycin resistance gene. The HA properties of the wild type and the hsa mutant were assessed with human erythrocytes. RESULTS: The NR2 amino acid sequence of the NDU1118 Hsa homolog was almost identical to that of the S. gordonii M99 Hsa homolog, also known as GspB, and less similar to that of DL1 Hsa. The hsa mutation of NDU1118 induced reduction of HA activity in untreated erythrocytes, but surprisingly increased lactose-inhibitable HA activity in neuraminidase-treated erythrocytes. CONCLUSIONS: The results suggest the existence of an adhesin other than the Hsa homolog on the cell surface of NDU1118.

Expression and diversity of the sialic acid-binding adhesin and its homologs associated with oral streptococcal infection.

Streptococcus gordonii, one of the early colonizers of oral biofilms, is involved in the development of dental caries, periodontal disease, and infective endocarditis. The Hsa adhesin of S. gordonii DL1 has the ability to bind strongly to the terminal sialic acid groups of host glycoproteins via the binding region, nonrepetitive region 2 (NR2), which is important for the pathogenicity of S. gordonii DL1. Low similarity with the NR2 of Hsa homologs among other streptococcal species has been reported. However, the reports have been limited to certain strains. This study attempted to assess frequency of the expression on the bacterial cell surface and to analyze the diversity of Hsa homologs among different wild strains of oral streptococci. We isolated 186 wild-type strains of oral streptococci from healthy volunteers and analyzed their hemagglutinating (HA) activity on human erythrocytes and their Hsa homologs and NR2 homologous regions by dot immunoblotting using anti-Hsa and anti-NR2 antisera, respectively. We found 30 strains reacted with anti-NR2 antiserum (NR2 positive) and determined the sequence of the NR2 regions. Many strains with high HA activity were also NR2 positive, suggesting that the NR2 region may be associated with HA activity. Among the NR2-positive strains, four different amino acid sequence patterns were observed, demonstrating diversity in the NR2 region. Notably, S. gordonii strains frequently possessed Hsa homologs and NR2-like antigens compared with other streptococci. It is speculated that the possessing frequency of Hsa homologs and the amino acid sequence of NR2 region may vary among streptococcal species.

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.

Transcriptomic Responses to Coaggregation between Streptococcus gordonii and Streptococcus oralis.

Cell-cell adhesion between oral bacteria plays a key role in the development of polymicrobial communities such as dental plaque. Oral streptococci such as Streptococcus gordonii and Streptococcus oralis are important early colonizers of dental plaque and bind to a wide range of different oral microorganisms, forming multispecies clumps or "coaggregates." S. gordonii actively responds to coaggregation by regulating gene expression. To further understand these responses, we assessed gene regulation in S. gordonii and S. oralis following coaggregation in 25% human saliva. Coaggregates were formed by mixing, and after 30 min, RNA was extracted for dual transcriptome sequencing (RNA-Seq) analysis. In S. oralis, 18 genes (6 upregulated and 12 downregulated) were regulated by coaggregation. Significantly downregulated genes encoded functions such as amino acid and antibiotic biosynthesis, ribosome, and central carbon metabolism. In total, 28 genes were differentially regulated in Streptococcus gordonii (25 upregulated and 3 downregulated). Many genes associated with transporters and a two-component (NisK/SpaK) regulatory system were upregulated following coaggregation. Our comparative analyses of S. gordonii-S. oralis with different previously published S. gordonii pairings (S. gordonii-Fusobacterium nucleatum and S. gordonii-Veillonella parvula) suggest that the gene regulation is specific to each pairing, and responses do not appear to be conserved. This ability to distinguish between neighboring bacteria may be important for S. gordonii to adapt appropriately during the development of complex biofilms such as dental plaque. IMPORTANCE Dental plaque is responsible for two of the most prevalent diseases in humans, dental caries and periodontitis. Controlling the formation of dental plaque and preventing the transition from oral health to disease requires a detailed understanding of microbial colonization and biofilm development. Streptococci are among the most common colonizers of dental plaque. This study identifies key genes that are regulated when oral streptococci bind to one another, as they do in the early stages of dental plaque formation. We show that specific genes are regulated in two different oral streptococci following the formation of mixed-species aggregates. The specific responses of S. gordonii to coaggregation with S. oralis are different from those to coaggregation with other oral bacteria. Targeting the key genes that are upregulated during interspecies interactions may be a powerful approach to control the development of biofilm and maintain oral health.

Periscope Proteins are variable-length regulators of bacterial cell surface interactions.

Changes at the cell surface enable bacteria to survive in dynamic environments, such as diverse niches of the human host. Here, we reveal "Periscope Proteins" as a widespread mechanism of bacterial surface alteration mediated through protein length variation. Tandem arrays of highly similar folded domains can form an elongated rod-like structure; thus, variation in the number of domains determines how far an N-terminal host ligand binding domain projects from the cell surface. Supported by newly available long-read genome sequencing data, we propose that this class could contain over 50 distinct proteins, including those implicated in host colonization and biofilm formation by human pathogens. In large multidomain proteins, sequence divergence between adjacent domains appears to reduce interdomain misfolding. Periscope Proteins break this "rule," suggesting that their length variability plays an important role in regulating bacterial interactions with host surfaces, other bacteria, and the immune system.

