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The oral cavity is thought to be one of the portals for SARS-CoV-2 entry. Because there is limited evidence of active oral infection by SARS-CoV-2 viruses, we assessed the capacity of SARS-CoV-2 to infect and replicate in oral epithelial cells. Oral gingival epithelial cells (hTERT TIGKs), salivary gland epithelial cells (A-253), and oral buccal epithelial cells (TR146), which occupy different regions of the oral cavity, were challenged with replication competent SARS-CoV-2 viruses and with pseudo-typed viruses expressing SARS-CoV-2 spike proteins. All oral epithelial cells expressing undetectable or low levels of human angiotensin-converting enzyme 2 (hACE2) but high levels of the alternative receptor CD147 were susceptible to SARS-CoV-2 infection. Viral dynamics in hTERT TIGKs were different from those in A-253 and TR146 cells. For example, levels of viral transcripts were sustained in hTERT TIGKs but were significantly decreased in A-253 and TR146 cells at day 3 after infection. Analysis of oral epithelial cells infected by replication competent SARS-CoV-2 viruses expressing GFP showed that the signals of GFP and SARS-CoV-2 mRNAs were not evenly distributed. Taken together, our results demonstrated oral epithelial cells were susceptible to SARS-CoV-2 viruses despite of low or undetectable levels of hACE2, suggesting that alternative receptors contribute to SARS-CoV-2 infection and may be considered for development of future vaccines and therapeutics.

Mechanical cleaning remains the standard of care for maintaining oral hygiene. However, mechanical cleaning is often augmented with active therapeutics that further promote oral health. A dentifrice, consisting of the "Dual Zinc plus Arginine" (DZA) technology, was found to be effective at controlling bacteria using in vitro laboratory studies, translating to clinical efficacy to deliver plaque and gingivitis reduction benefits. Here, we used biophysical analyses and confocal laser scanning microscopy to understand how a DZA dentifrice impacted the mechanical properties of dental plaque biofilms and determine if changes to biofilm rheology enhanced the removal of dental plaque. Using both uniaxial mechanical indentation and an adapted rotating-disc rheometry assay, it was found that DZA treatment compromised biofilm mechanical integrity, resulting in the biofilm being more susceptible to removal by shear forces compared to treatment with either arginine or zinc alone. Confocal laser scanning microscopy revealed that DZA treatment reduced the amount of extracellular polymeric slime within the biofilm, likely accounting for the reduced mechanical properties. We propose a model where arginine facilitates the entry of zinc into the biofilm, resulting in additive effects of the two activities toward dental plaque biofilms. Together, our results support the use of a dentifrice containing Dual Zinc plus Arginine as part of daily oral hygiene regimens. IMPORTANCE Mechanical removal of dental plaque is augmented with therapeutic compounds to promote oral health. A dentifrice containing the ingredients zinc and arginine has shown efficacy at reducing dental plaque both in vitro and in vivo. However, how these active compounds interact together to facilitate dental plaque removal is unclear. Here, we used a combination of biophysical analyses and microscopy to demonstrate that combined treatment with zinc and arginine targets the matrix of dental plaque biofilms, which destabilized the mechanical integrity of these microbial communities, making them more susceptible to removal by shear forces. Published version

Aims We present a dynamic typodont biofilm model (DTBM) incorporating (1) human dentition anatomy, (2) fluid flow over intermittently fluid bathed tooth surfaces and (3) an oxic headspace to allow aerobic and anaerobic niches to develop naturally, as a screening tool to assess the effect of stannous fluoride (SnF2) toothpaste against a simulated human plaque biofilm (SPB). Methods and results First, hydroxyapatite (HA) coupons were inoculated with human saliva/plaque and cultured at 37°C under air. Selected species representative of common commensal and anaerobic pathogens were quantified for relative abundance changes over 4 days by PCR densitometry to confirm the culture conditions allowed the proliferation of these species. A continuous culture DTBM reactor on a rocker table was inoculated with saliva/plaque and incubated at 37°C for 24 h. Tooth shear stress was estimated by particle tracking. A SnF2 toothpaste solution, or a sham rise was administered twice daily for 3 days to mimic routine oral hygiene. SPB biomass was assessed by total bacterial DNA and methylene blue (MB) staining. Early colonizer aerobes and late colonizer anaerobes species were detected in the HA and DTBM, and the trends in changing abundance were consistent with those seen clinically. Conclusions Treatment with the SnF2 solution showed significant reductions of 53.05% and 54.4% in the SPB by MB staining and DNA, respectively. Significance and impact of study: The model has potential for assessing dentition anatomy and fluid flow on the efficacy of antimicrobial efficacy against localized SPB and may be amenable to the plaque index clinical evaluation.

