Pathogenic mechanisms of Candida glabrata in oral candidiasis, especially because of its inability to form hyphae, are understudied. Since both Candida albicans and C. glabrata are frequently co-isolated in oropharyngeal candidiasis (OPC), we examined their co-adhesion in vitro and observed adhesion of C. glabrata only to C. albicans hyphae microscopically. Mice were infected sublingually with C. albicans or C. glabrata individually, or with both species concurrently, to study their ability to cause OPC. Infection with C. glabrata alone resulted in negligible infection of tongues; however, colonization by C. glabrata was increased by co-infection or a pre-established infection with C. albicans. Furthermore, C. glabrata required C. albicans for colonization of tongues, since decreasing C. albicans burden with fluconazole also reduced C. glabrata. C. albicans hyphal wall adhesins Als1 and Als3 were important for in vitro adhesion of C. glabrata and to establish OPC. C. glabrata cell wall protein coding genes EPA8, EPA19, AWP2, AWP7, and CAGL0F00181 were implicated in mediating adhesion to C. albicans hyphae and remarkably, their expression was induced by incubation with germinated C. albicans. Thus, we found a near essential requirement for the presence of C. albicans for both initial colonization and establishment of OPC infection by C. glabrata.

Perception of external stimuli and generation of an appropriate response are crucial for host colonization by pathogens. In pathogenic fungi, mitogen activated protein kinase (MAPK) pathways regulate dimorphism, biofilm/mat formation, and virulence. Signaling mucins, characterized by a heavily glycosylated extracellular domain, a transmembrane domain, and a small cytoplasmic domain, are known to regulate various signaling pathways. In Candida albicans, the mucin Msb2 regulates the Cek1 MAPK pathway. We show here that Msb2 is localized to the yeast cell wall and is further enriched on hyphal surfaces. A msb2Δ/Δ strain formed normal hyphae but had biofilm defects. Cek1 (but not Mkc1) phosphorylation was absent in the msb2Δ/Δ mutant. The extracellular domain of Msb2 was shed in cells exposed to elevated temperature and carbon source limitation, concomitant with germination and Cek1 phosphorylation. Msb2 shedding occurred differentially in cells grown planktonically or on solid surfaces in the presence of cell wall and osmotic stressors. We further show that Msb2 shedding and Cek1 phosphorylation were inhibited by addition of Pepstatin A (PA), a selective inhibitor of aspartic proteases (Saps). Analysis of combinations of Sap protease mutants identified a sap8Δ/Δ mutant with reduced MAPK signaling along with defects in biofilm formation, thereby suggesting that Sap8 potentially serves as a major regulator of Msb2 processing. We further show that loss of either Msb2 (msb2Δ/Δ) or Sap8 (sap8Δ/Δ) resulted in higher C. albicans surface β-glucan exposure and msb2Δ/Δ showed attenuated virulence in a murine model of oral candidiasis. Thus, Sap-mediated proteolytic cleavage of Msb2 is required for activation of the Cek1 MAPK pathway in response to environmental cues including those that induce germination. Inhibition of Msb2 processing at the level of Saps may provide a means of attenuating MAPK signaling and reducing C. albicans virulence.

The oral cavity is a unique niche where Candida albicans infections occur in immunocompetent as well as immunosuppressed individuals. Here we critically review the significance of human innate immune response in preventing oral candidiasis. One important line of defense against oropharyngeal candidiasis is the oral microbiota that prevents infection by competing for space and nutrients as well as by secreting antagonistic molecules and triggering local inflammatory responses. C. albicans is able to induce mucosal defenses through activation of immune cells and production of cytokines. Also, saliva contains various proteins that affect C. albicans growth positively by promoting mucosal adherence and negatively through immune exclusion and direct fungicidal activity. We further discuss the role of saliva in unifying host innate immune defenses against C. albicans as a communicating medium and how C. albicans overgrowth in the oral cavity may be a result of aberrations ranging from microbial dysbiosis and salivary dysfunction to epithelial damage. Last we underscore select oral diseases in which C. albicans is a contributory microorganism in immune-competent individuals.

