Discovering Antifungal Resistance Mechanisms and Drug Targets of Candida Biofilms

Candida inhabits 50% of human oral cavities and cause opportunistic infections ranging from superficial mucosal diseases to life-threatening systemic mycoses. Oral candidiasis is a frequent problem in compromised population groups and, also among denture wearers. In these patients Candida colonise host surfaces and live in biofilm communities that are difficult to eradicate due to their high antifungal resistance. Hence, elucidation of drug resistance mechanisms in Candida biofilms has major health and economic implications.

Objectives: To uncover the mechanism/s underlying antifungal resistance of Candida biofilms and thereby discern possible drug targets.

Methods: First, C. albicans biofilms were characterized in terms of their development, architecture and cellular viability using standard growth methodologies and, SEM, confocal scanning laser microscopy (CSLM) and, novel Confocal-COMSTAT analysis. Then, antifungal susceptibility of planktonic vs. biofilm mode Candida was studied to elucidate factors affecting antifungal resistance, using differential genomic and proteomic tools. A “top down” proteomic approach using two-dimensional gel electrophoresis, tandem mass spectrometry and bioinformatics tools were employed to characterize differentially expressed protein biomarkers in the two growth modes. Expression patterns of clinically significant biomarkers were further investigated for transcriptomic regulation and oxygen species reactivity.

Results: SEM and Confocal-COMSTAT analysis revealed the detailed architecture and cellular viability of the developing Candida biofilms (upto 72h). Antioxidant protein biomarkers were significantly upregulated in the biofilm mode compared with planktonic mode Candida (controls) illustrating a novel mechanism that could contribute to higher antifungal resistance. Interestingly, a protein biomarker, Pil1p, unique to fungi, with an echinocandin binding drug target was highly expressed especially in the biofilm mode of Candida.

Conclusion: This study uncovers, for the first time, a key mechanism underlying higher antifungal resistance in Candida biofilms and, elucidates a potential antifungal drug target that could be commercially exploited.

Supported by the Research Grants Council of Hong Kong #HKU7624/06M.