We present a dynamic typodont biofilm model (DTBM) incorporating (1) human dentition anatomy, (2) fluid flow over intermittently fluid bathed tooth surfaces and (3) an oxic headspace to allow aerobic and anaerobic niches to develop naturally, as a screening tool to assess the effect of stannous fluoride (SnFFirst, hydroxyapatite (HA) coupons were inoculated with human saliva/plaque and cultured at 37°C under air. Selected species representative of common commensal and anaerobic pathogens were quantified for relative abundance changes over 4 days by PCR densitometry to confirm the culture conditions allowed the proliferation of these species. A continuous culture DTBM reactor on a rocker table was inoculated with saliva/plaque and incubated at 37°C for 24 h. Tooth shear stress was estimated by particle tracking. A SnFTreatment with the SnFThe model has potential for assessing dentition anatomy and fluid flow on the efficacy of antimicrobial efficacy against localized SPB and may be amenable to the plaque index clinical evaluation.

Oral diseases are one of the most common pathologies affecting human health. These diseases are typically associated with dental plaque-biofilms, through either build-up of the biofilm or dysbiosis of the microbial community. Arginine can disrupt dental plaque-biofilms, and maintain plaque homeostasis, making it an ideal therapeutic to combat the development of oral disease. Despite our understanding of the actions of arginine towards dental plaque-biofilms, it is still unclear how or if arginine effects the mechanical integrity of the dental plaque-biofilm. Here we adapted a rotating-disc rheometry assay, a method used to quantify marine biofilm fouling, to study how arginine treatment of Streptococcus gordonii biofilms influences biofilm detachment from surfaces. We demonstrate that the assay is highly sensitive at quantifying the presence of biofilm and the detachment or rearrangement of the biofilm structure as a function of shear stress. We demonstrate that arginine treatment leads to earlier detachment of the biofilm, indicating that arginine treatment weakens the biofilm, making it more susceptible to removal by shear stresses. Finally, we demonstrate that the biofilm disrupting affect is specific to arginine, and not a general property of amino acids, as S. gordonii biofilms treated with either glycine or lysine had mechanical properties similar to untreated biofilms. Our results add to the understanding that arginine targets biofilms by multifaceted mechanisms, both metabolic and physical, further promoting the potential of arginine as an active compound in dentifrices to maintain oral health.

Oral diseases are one of the most common pathologies affecting human health. These diseases are typically associated with dental plaque-biofilms, through either build-up of the biofilm or dysbiosis of the microbial community. Arginine can disrupt dental plaque-biofilms, and maintain plaque homeostasis, making it an ideal therapeutic to combat the development of oral disease. Despite our understanding of the actions of arginine towards dental plaque-biofilms, it is still unclear how or if arginine effects the mechanical integrity of the dental plaque-biofilm. Here we adapted a rotating-disc rheometry assay, a method used to quantify marine biofilm fouling, to study how arginine treatment of Streptococcus gordonii biofilms influences biofilm detachment from surfaces. We demonstrate that the assay is highly sensitive at quantifying the presence of biofilm and the detachment or rearrangement of the biofilm structure as a function of shear stress. We demonstrate that arginine treatment leads to earlier detachment of the biofilm, indicating that arginine treatment weakens the biofilm, making it more susceptible to removal by shear stresses. Finally, we demonstrate that the biofilm disrupting affect is specific to arginine, and not a general property of amino acids, as S. gordonii biofilms treated with either glycine or lysine had mechanical properties similar to untreated biofilms. Our results add to the understanding that arginine targets biofilms by multifaceted mechanisms, both metabolic and physical, further promoting the potential of arginine as an active compound in dentifrices to maintain oral health.

