Candida albicans binds to saliva proteins selectively adsorbed to silicone

Explanted voice prostheses obtained from 5 patients at the time of prosthesis replacement were consistently colonized by yeast, in particular Candida albicans. A simple, reproducible, in vitro model of C. albicans adherence to saliva-coated voice prosthesis silicone was developed. Whole saliva promoted adherence of C. albicans to silicone in a dose-dependent manner. Saliva rinses from voice prosthesis patients also promoted binding of C. albicans to silicone in vitro (mean adherence 14.9% +/- 2.8% of input C. albicans cells). This was significantly higher than C. albicans adherence to silicone in the absence of saliva (P < .001) or adherence promoted by saliva rinses from healthy volunteers (P < .005). Polyacrylamide gel electrophoresis analysis and a blot overlay adherence assay revealed that certain salivary proteins were selectively adsorbed to silicone and that C. albicans yeast cells adhered specifically to the adsorbed salivary proteins.     

Induction of apoptosis in oral epithelial cells by Candida albicans

During infection, interactions between Candida albicans and oral epithelial cells result in oral epithelial cell death. This is clinically manifested by the development of oral mucosal ulcerations generally associated with discomfort. In vitro studies have shown that C. albicans induces early apoptotic alterations in oral epithelial cells; however, these studies have also shown that treatment of infected cells with caspase inhibitors does not prevent their death. The reasons for these contradictory results are unknown and it is still not clear if C. albicans stimulates oral epithelial signaling pathways that promote apoptotic cell death. Activation of specific death pathways in response to microbial organisms plays an essential role in modulating the pathogenesis of a variety of infectious diseases. The aim of this study was to (i) characterize C. albicans‐induced apoptotic morphological alterations in oral epithelial cells, and (ii) investigate the activation of apoptotic signaling pathways and expression of apoptotic genes during infection. Candida albicans induced early apoptotic changes in over 50% of oral epithelial cells. However, only 15% of those showed mid‐late apoptotic alterations. At the molecular level, C. albicans caused a loss of the mitochondrial transmembrane potential and translocation of mitochondrial cytochrome c. Caspase‐3/9 activities increased only during the first hours of infection. Moreover, poly[ADP ribose] polymerase 1 was cleaved into apoptotic and necrotic‐like fragments. Finally, five anti‐apoptotic genes were significantly upregulated and two pro‐apoptotic genes were downregulated during infection. Altogether, these findings indicate that epithelial apoptotic pathways are activated in response to C. albicans, but fail to progress and promote apoptotic cell death.     

Antigen I/II mediates interactions between Streptococcus mutans and Candida albicans

Streptococcus mutans and Candida albicans are frequently co‐isolated from dental plaque of children with early childhood caries (ECC) and are only rarely found in children without ECC, suggesting that these species interact in a manner that contributes to the pathogenesis of ECC. Previous studies have demonstrated that glucans produced by S. mutans are crucial for promoting the formation of biofilm and cariogenicity with C. albicans; however, it is unclear how non‐glucan S. mutans biofilm factors contribute to increased biofilm formation in the presence of C. albicans. In this study we examined the role of S. mutans antigen I/II in two‐species biofilms with C. albicans, and determined that antigen I/II is important for the incorporation of C. albicans into the two‐species biofilm and is also required for increased acid production. The interaction is independent of the proteins Als1 and Als3, which are known streptococcal receptors of C. albicans. Moreover, antigen I/II is required for the colonization of both S. mutans and C. albicans during co‐infection of Drosophila melanogaster in vivo. Taken together, these results demonstrate that antigen I/II mediates the increase of C. albicans numbers and acid production in the two‐species biofilm, representing new activities associated with this known S. mutans adhesin.     

