High iron-mediated increased oral fungal burden, oral-to-gut transmission, and changes to pathogenicity of Candida albicans in oropharyngeal candidiasis.

BACKGROUND: Iron affects the diversity of the oral microbial landscape. Laboratory-strain CAI4 of Candida albicans that causes oropharyngeal candidiasis (OPC) exhibits iron-induced changes to the cell wall, impacting phagocytosis (by macrophages) and susceptibility of fungal cells to cell wall-perturbing antifungals, in vitro. AIM: To understand the effect of iron on the CAI4-strain, wild type (WT) SC5314-strain, and oral isolates of C. albicans. METHODS: An immunosuppressed murine model of OPC was used to assess the effect of iron on oral-to-gut infection and antifungal susceptibility of the CAI4-strain. In vitro antifungal susceptibility, cell wall analysis, and phagocytic assays were performed under low and high iron, for the SC5314-strain and oral isolates. RESULTS: High iron enhanced oral and gut fungal levels for the CAI4-strain in mice; CAI4 cells from low iron mice were more susceptible to antifungals. The SC5314-strain and oral isolates showed enhanced antifungal-resistance towards most antifungals tested, under high iron. Iron-mediated cell wall changes and phagocytic response in the SC5315-strain were similar to CAI4; oral isolates showed a variable response. CONCLUSION: Host iron can potentially alter infection severity and dissemination, efficacy of antifungal treatment, and host immune response during OPC. Clinical isolates showed most of these effects of iron, despite exhibiting a varied cell wall composition-change response to iron.

Supragingival mycobiome and inter-kingdom interactions in dental caries.

Background: Recent studies have reveled the presence of a complex fungal community (mycobiome) in the oral cavity. However, the role of oral mycobiome in dental caries and its interaction with caries-associated bacteria is not yet clear. Methods: Whole-mouth supragingival plaque samples from 30 children (6-10 years old) with no caries, early caries, or advanced caries were sequenced for internal transcribed spacer 2 (ITS-2). The mycobiome profiles were correlated with previously published bacteriome counterparts. Interaction among selected fungal and bacterial species was assessed by co-culture or spent media experiments. Results: Fungal load was extremely low. Candida, Malassezia, Cryptococcus, and Trichoderma spp. were the most prevalent/abundant taxa. Advanced caries was associated with significantly higher fungal load and prevalence/abundance of Candida albicans. Cryptococcus neoformans and Candida sake were significantly over-abundant in early caries, while Malassezia globosa was significantly enriched in caries-free subjects. C. albicans correlated with Streptococcus mutans and Scardovia wiggsiae among other caries-associated bacteria, while M. globosa inversely correlated with caries-associated bacteria. In-vitro, M. globosa demonstrated inhibitory properties against S. mutans. Conclusions: the results substantiate the potential role of the oral mycobiome, primarily Candida species, in dental caries. Inter-kingdom correlations and inhibition of S. mutans by M. globosa are worth further investigation.

Improved Tet-On and Tet-Off systems for tetracycline-regulated expression of genes in Candida.

Tetracycline-regulated expression of genes is often used for functional analysis of Candida albicans genes. However, the widely used Tet-On system has certain limitations such as prolonged lag time (up to 8 h) for induction and non-uniform expression among the cells. We speculated that poor expression of tetracycline-controlled transactivator (Tet-transactivator) from CaADH1 promoter could be responsible for this, and thus compared the effect of expressing this protein under the control of CaADH1, CaTDH3 and CaRP10 promoters on the expression of GFP from the TET promoter. Only CaRP10 promoter facilitated a more uniform and rapid induction of GFP. However, a high concentration of doxycycline was needed for induction, which is not desirable for assessing certain phenotypes. Tet-Off systems are known to require a low concentration of doxycycline, but a limitation of the widely used Tet-Off system for C. albicans is the use of CaENO1 promoter, which is known to be repressed in the presence of gluconeogenic carbon source, for expression of transactivator. Thus, we have converted the above-mentioned Tet-On systems to Tet-Off systems by site-directed mutagenesis of the Tet-transactivator. Compared to the Tet-On systems, the Tet-Off systems required about 200-fold less concentration of doxycycline for modulation of gene expression. Only the Tet-Off system with CaRP10 promoter driving the expression of transactivator allowed rapid and high level expression of GFP compared to those with CaADH1 or CaTDH3 promoters. The utility of CaRP10 based Tet-On and Tet-Off systems was further validated by the conditional expression of the CaTUP1 gene. We have also adapted these systems for use with Candida tropicalis and find that the Tet-Off system is functional in this species. The Tet systems reported here will be useful for conditional expression of genes in C. albicans as well as C. tropicalis.

Critical role for CaFEN1 and CaFEN12 of Candida albicans in cell wall integrity and biofilm formation.

Sphingolipids are involved in several cellular functions, including maintenance of cell wall integrity. To gain insight into the role of individual genes of sphingolipid biosynthetic pathway, we have screened Saccharomyces cerevisiae strains deleted in these genes for sensitivity to cell wall perturbing agents calcofluor white and congo red. Only deletants of FEN1 and SUR4 genes were found to be sensitive to both these agents. Candida albicans strains deleted in their orthologs, CaFEN1 and CaFEN12, respectively, also showed comparable phenotypes, and a strain deleted for both these genes was extremely sensitive to cell wall perturbing agents. Deletion of these genes was reported earlier to sensitise cells to amphotericin B (AmB), which is a polyene drug that kills the cells mainly by binding and sequestering ergosterol from the plasma membrane. Here we show that their AmB sensitivity is likely due to their cell wall defect. Further, we show that double deletant of C. albicans is defective in hyphae formation as well as biofilm development. Together this study reveals that deletion of FEN1 and SUR4 orthologs of C. albicans leads to impaired cell wall integrity and biofilm formation, which in turn sensitise cells to AmB.