To investigate bioavailability enhancement of zinc on model oral surfaces and in oral biofilms in vitro through strategic formulation with two sources of zinc and L-arginine.To modulate the bioavailability of active zinc ions in a zinc citrate dentifrice, an additive research strategy was pursued. A series of zinc citrate dentifrice formulations were prepared with increasing replacement of zinc citrate with zinc oxide (a water insoluble source of zinc ions) to generate a Dual Zinc active system. A screening of isolated zinc and amino acid effects in simple solutions using zeta potential and uptake to model oral surfaces was performed in an effort to determine the effect of particle charge on zinc bioavailability. Zinc delivery and antibacterial efficacy of the Dual Zinc plus Arginine dentifrice formula were tested using in vitro oral epithelial tissue and saliva-derived biofilm models. Furthermore, zinc penetration and retention were determined by subjecting in vitro biofilms to dynamic flow after treatment with the Dual Zinc plus Arginine dentifrice with treated biofilms evaluated for zinc using imaging mass spectrometry (I-MS). Bacterial adhesion to gingival epithelial cells treated with the Dual Zinc plus Arginine dentifrice was imaged upon challenging with Streptococcus gordonii.Addition of zinc oxide into a zinc citrate dentifrice formula enhanced the efficacy of the system against anaerobic biofilms in a concentration- dependent manner. L-arginine further provided a significant positive charge (+36 mV) to the zinc oxide suspension (+16 mV) as measured by zeta potential. Simple solutions of the Dual Zinc active showed increased zinc uptake on model oral surfaces as a direct function of L-arginine concentration. Antibacterial efficacy of a Dual Zinc plus Arginine dentifrice was evaluated through multiple mechanisms. Enhanced antibacterial performance was observed through significant reductions in metabolic activity as measured through bacterial glycolytic function (p = 0.0001) and total oxygen consumption (p = 0.0001). Greater penetration and retention of zinc was observed in bacterial biofilms treated with the Dual Zinc plus Arginine dentifrice in comparison to treatment with a Dual Zinc dentifrice after twelve hours of dynamic flow (10 mL/hour) in an in vitro drip flow biofilm culture. Confocal microscopy showed adherent bacteria on cheek cells treated with the Dual Zinc plus Arginine dentifrice formula.The combination of zinc citrate, zinc oxide, and the amino acid L-arginine in a dentifrice formula enhances the bioavailability of zinc to model oral tissue surfaces, resulting in unique physicochemical effects. The significant antimicrobial control associated with the Dual Zinc plus Arginine dentifrice provides a unique vehicle toward achieving whole mouth health.

The interaction of the periodontal pathogen, Porphyromonas gingivalis, with oral streptococci such as Streptococcus gordonii precedes colonization of the subgingival pocket and represents a target for limiting P. gingivalis colonization of the oral cavity. Previous studies showed that a synthetic peptide (designated BAR) derived from the antigen I/II protein of S. gordonii was a potent competitive inhibitor of P. gingivalis adherence to S. gordonii and subsequent biofilm formation. Here we show that despite its inhibitory activity, BAR is rapidly degraded by intact P. gingivalis cells in vitro. However, in the presence of soluble Mfa protein, the P. gingivalis receptor for BAR, the peptide is protected from proteolytic degradation suggesting that the affinity of BAR for Mfa is higher than for P. gingivalis proteases. The rate of BAR degradation was reduced when the P. gingivalis lysine-specific gingipain was inhibited using the specific protease inhibitor, z-FKcK, or when the gene encoding the Lys-gingipain was inactivated. In addition, substituting d -Lys for l -Lys residues in BAR prevented degradation of the peptide when incubated with the Lys-gingipain and increased its specific adherence inhibitory activity in a S. gordonii–P. gingivalis dual species biofilm model. These results suggest that Lys-gingipain accounts in large part for P. gingivalis-mediated degradation of BAR and that more effective peptide inhibitors of P. gingivalis adherence to streptococci can be produced by introducing modifications that limit the susceptibility of BAR to the Lys-gingipain and other P. gingivalis associated proteases.