Candida albicans induces early apoptosis followed by secondary necrosis in oral epithelial cells

The capacity of Candida albicans to invade and damage oral epithelial cells is critical for its ability to establish and maintain symptomatic oropharyngeal infection. Although oral epithelial cells are reported dead after 18 h of candidal infection, activation of specific epithelial cell‐death pathways in response to C. albicans infection has not yet been demonstrated. Considering the key role of oral epithelial cell damage in the pathogenesis of oropharyngeal candidiasis, the aim of this study was to characterize this event during infection. Using an oral epithelial–C. albicans co‐culture system, we examined the ability of C. albicans to induce classic necrotic, pyroptotic and apoptotic cellular alterations in oral epithelial cells such as osmotic lysis, exposure of phosphatidylserine on the epithelial cell plasma membrane and internucleosomal DNA fragmentation. It was found that the ability of C. albicans to kill oral epithelial cells depends on its capacity to physically interact with and invade these cells. Caspase‐dependent apoptotic pathways were activated early during C. albicans infection and contributed to C. albicans‐induced oral epithelial cell death. Earlier apoptotic events were followed by necrotic death of infected oral epithelial cells. Hence, C. albicans stimulates oral epithelial signaling pathways that promote early apoptotic cell death through the activation of cellular caspases, followed by late necrosis.     

Oral candidosis in relation to oral immunity

Symptomatic oral infection with Candida albicans is characterized by invasion of the oral epithelium by virulent hyphae that cause tissue damage releasing the inflammatory mediators that initiate and sustain local inflammation. Candida albicans triggers pattern‐recognition receptors of keratinocytes, macrophages, monocytes and dendritic cells, stimulating the production of IL‐1β, IL‐6 and IL‐23. These cytokines induce the differentiation of Th17 cells and the generation of IL‐17‐ and/or IL‐22‐mediated antifungal protective immuno‐inflammatory responses in infected mucosa. Some immune cells including NKT cells, γδ T cells and lymphoid cells that are innate to the oral mucosa have the capacity to produce large quantities of IL‐17 in response to C. albicans, sufficient to mediate effective protective immunity against C. albicans. On the other hand, molecular structures of commensal C. albicans blastoconidia, although detected by pattern‐recognition receptors, are avirulent, do not invade the oral epithelium, do not elicit inflammatory responses in a healthy host, but induce regulatory immune responses that maintain tissue tolerance to the commensal fungi. The type, specificity and sensitivity of the protective immune response towards C. albicans is determined by the outcome of the integrated interactions between the intracellular signalling pathways of specific combinations of activated pattern‐recognition receptors (TLR2, TLR4, Dectin‐1 and Dectin‐2). IL‐17‐mediated protective immune response is essential for oral mucosal immunity to C. albicans infection.     

Differences between salivary and blood neutrophils from elderly and young denture wearers

Summary We previously showed that neutrophils from patients with Candida‐related denture stomatitis exhibited damaged function, and the advance in age intensified this condition. Because such alterations had been determined in elderly people that were not denture wearers, the purpose of this study was to clarify functional and phenotypic characteristics of neutrophils from elderly denture wearers (EDW) and young denture wearers (YDW) without oral lesion. We enrolled 20 denture wearers (12 EDW and 8 YDW) and determined the positivity of Candida species on maxillary prosthesis and palate. Additionally, blood and salivary neutrophils were evaluated. Furthermore, cytokines and chemokines salivary levels were detected. YDW presented higher positivity of Candida albicans than elderly ones. However, blood neutrophils from EDW expressed less CXCR1, CD62L and CD11b and had lower C. albicans phagocytosis than YDW. Although myeloperoxidase and elastase activity was significantly higher in C. albicans‐stimulated blood neutrophils from elderly, they produced high levels of IL‐10 and low levels of Granulocyte macrophage‐colony stimulating factor (GM‐CSF). Despite apoptosis rate of salivary neutrophils was enhanced, these cells were at a high number in YDW. GM‐CSF and IL10 were lower in saliva from elderly group. These data confirmed that ageing affects blood and salivary neutrophils and could predispose elderly to persistent oral infections.     

Differential profiles of salivary proteins with affinity to Streptococcus mutans lipoteichoic acid in caries‐free and caries‐positive human subjects

