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.     

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.     

Granulocyte-macrophage colony-stimulating factor responses of oral epithelial cells to Candida albicans

Candida albicans is the principal fungal species responsible for oropharyngeal candidiasis, the most frequent opportunistic infection associated with immune deficiencies. Cytokines, such as granulocyte-macrophage colony-stimulating factor (GM-CSF), are important in the generation of effective immunity to C. albicans. The purposes of this investigation were to determine whether C. albicans triggers secretion of GM-CSF by oral epithelial cells in vitro and to investigate mechanisms of host cell-fungal interactions that trigger such responses. Oral epithelial cell lines as well as primary oral mucosal epithelial cells were challenged with stationary phase viable C. albicans, added to human cell cultures at varying yeast:oral cell ratios. Yeast were allowed to germinate for up to 48 h and supernatants were analyzed for GM-CSF by ELISA. Fixed organisms, germination-deficient mutants and separation of yeast from epithelial cells using cell culture inserts were used to assess the effects of viability, germination and physical contact, respectively, on the GM-CSF responses of these cells. Two out of three cell lines and three out of six primary cultures responded to C. albicans with an increase in GM-CSF secretion. GM-CSF responses were contact-dependent, strain-dependent, required yeast viability and were optimal when the yeast germinated into hyphae.     

Role of Candida albicans polymorphism in interactions with oral epithelial cells

BACKGROUND: Candida albicans is a polymorphic organism which undergoes morphologic transition between yeast, pseudohyphal and hyphal forms. The ability of C. albicans to change from yeast to filamentous types is a major virulence determinant of this organism. However, the exact role of hyphal transformation in establishing oral mucosal infection is still poorly understood. METHODS: In this study we used mutants with defects in filamentation, as well as oral strains, which differ in their capacity to form true hyphae, to examine the role of hyphal transformation in the interactions of C. albicans with oral epithelial cells in vitro. These interactions included the ability of these strains to adhere to and injure epithelial cells, as well as their ability to trigger a proinflammatory cytokine response. RESULTS: We found that strains SC5314 and ATCC28366 formed true hyphae on epithelial cells, whereas strain ATCC32077 and the tup1/tup1 mutant formed only pseudohyphae. Double mutant efg1/efg1cph1/cph1 grew exclusively as blastospores. We also found that yeast and pseudohyphal strains showed reduced adherence capacity to oral keratinocytes and caused minimal cell damage. Moreover, we showed that both yeast and pseudohyphal forms have a strongly attenuated proinflammatory phenotype, since they failed to induce significant interleukin (IL)-1alpha and IL-8 responses by oral epithelial cells. CONCLUSIONS: Germination of C. albicans into true hyphae is particularly important in the interactions with oral epithelial cells in vitro.     

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.     

Granulocyte‐macrophage colony‐stimulating factor responses of oral epithelial cells to Candida albicans

Candida albicans is the principal fungal species responsible for oropharyngeal candidiasis, the most frequent opportunistic infection associated with immune deficiencies. Cytokines, such as granulocyte‐macrophage colony‐stimulating factor (GM‐CSF), are important in the generation of effective immunity to C. albicans. The purposes of this investigation were to determine whether C. albicans triggers secretion of GM‐CSF by oral epithelial cells in vitro and to investigate mechanisms of host cell–fungal interactions that trigger such responses. Oral epithelial cell lines as well as primary oral mucosal epithelial cells were challenged with stationary phase viable C. albicans, added to human cell cultures at varying yeast:oral cell ratios. Yeast were allowed to germinate for up to 48 h and supernatants were analyzed for GM‐CSF by ELISA. Fixed organisms, germination‐deficient mutants and separation of yeast from epithelial cells using cell culture inserts were used to assess the effects of viability, germination and physical contact, respectively, on the GM‐CSF responses of these cells. Two out of three cell lines and three out of six primary cultures responded to C. albicans with an increase in GM‐CSF secretion. GM‐CSF responses were contact‐dependent, strain‐dependent, required yeast viability and were optimal when the yeast germinated into hyphae.     