O-acetylation controls the glycosylation of bacterial serine-rich repeat glycoproteins.

The serine-rich repeat (SRR) glycoproteins of gram-positive bacteria are a family of adhesins that bind to a wide range of host ligands, and expression of SRR glycoproteins is linked with enhanced bacterial virulence. The biogenesis of these surface glycoproteins involves their intracellular glycosylation and export via the accessory Sec system. Although all accessory Sec components are required for SRR glycoprotein export, Asp2 of Streptococcus gordonii also functions as an O-acetyltransferase that modifies GlcNAc residues on the SRR adhesin gordonii surface protein B (GspB). Because these GlcNAc residues can also be modified by the glycosyltransferases Nss and Gly, it has been unclear whether the post-translational modification of GspB is coordinated. We now report that acetylation modulates the glycosylation of exported GspB. Loss of O-acetylation due to aps2 mutagenesis led to the export of GspB glycoforms with increased glucosylation of the GlcNAc moieties. Linkage analysis of the GspB glycan revealed that both O-acetylation and glucosylation occurred at the same C6 position on GlcNAc residues and that O-acetylation prevented Glc deposition. Whereas streptococci expressing nonacetylated GspB with increased glucosylation were significantly reduced in their ability to bind human platelets in vitro, deletion of the glycosyltransferases nss and gly in the asp2 mutant restored platelet binding to WT levels. These findings demonstrate that GlcNAc O-acetylation controls GspB glycosylation, such that binding via this adhesin is optimized. Moreover, because O-acetylation has comparable effects on the glycosylation of other SRR adhesins, acetylation may represent a conserved regulatory mechanism for the post-translational modification of the SRR glycoprotein family.

Starvation Survival and Biofilm Formation under Subminimum Inhibitory Concentration of QAMs.

Quaternary ammonium methacrylates (QAMs) are useful antimicrobial compounds against oral bacteria. Here, we investigated the effects of two QAMs, dimethylaminododecyl methacrylate (DMADDM) and dimethylaminohexadecyl methacrylate (DMAHDM), on biofilm formation, survival and development of tolerance by biofilm, and survival and development of tolerance against QAMs after prolonged starvation. Enterococcus faecalis (E. faecalis), Streptococcus gordonii (S. gordonii), Lactobacillus acidophilus (L. acidophilus), and Actinomyces naeslundii (A. naeslundii) were used. Minimum inhibitory concentration (MIC) of QAMs against multispecies biofilm was determined. Biofilm formed under sub-MIC was observed by crystal violet staining and confocal laser scanning microscopy (CLSM). Metabolic activity was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and lactic acid production measurement. Development of tolerance was determined by MIC values before and after exposure to QAMs or after prolonged starvation. It was found that E. faecalis and S. gordonii could survive and form biofilm under sub-MIC of QAMs. Lactic acid production from biofilms formed under sub-MIC was significantly higher than control specimens (p < 0.05). The exposure to sub-MIC of QAMs promoted biofilm formation, and prolonged starvation or prolonged contact with sub-MIC helped bacteria develop tolerance against killing by QAMs.

Interactions Between Streptococcus gordonii and Fusobacterium nucleatum Altered Bacterial Transcriptional Profiling and Attenuated the Immune Responses of Macrophages.

Interspecies coaggregation promotes transcriptional changes in oral bacteria, affecting bacterial pathogenicity. Streptococcus gordonii (S. gordonii) and Fusobacterium nucleatum (F. nucleatum) are common oral inhabitants. The present study investigated the transcriptional profiling of S. gordonii and F. nucleatum subsp. polymorphum in response to the dual-species coaggregation using RNA-seq. Macrophages were infected with both species to explore the influence of bacterial coaggregation on both species' abilities to survive within macrophages and induce inflammatory responses. Results indicated that, after the 30-min dual-species coaggregation, 116 genes were significantly up-regulated, and 151 genes were significantly down-regulated in S. gordonii; 97 genes were significantly down-regulated, and 114 genes were significantly up-regulated in F. nucleatum subsp. polymorphum. Multiple S. gordonii genes were involved in the biosynthesis and export of cell-wall proteins and carbohydrate metabolism. F. nucleatum subsp. polymorphum genes were mostly associated with translation and protein export. The coaggregation led to decreased expression levels of genes associated with lipopolysaccharide and peptidoglycan biosynthesis. Coaggregation between S. gordonii and F. nucleatum subsp. polymorphum significantly promoted both species' intracellular survival within macrophages and attenuated the production of pro-inflammatory cytokines IL-6 and IL-1ß. Physical interactions between these two species promoted a symbiotic lifestyle and repressed macrophage's killing and pro-inflammatory responses.

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.

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.