Streptococcus mutans is a representative oral pathogen that causes dental caries and pulpal inflammation. Its lipoteichoic acid (Sm.LTA) is known to be an important cell‐wall virulence factor involved in bacterial adhesion and induction of inflammation. Since Sm.LTA‐binding proteins (Sm.LTA‐BPs) might play an important role in pathogenesis and host immunity, we identified the Sm.LTA‐BPs in the saliva of caries‐free and caries‐positive human subjects using Sm.LTA‐conjugated beads and LTQ‐Orbitrap hybrid Fourier transform mass spectrometry. Sm.LTA was conjugated to N‐hydroxysuccinimidyl‐Sepharose^® 4 Fast Flow beads (Sm.LTA‐beads). Sm.LTA retained its biological properties during conjugation, as determined by the expression of nitric oxide and interferon‐γ‐inducible protein 10 in a murine macrophage cell line and activation of Toll‐like receptor 2 (TLR2) in CHO/CD14/TLR2 cells. Sm.LTA‐BPs were isolated from pooled saliva prepared from 10 caries‐free or caries‐positive human subjects each, electrophoresed to see their differential expression in each group, and further identified by high‐resolution mass spectrometry. A total of 8 and 12 Sm.LTA‐BPs were identified with statistical significance in the pooled saliva from the caries‐free and caries‐positive human subjects, respectively. Unique Sm.LTA‐BPs found in caries‐free saliva included histone H4, profilin‐1 and neutrophil defensin‐1, and those in caries‐positive saliva included cystatin‐C, cystatin‐SN, cystatin‐S, cystatin‐D, lysozyme C, calmodulin‐like protein 3 and β‐actin. The Sm.LTA‐BPs found in both groups were hemoglobin subunits α and β, prolactin‐inducible protein, protein S100‐A9, and SPLUNC2. Collectively, we identified Sm.LTA‐BPs in the saliva of caries‐free and caries‐positive subjects, which exhibit differential protein profiles.     

Fermentative 2‐carbon metabolism produces carcinogenic levels of acetaldehyde in Candida albicans

Acetaldehyde is a carcinogenic product of alcohol fermentation and metabolism in microbes associated with cancers of the upper digestive tract. In yeast acetaldehyde is a by‐product of the pyruvate bypass that converts pyruvate into acetyl‐Coenzyme A (CoA) during fermentation. The aims of our study were: (i) to determine the levels of acetaldehyde produced by Candida albicans in the presence of glucose in low oxygen tension in vitro; (ii) to analyse the expression levels of genes involved in the pyruvate‐bypass and acetaldehyde production; and (iii) to analyse whether any correlations exist between acetaldehyde levels, alcohol dehydrogenase enzyme activity or expression of the genes involved in the pyruvate‐bypass. Candida albicans strains were isolated from patients with oral squamous cell carcinoma (n = 5), autoimmune polyendocrinopathy–candidiasis–ectodermal dystrophy (APECED) patients with chronic oral candidosis (n = 5), and control patients (n = 5). The acetaldehyde and ethanol production by these isolates grown under low oxygen tension in the presence of glucose was determined, and the expression of alcohol dehydrogenase (ADH1 and ADH2), pyruvate decarboxylase (PDC11), aldehyde dehydrogenase (ALD6) and acetyl‐CoA synthetase (ACS1 and ACS2) and Adh enzyme activity were analysed. The C. albicans isolates produced high levels of acetaldehyde from glucose under low oxygen tension. The acetaldehyde levels did not correlate with the expression of ADH1, ADH2 or PDC11 but correlated with the expression of down‐stream genes ALD6 and ACS1. Significant differences in the gene expressions were measured between strains isolated from different patient groups. Under low oxygen tension ALD6 and ACS1, instead of ADH1 or ADH2, appear the most reliable indicators of candidal acetaldehyde production from glucose.     

Inhibition of Candida albicans by the peroxidase/SCN−/H2O2 system

Effects of the salivary peroxidase (SPO) system on the growth, glucose uptake and metabolic activities of oral bacteria are well documented but the effects on oral fungi are virtually unknown. Therefore, the viability of Candida albicans (ATCC 28366) exposed to the peroxidase/SCN^?/H₂O₂: system was studied in sterilized saliva, in phosphate-buffered saline (PBS) and in potassium chloride. The growth of C. albicans in glucose-supplemented saliva was faster at pH 5.5 than at pH 7. The addition of the complete SPO (or lactoperoxidase) system to either sterilized saliva, KC1 (50 μM) or PBS at pH 5.5 inhibited dose-dependently the viability of C. albicans in KC1, but no inhibition was found in PBS or saliva. Maximal inhibition was achieved in 2 h and with > 320 μM of peroxidase-generated HOSCN/OSCN^?. However, physiological salivary concentrations of phosphate (> 1.0 μM) and PBS blocked the antifungal effect of HOSCN/OSCN^?. The relative proportions of SCN^? and H₂O₂: were critical to the antifungal effects. With 0.2 mM KSCN, a complete loss of viability was achieved, though the HOSCN/OSCN^? concentrations did not exceed 100 μM. It is concluded that C. albicans is sensitive to HOSCN/OSCN^? but salivary concentrations of phosphate block the antifungal effect of the peroxidase systems.     

