SARS-CoV-2 Infection of Salivary Glands Compromises Oral Antifungal Innate Immunity and Predisposes to Oral Candidiasis.

Saliva contains antimicrobial peptides considered integral components of host innate immunity, and crucial for protection against colonizing microbial species. Most notable is histatin-5 which is exclusively produced in salivary glands with uniquely potent antifungal activity against the opportunistic pathogen Candida albicans. Recently, SARS-CoV-2 was shown to replicate in salivary gland acinar cells eliciting local immune cell activation. In this study, we performed mechanistic and clinical studies to investigate the implications of SARS-CoV-2 infection on salivary histatin-5 production and Candida colonization. Bulk RNA-sequencing of parotid salivary glands from COVID-19 autopsies demonstrated statistically significant decreased expression of histatin genes. In situ hybridization, coupled with immunofluorescence for co-localization of SARS-CoV-2 spike and histatin in salivary gland cells, showed that histatin was absent or minimally present in acinar cells with replicating viruses. To investigate the clinical implications of these findings, salivary histatin-5 levels and oral Candida burden in saliva samples from three independent cohorts of mild and severe COVID-19 patients and matched healthy controls were evaluated. Results revealed significantly reduced histatin-5 in SARS-CoV-2 infected subjects, concomitant with enhanced prevalence of C. albicans. Analysis of prospectively recovered samples indicated that the decrease in histatin-5 is likely reversible in mild-moderate disease as concentrations tended to increase during the post-acute phase. Importantly, salivary cytokine profiling demonstrated correlations between activation of the Th17 inflammatory pathway, changes in histatin-5 concentrations, and subsequent clearance of C. albicans in a heavily colonized subject. The importance of salivary histatin-5 in controlling the proliferation of C. albicans was demonstrated using an ex vivo assay where C. albicans was able to proliferate in COVID-19 saliva with low histatin-5, but not with high histatin-5. Taken together, the findings from this study provide direct evidence implicating SARS-CoV-2 infection of salivary glands with compromised oral innate immunity, and potential predisposition to oral candidiasis.

Functional Redundancy in Candida auris Cell Surface Adhesins Crucial for Cell-Cell Interaction and Aggregation.

Candida auris is an emerging nosocomial fungal pathogen associated with life-threatening invasive disease due to its persistent colonization, high level of transmissibility and multi-drug resistance. Aggregative and non-aggregative growth phenotypes for C. auris strains with different biofilm forming abilities, drug susceptibilities and virulence characteristics have been described. Using comprehensive transcriptional analysis we identified key cell surface adhesins that were highly upregulated in the aggregative phenotype during in vitro and in vivo grown biofilms using a mouse model of catheter infection. Phenotypic and functional evaluations of generated null mutants demonstrated crucial roles for the adhesins Als5 and Scf1 in mediating cell-cell adherence, coaggregation and biofilm formation. While individual mutants were largely non-aggregative, in combination cells were able to co-adhere and aggregate, as directly demonstrated by measuring cell adhesion forces using single-cell atomic force spectroscopy. This co-adherence indicates their role as complementary adhesins, which despite their limited similarity, may function redundantly to promote cell-cell interaction and biofilm formation. Functional diversity of cell wall proteins may be a form of regulation that provides the aggregative phenotype of C. auris with flexibility and rapid adaptation to the environment, potentially impacting persistence and virulence.

Functional Redundancy in Candida auris Cell Surface Adhesins Crucial for Cell-Cell Interaction and Aggregation.

Candida auris is an emerging nosocomial fungal pathogen associated with life-threatening invasive disease due to its persistent colonization, high level of transmissibility and multi-drug resistance. Aggregative and non-aggregative growth phenotypes for C. auris strains with different biofilm forming abilities, drug susceptibilities and virulence characteristics have been described. Using comprehensive transcriptional analysis we identified key cell surface adhesins that were highly upregulated in the aggregative phenotype during in vitro and in vivo grown biofilms using a mouse model of catheter infection. Phenotypic and functional evaluations of generated null mutants demonstrated crucial roles for the adhesins Als5 and Scf1 in mediating cell-cell adherence, coaggregation and biofilm formation. While individual mutants were largely non-aggregative, in combination cells were able to co-adhere and aggregate, as directly demonstrated by measuring cell adhesion forces using single-cell atomic force spectroscopy. This co-adherence indicates their role as complementary adhesins, which despite their limited similarity, may function redundantly to promote cell-cell interaction and biofilm formation. Functional diversity of cell wall proteins may be a form of regulation that provides the aggregative phenotype of C. auris with flexibility and rapid adaptation to the environment, potentially impacting persistence and virulence.