Porphyromonas gingivalis initially colonizes the oral cavity by interacting with organisms in supragingival plaque, such as the oralis group of oral streptococci. This interaction involves the association of the streptococcal antigen I/II with the minor fimbrial antigen (Mfa1) of P. gingivalis . Our previous studies showed that a peptide (BAR) derived from antigen I/II inhibits P. gingivalis adherence and subsequent biofilm formation on streptococcal substrates. In addition, screening a combinatorial peptide library identified select amino acid substitutions in the NITVK active region of BAR that increased the adherence of P. gingivalis to streptococci. Here we report that incorporating these residues in a synthetic peptide results in more-potent inhibition of P. gingivalis adherence and biofilm formation (I 50 [50% inhibition] at 0.52 μM versus I 50 at 1.25 μM for BAR). In addition, a second structural motif in BAR, comprised of the amino acids KKVQDLLKK, was shown to contribute to P. gingivalis adherence to streptococci. Consistent with this, the KKVQDLLKK and NITVK motifs are conserved only in antigen I/II proteins expressed by the oralis group of streptococci, which interact with P. gingivalis . Interestingly, the primary and secondary structures and the functional characteristics of the amphipathic VQDLL core α-helix resemble the consensus nuclear receptor (NR) box protein-protein interacting domain sequence (LXXLL) of eukaryotes. BAR peptides containing amino acid substitutions with the potential to disrupt the secondary structure of VQDLL were less-effective inhibitors of P. gingivalis adherence and biofilm formation, suggesting that the α-helical character of VQDLL is important. Furthermore, replacing the lysines that flank VQDLL with acidic amino acids also reduced inhibitory activity, suggesting that the association of VQDLL with Mfa1 may be stabilized by a charge clamp. These results indicate that the Mfa1-interacting interface of streptococcal antigen I/II encompasses both the KKVQDLLKK and NITVK motif and suggest that the adherence of P. gingivalis to streptococci is driven by a protein-protein interaction domain that resembles the eukaryotic NR box. Thus, both motifs must be taken into account in designing potential peptidomimetics that target P. gingivalis adherence and biofilm formation.

The flaviviruses dengue, West Nile, and Japanese encephalitis represent three major mosquito-borne viruses worldwide. These pathogens impact the lives of millions of individuals and potentially could affect non-endemic areas already colonized by mosquito vectors. Unintentional transport of infected vectors (Aedes and Culex spp.), traveling within endemic areas, rapid adaptation of the insects into new geographic locations, climate change, and lack of medical surveillance have greatly contributed to the increase in flaviviral infections worldwide. The mechanisms by which flaviviruses alter the immune and the central nervous system have only recently been examined despite the alarming number of infections, related deaths, and increasing global distribution. In this review, we will discuss the expansion of the geographic areas affected by flaviviruses, the potential threats to previously unaffected countries, the mechanisms of pathogenesis, and the potential therapeutic interventions to limit the devastating consequences of these viruses.

The interaction of the minor fimbrial antigen (Mfa) with streptococcal antigen I/II (e.g., SspB) facilitates colonization of the dental biofilm by Porphyromonas gingivalis. We previously showed that a 27-mer peptide derived from SspB (designated BAR) resembles the nuclear receptor (NR) box protein-protein interacting domain and potently inhibits this interaction in vitro . Here, we show that the EXXP motif upstream of the NR core α-helix contributes to the Mfa-SspB interaction and that BAR reduces P. gingivalis colonization and alveolar bone loss in vivo in a murine model of periodontitis. Substitution of Gln for Pro 1171 or Glu 1168 increased the α-helicity of BAR and reduced its inhibitory activity in vitro by 10-fold and 2-fold, respectively. To determine if BAR prevents P. gingivalis infection in vivo , mice were first infected with Streptococcus gordonii and then challenged with P. gingivalis in the absence and presence of BAR. Animals that were infected with either 10 9 CFU of S. gordonii DL-1 or 10 7 CFU of P. gingivalis 33277 did not show a statistically significant increase in alveolar bone resorption over sham-infected controls. However, infection with 10 9 CFU of S. gordonii followed by 10 7 CFU of P. gingivalis induced significantly greater bone loss ( P < 0.01) than sham infection or infection of mice with either organism alone. S. gordonii -infected mice that were subsequently challenged with 10 7 CFU of P. gingivalis in the presence of BAR exhibited levels of bone resorption similar to those of sham-infected animals. Together, these results indicate that both EXXP and the NR box are important for the Mfa-SspB interaction and that BAR peptide represents a potential therapeutic that may limit colonization of the oral cavity by P. gingivalis .