Polymicrobial interactions of Candida albicans and its role in oral carcinogenesis

The oral microbiome is composed of microorganisms residing in the oral cavity, which are critical components of health and disease. Disruption of the oral microbiome has been proven to influence the course of oral diseases, especially among immunocompromised patients. Oral microbiome is comprised of inter‐kingdom microorganisms, including yeasts such as Candida albicans, bacteria, archaea and viruses. These microorganisms can interact synergistically, mutualistically and antagonistically, wherein the sum of these interactions dictates the composition of the oral microbiome. For instance, polymicrobial interactions can improve the ability of C albicans to form biofilm, which subsequently increases the colonisation of oral mucosa by the yeast. Polymicrobial interactions of C albicans with other members of the oral microbiome have been reported to enhance the malignant phenotype of oral cancer cells, such as the attachment to extracellular matrix molecules (ECM) and epithelial‐mesenchymal transition (EMT). Polymicrobial interactions may also exacerbate an inflammatory response in oral epithelial cells, which may play a role in carcinogenesis. This review focuses on the role of polymicrobial interactions between C albicans and other oral microorganisms, including its role in promoting oral carcinogenesis.     

Candida–streptococcal mucosal biofilms display distinct structural and virulence characteristics depending on growth conditions and hyphal morphotypes

Candida albicans and streptococci of the mitis group form communities in multiple oral sites, where moisture and nutrient availability can change spatially or temporally. This study evaluated structural and virulence characteristics of Candida–streptococcal biofilms formed on moist or semidry mucosal surfaces, and tested the effects of nutrient availability and hyphal morphotype on dual‐species biofilms. Three‐dimensional models of the oral mucosa formed by immortalized keratinocytes on a fibroblast‐embedded collagenous matrix were used. Infections were carried out using Streptococcus oralis strain 34, in combination with a C. albicans wild‐type strain, or pseudohyphal‐forming mutant strains. Increased moisture promoted a homogeneous surface biofilm by C. albicans. Dual biofilms had a stratified structure, with streptococci growing in close contact with the mucosa and fungi growing on the bacterial surface. Under semidry conditions, Candida formed localized foci of dense growth, which promoted focal growth of streptococci in mixed biofilms. Candida biofilm biovolume was greater under moist conditions, albeit with minimal tissue invasion, compared with semidry conditions. Supplementing the infection medium with nutrients under semidry conditions intensified growth, biofilm biovolume and tissue invasion/damage, without changing biofilm structure. Under these conditions, the pseudohyphal mutants and S. oralis formed defective superficial biofilms, with most bacteria in contact with the epithelial surface, below a pseudohyphal mass, resembling biofilms growing in a moist environment. The presence of S. oralis promoted fungal invasion and tissue damage under all conditions. We conclude that moisture, nutrient availability, hyphal morphotype and the presence of commensal bacteria influence the architecture and virulence characteristics of mucosal fungal biofilms.     

Clonal identity of Candida albicans in the oral cavity and the gastrointestinal tract of pre‐school children

The clonal relationship between oral and fecal Candida albicans isolated from children of pre‐school age was examined using RAPD analysis. Significantly higher levels of C. albicans were found in saliva, dental plaque, carious specimens and stools of 56 patients with severe caries as compared to 52 healthy control subjects. The highest prevalence was found in carious specimens and a strong correlation was observed between its presence in saliva, dental plaque, carious specimen and feces. RAPD analysis of isolates from 23 patients with simultaneous oral and fecal C. albicans revealed clonal counterparts present in both oral and stool samples in 15 cases; five patients harbored closely related strains; and three patients harbored unrelated strains. Our results demonstrate a strong correlation between oral and gastrointestinal C. albicans colonization. We assume that carious teeth may constitute an ecologic niche for C. albicans potentially responsible for recurrent oral and non‐oral candidiasis.     