In vitro demineralization prevention by fluoride and silver nanoparticles when applied to sound enamel and enamel caries-like lesions of varying severities.

OBJECTIVES: To investigate the effect of fluoride and silver nanoparticles on the prevention of in vitro demineralization of sound enamel and enamel caries-like lesions of varying severities. METHODS: Caries-like lesions of different severities (1/6/15 days) were created in bovine enamel specimens. One group remained sound. All specimens were demineralized again using a partially saturated acetic acid solution. Mimicking the intra-oral retention of fluoride and silver in vitro, this solution was supplemented with fluoride (0/1/10 ppm) and/or silver nanoparticles (0/10 ppm) in a factorial design. Changes in lesion depth (ΔL) and integrated mineral loss (ΔΔZ) were evaluated by digital transverse microradiography. Data was analyzed using three-way ANOVA. RESULTS: Lesion severity significantly affected ΔΔZ and ΔL, after no treatment and after the treatment of fluoride and silver independently (p = 0.012 and p = 0.037, respectively). Fluoride and the fluoride × lesion severity interaction were shown to be significant (p < 0.001) on ΔΔZ and ΔL. Silver nanoparticles significantly affected ΔΔZ (p = 0.041), but not ΔL (p = 0.15). The silver nanoparticles × lesion severity interaction was significant for ΔΔZ and ΔL (p = 0.032 and p = 0.024, respectively). No interaction was observed for ΔΔZ and ΔL between fluoride and silver (p = 0.962 and p = 0.971, respectively) as well as lesion severity and the use of fluoride and silver combined (p = 0.722 and p = 0.158, respectively). CONCLUSION: Fluoride and silver nanoparticles had a significant effect on the prevention of in vitro demineralization of sound enamel and enamel caries-like lesions of varying severities. CLINICAL SIGNIFICANCE: Fluoride and silver nanoparticles may potentially allow for more tailored caries prevention.

Impact of caffeine on metabolic activity and biofilm formation of Candida albicans on acrylic denture resin in the presence of nicotine.

STATEMENT OF PROBLEM: Candida albicans has been implicated in denture stomatitis, and this effect is exacerbated by nicotine exposure. However, studies have also suggested that caffeine exposure inhibits the growth of C. albicans. The interaction effects of nicotine and caffeine are not yet clear on the growth of C. albicans. PURPOSE: The purpose of this in vitro study was to determine the effect of caffeine on metabolic activity and biofilm formation of C. albicans growing on acrylic denture resin while simultaneously exposed to nicotine and, if an effect were to be identified, whether this effect would vary depending on the caffeine concentration. MATERIAL AND METHODS: A total of 240 acrylic resin specimens were divided into 2 equal groups (120 each). Specimens in one group were processed to measure C. albicans metabolic activity, and those in the other group were processed to measure C. albicans biofilm attachment. Ten subgroups (n=12) were established within each group with different concentration combinations of nicotine and caffeine to test the interaction effect. The first subgroup was designed as a negative control, containing 0 mg/mL of nicotine and caffeine. The following subgroups all contained 8.00 mg/mL of nicotine, and the caffeine concentrations were prepared at the following 9 levels: 0, 0.25, 0.50, 1.00, 2.00, 4.00, 8.00, 16.00, and 32.00 mg/mL. Metabolic activity was measured by using a 2,3-bis (2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-carboxanilide (XTT) assay. Biofilm attachment was measured by using spiral plating and calculated in terms of the number of colony-forming units (CFUs)/mL. Descriptive statistics and a 2-way ANOVA were conducted to determine whether the concentrations of nicotine and caffeine used affected the biofilm attachment and metabolic activity of C. albicans (α=.05). RESULTS: The presence of 8 mg/mL of nicotine increased the metabolic activity and biofilm formation of C. albicans. When compared with the 0 mg/mL of caffeine and 8.00 mg/mL of nicotine group, caffeine from 1.00 to 4.00 mg/mL significantly increased C. albicans biofilm metabolic activity. Caffeine at 16.00 and 32.00 mg/mL significantly decreased C. albicans biofilm metabolic activity in the presence of 8 mg/mL of nicotine. Caffeine from 1.00 to 32.00 mg/mL significantly decreased the biofilm formation of C. albicans in the presence of 8 mg/mL of nicotine. CONCLUSIONS: The presence of 8 mg/mL of nicotine alone increased the metabolic activity and biofilm formation of C. albicans. In the presence of 8 mg/mL of nicotine with different caffeine concentrations, the results suggest that, overall, caffeine at higher concentrations (16 and 32 mg/mL) inhibited the metabolic activity and biofilm formation of C. albicans on acrylic denture resin most.