Salivary protein histatin 5 (Hst 5) is fungicidal toward Candida albicans, the causative agent of oropharyngeal candidiasis. However, its activity in saliva is compromised by salivary protease-mediated degradation and interaction with salivary salts. Hst 5 has also been shown to bind various metals in saliva-namely, Zn, Cu, and Ni. Surprisingly, interactions of Hst 5 with Fe have not been studied, although iron is one of the most abundant metals present in saliva. Using circular dichroism, we show that Hst 5 can bind up to 10 equivalents of iron as measured by loss of its alpha-helical secondary structure that is normally observed for it in trifluoroethylene. A significant decrease in the candidacidal ability of Hst 5 was observed upon iron binding, with increasing iron concentrations being inversely proportional to Hst 5 killing activity. Binding assays showed that the decrease in killing was likely a result of reduced binding (10-fold reduction) of Fe-Hst 5 to C. albicans cells. Protease stability analysis showed that Fe-Hst 5 was completely resistant to trypsin digestion. In contrast, zinc binding had limited effects on Hst 5 fungicidal activity or protease susceptibility. RNA sequencing results identified changes in iron uptake genes in Hst 5-treated C. albicans cells. Our findings thus suggest that consequences of Hst 5 binding iron not only affect candidacidal ability and proteolyic stability of Hst 5, but may also contribute to a novel killing mechanism involving interference with cellular iron metabolism.

Candida albicans, a commensal fungus of the oral microbiome, causes oral candidiasis in humans with localized or systemic immune deficiencies. Secreted aspartic proteinases (Saps) are a family of 10 related proteases and are virulence factors due to their proteolytic activity, as well as their roles in adherence and colonization of host tissues. We found that mice infected sublingually with C. albicans cells overexpressing Sap6 (SAP6 OE and a ⌬sap8 strain) had thicker fungal plaques and more severe oral infection, while infection with the ⌬sap6 strain was attenuated. These hypervirulent strains had highly aggregative colony structure in vitro and higher secreted proteinase activity; however, the levels of proteinase activity of C. albicans Saps did not uniformly match their abilities to damage cultured oral epithelial cells (SCC-15 cells). Hyphal induction in cells overexpressing Sap6 (SAP6 OE and ⌬sap8 cells) resulted in formation of large cell-cell aggregates. These aggregates could be produced in germinated wild-type cells by addition of native or heat-inactivated Sap6. Sap6 bound only to germinated cells and increased C. albicans adhesion to oral epithelial cells. The adhesion properties of Sap6 were lost upon deletion of its integrin-binding motif (RGD) and could be inhibited by addition of RGD peptide or anti-integrin antibodies. Thus, Sap6 (but not Sap5) has an alternative novel function in cell-cell aggregation, independent of its proteinase activity, to promote infection and virulence in oral candidiasis. Candida albicans is a commensal fungus that is often part of the oral microflora of healthy people. Loss of host immunity, HIV infection, corticosteroid use, or alteration of the oral microflora following antibiotic therapies permits a pathogenic transition of C. albicans to cause oropharyngeal candidiasis (OPC) (1, 2). Acute pseudomembranous candidiasis is one of the most common forms of OPC, in which C. albicans forms white patches on the surface of the buccal mucosa, tongue, or soft palate. These superficial fungal plaques can be lifted from underlying tissues for purposes of clinical diagnosis and analysis (3).C. albicans expresses specific sets of virulence factors that promote hypha formation and adhesion and invasion of host tissues (4). Secreted aspartyl proteinases (Saps) are recognized virulence factors because they degrade host proteins to provide nitrogen for fungal cell metabolism, contribute to adherence, facilitate fungal epithelial and endothelial penetration, and are immunogenic during infection (5-7). Microbial proteinases are classified as serine, cysteine, metallo-, or aspartyl proteinases according to the site of catalytic hydrolysis of substrate peptide bonds; however, C. albicans produces only aspartyl proteinases (5, 6).C. albicans expresses a family of 10 SAP genes that are clustered into groups SAP1 to SAP3, SAP4 to SAP6, SAP7, SAP8, and SAP9 and SAP10 based upon their sequence homologies and pH activities (8, 9). Sap1 through Sap8 are processed and transported via the secreto...