Biofilm formation by the periodontal pathogen Aggregatibacter actinomycetemcomitans is dependent upon autoinducer-2 (AI-2)-mediated quorum sensing. However, the components that link the detection of the AI-2 signal to downstream gene expression have not been determined. One potential regulator is the QseBC two-component system, which is part of the AI-2-dependent response pathway that controls biofilm formation in Escherichia coli . Here we show that the expression of QseBC in A. actinomycetemcomitans is induced by AI-2 and that induction requires the AI-2 receptors, LsrB and/or RbsB. Additionally, inactivation of qseC resulted in reduced biofilm growth. Since the ability to grow in biofilms is essential for A. actinomycetemcomitans virulence, strains that were deficient in QseC or the AI-2 receptors were examined in an in vivo mouse model of periodontitis. The Δ qseC mutant induced significantly less alveolar bone resorption than the wild-type strain ( P < 0.02). Bone loss in animals infected with the Δ qseC strain was similar to that in sham-infected animals. The Δ lsrB , Δ rbsB , and Δ lsrB Δ rbsB strains also induced significantly less alveolar bone resorption than the wild type ( P < 0.03, P < 0.02, and P < 0.01, respectively). However, bone loss induced by a Δ luxS strain was indistinguishable from that induced by the wild type, suggesting that AI-2 produced by indigenous microflora in the murine oral cavity may complement the Δ luxS mutation. Together, these results suggest that the QseBC two-component system is part of the AI-2 regulon and may link the detection of AI-2 to the regulation of downstream cellular processes that are involved in biofilm formation and virulence of A. actinomycetemcomitans .

Porphyromonas gingivalis is a periodontal pathogen whose primary niche is the anaerobic environment of subgingival dental plaque, but initial colonization of the oral cavity is likely to occur on supragingival surfaces that already support robust biofilm communities. Our studies have shown that P. gingivalis adheres to Streptococcus gordonii through interaction of the minor fimbrial antigen Mfa1 with a specific region of the streptococcal SspB polypeptide (residues 1167 to 1193) designated BAR. We show that a synthetic peptide comprising the BAR sequence potently inhibits P. gingivalis adherence to S. gordonii (50% inhibitory concentration = 1.3 μM) and prevents the development of P. gingivalis biofilms. However, a retroinverso peptide that possessed the same side chain topology as that of BAR was inactive, suggesting that interactions of Mfa1 with the peptide backbone of BAR are important for binding. A conformationally constrained analog of BAR inhibited P. gingivalis adherence and biofilm formation but at a lower specific activity than that of BAR. Therefore, to further define the structural features of the Mfa1-BAR interaction, we functionally screened combinatorial libraries of BAR in which active site residues (Asn 1182 , Thr 1184 , and Val 1185 ) were replaced with each of the 19 common amino acids. Peptides containing positively charged amino acids at position 1182 or hydrophobic residues at position 1185 bound P. gingivalis more efficiently than did control peptides containing Asn and Val at these positions, suggesting that electrostatic and hydrophobic interactions may contribute to Mfa1-SspB binding. In contrast, replacement of Pro or Gly at these positions was detrimental to adherence, suggesting that perturbation of the BAR secondary structure influences activity. The net effect of substitutions for Thr 1184 was less pronounced either positively or negatively than that at the other sites. These results define physicochemical characteristics of the interacting interface of Mfa1 and SspB and suggest that peptides or peptidomimetics with greater specific inhibitory activity than that of BAR can be developed. These compounds may represent potential therapeutics that target some of the first molecular interactions that allow P. gingivalis to colonize the oral cavity.

Smoking is responsible for the majority of periodontitis cases in the US and smokers are more susceptible than non-smokers to infection by the periodontal pathogen Porphyromonas gingivalis. P. gingivalis colonization of the oral cavity is dependent upon its interaction with other plaque bacteria, including Streptococcus gordonii. Microarray analysis suggested that exposure of P. gingivalis to cigarette smoke extract (CSE) increased the expression of the major fimbrial antigen (FimA), but not the minor fimbrial antigen (Mfa1). Therefore, we hypothesized that CSE promotes P. gingivalis-S. gordonii biofilm formation in a FimA-dependent manner. FimA total protein and cell surface expression were increased upon exposure to CSE whereas Mfa1 was unaffected. CSE exposure did not induce P. gingivalis auto-aggregation but did promote dual species biofilm formation, monitored by microcolony numbers and depth (both, p