The development and validation of a rapid genetic method for species identification and genotyping of medically important fungal pathogens using high‐resolution melting curve analysis

Accurate, rapid and economical fungal species identification has been a major aim in mycology. In this study, our goal was to examine the feasibility of a high‐resolution melting curve analysis (HRMA) of internal transcribed regions ITS1 and ITS2 in ribosomal DNA (rDNA) for a rapid, simple and inexpensive differentiation of eight clinically relevant Candida species (Candida albicans, Candida glabrata, Candida parapsilosis, Candida krusei, Candida tropicalis, Candida guilliermondii, Candida dubliniensis and Candida lusitaniae). In addition, for the first time, we tested the applicability of HRMA to classify C. albicans strains into four previously described genotypes (A, B, C and D) using a primer set that spans the transposable intron region of 25S of rDNA. Type and unknown clinical oral isolates were used in this study and the melting curve analysis was compared with both amplicons' sequencing and agarose gel electrophoresis analysis. Real‐time PCR and subsequent HRMA of the two described rDNA regions generated distinct melting curve profiles that were in accord with sequencing and gel electrophoresis analysis, highly reproducible, and characteristic of each of the eight Candida species and C. albicans genotypes. Moreover, results were obtained in 4 h and without the need for any post‐amplification handling, so reducing time and cost. Owing to its simplicity and speed, this technique is a good fit for genotypic analysis of hundreds of clinical strains in large epidemiological settings.     

Interactions between Streptococcus oralis,Actinomyces oris,and Candida albicans in the development of multispecies oral microbial biofilms on salivary pellicle

The fungus Candida albicans is carried orally and causes a range of superficial infections that may become systemic. Oral bacteria Actinomyces oris and Streptococcus oralis are abundant in early dental plaque and on oral mucosa. The aims of this study were to determine the mechanisms by which S. oralis and A. oris interact with each other and with C. albicans in biofilm development. Spatial distribution of microorganisms was visualized by confocal laser scanning microscopy of biofilms labeled by differential fluorescence or by fluorescence in situ hybridization (FISH). Actinomyces oris and S. oralis formed robust dual‐species biofilms, or three‐species biofilms with C. albicans. The bacterial components tended to dominate the lower levels of the biofilms while C. albicans occupied the upper levels. Non‐fimbriated A. oris was compromised in biofilm formation in the absence or presence of streptococci, but was incorporated into upper biofilm layers through binding to C. albicans. Biofilm growth and hyphal filament production by C. albicans was enhanced by S. oralis. It is suggested that the interkingdom biofilms are metabolically coordinated to house all three components, and this study demonstrates that adhesive interactions between them determine spatial distribution and biofilm architecture. The physical and chemical communication processes occurring in these communities potentially augment C. albicans persistence at multiple oral cavity sites.     

Adhesion of Candida albicans,but not Candida krusei,to salivary statherin and mimicking host molecules

The aim of the present study was to identify salivary molecules affecting adhesion of Candida albicans and Candida krusei to salivary pellicles and epithelial cells. Strains of C. albicans (GDH18, GDH3339, CA1957, ATCC 28366 and ATCC 10321), but not C. krusei (strains ATCC 14243 and Ck9), bound to saliva‐coated hydroxyapatite and buccal epithelial cells. Parotid saliva fractions containing statherin, glycosylated proline‐rich proteins (PRP) and as yet unidentified components mediated adhesion of strain GDH18; Fucα1‐2Galβ1‐4Glc partly inhibited the adhesion to those fractions not containing statherin. Pure statherin, but not PRP‐1, mediated dose‐dependent adhesion of C. albicans strain GDH18 to hydroxyapatite beads. Candida isolates (GDH18, GDH3339 and CA1957) bound somewhat more avidly to statherin/saliva relative to ATCC strains 28366 and 10321, while the opposite was true for adhesion to buccal epithelial cells. Adhesion of C. albicans strain GDH18 to saliva‐coated hydroxyapatite and buccal epithelial cells was completely (93%) and partly (43%) blocked by statherin‐specific immunoglobulin G (IgG) antibodies, respectively. Control IgG antibodies did not block Candida adhesion. Blockage of Candida adhesion to epithelial cells also occurred with Fucα1‐2Galβ1‐4Glc (49%) and N‐acetylglucosamine (38%), while statherin specific IgG antibodies in combination with Fucα1‐2Galβ1‐4Glc almost completely eliminated Candida adhesion (79%). In addition, statherin in solution blocked the adhesion of strain GDH18 to epithelial cells by inducing aggregation of Candida cells.