In vivo photodynamic inactivation of Candida albicans using chloro‐aluminum phthalocyanine

This study evaluated the photoinactivation of Candida albicans in a murine model of oral candidiasis using chloro‐aluminum phthalocyanine (ClAlP) encapsulated in cationic nanoemulsions (NE) and chloro‐aluminum phthalocyanine (ClAlP) diluted in DMSO (DMSO) as photosensitizer (PS). Seventy‐five 6‐week‐old female Swiss mice were immunosuppressed and inoculated with C. albicans to induce oral candidiasis. PDT was performed on the tongue by the application of the photosensitizers and LED light (100 J cm^?2–660 nm). Twenty‐four hours and 7 days after treatments, microbiological evaluation was carried out by recovering C. albicans from the tongue of animals (CFU ml^?1). Then, mice were sacrificed and the tongues were surgically removed for histological and biomolecular analysis of pro‐ and anti‐inflammatory cytokines. Data were analyzed by ANOVA followed by Tukey's post hoc test. ClAlP‐NE‐mediated PDT reduced 2.26 log₁₀ of C. albicans recovered from the tongue when compared with the control group (P?L?) (P < 0.05). PDT did not promote adverse effects on the tongue tissue. Seven days after treatment, all animals were completely healthy. In summary, PDT mediated by chloro‐aluminum phthalocyanine entrapped in cationic nanoemulsions was effective in reducing C. albicans recovered from the oral lesions of immunocompromised mice.     

Identification of Candida species in the clinical laboratory: a review of conventional,commercial, and molecular techniques

In healthy individuals, Candida species are considered commensal yeasts of the oral cavity. However, these microorganisms can also act as opportunist pathogens, particularly the so‐called non‐albicans Candida species that are increasingly recognized as important agents of human infection. Several surveys have documented increased rates of C. glabrata, C. tropicalis, C. guilliermondii, C. dubliniensis, C. parapsilosis, and C. krusei in local and systemic fungal infections. Some of these species are resistant to antifungal agents. Consequently, rapid and correct identification of species can play an important role in the management of candidiasis. Conventional methods for identification of Candida species are based on morphological and physiological attributes. However, accurate identification of all isolates from clinical samples is often complex and time‐consuming. Hence, several manual and automated rapid commercial systems for identifying these organisms have been developed, some of which may have significant sensitivity issues. To overcome these limitations, newer molecular typing techniques have been developed that allow accurate and rapid identification of Candida species. This study reviewed the current state of identification methods for yeasts, particularly Candida species.     

Adherence of Candida albicans to silicone is promoted by the human salivary protein SPLUNC2/PSP/BPIFA2

Interactions between Candida albicans, saliva and saliva‐coated oral surfaces are initial events in the colonization of the oral cavity by this commensal yeast, which can cause oral diseases such as candidiasis and denture stomatitis. Candida albicans also colonizes silicone voice prostheses, and the microbial biofilm formed can impair valve function, necessitating frequent prosthesis replacement. We have previously shown that saliva promoted binding of C. albicans cells to silicone in vitro, and that the selective binding of specific salivary proteins to voice prosthesis silicone mediated attachment of C. albicans cells. The C. albicans cells adhered to a polypeptide (or polypeptides) of ~36 kDa eluted from saliva‐treated silicone. We show here that a protein of similar size was identified in replicate blots of the eluate from saliva‐treated silicone when the blots were probed with antibodies to human SPLUNC2, a salivary protein with reported microbial agglutination properties. In addition, SPLUNC2 was depleted from saliva that had been incubated with silicone coupons. To determine whether SPLUNC2 is a yeast‐binding protein, SPLUNC2 cDNA was expressed in Escherichia coli. Purified recombinant His‐tagged protein (SPLUNC2r) bound to silicone as demonstrated by immunoblot analysis of an eluate from SPLUNC2r‐treated silicone coupons and ³⁵S‐radiolabelled C. albicans cells adhered in a dose‐dependent manner to SPLUNC2r‐coated silicone. We conclude that SPLUNC2 binds to silicone and acts as a receptor for C. albicans adherence to, and subsequent colonization of, voice prosthesis silicone.