Development of a high-throughput fungal biofilm chip and associated screening methodology for the identification of novel antifungal drug candidates.

摘要

Candida albicans remains the most frequent causative agent of Candidiasis, the most frequent fungal infection and now the third most common nosocomial infection in the US hospitals. These infections have emerged as a growing threat to human health, especially for an increasing number of immunocompromised individuals who are at risk for opportunistic infections. The high mortality rate associated with these fungal infections is in part due to the limited arsenal of antifungal drugs. Another major reason is because most forms of candidiasis are associated with a biofilm mode of growth, and cells within these biofilms are intrinsically resistant to most antifungal agents. We have developed a high-density microarray platform consisting of nano-biofilms of C. albicans. Briefly, a robotic micro-arrayer is used to print yeast cells of C. albicans (strain SC5314) onto a solid substrate. During printing, the yeast cells are enclosed in a three dimensional (3D) matrix using a volume as low as 50 nL and immobilized on modified glass substrates. After initial printing, the slides are incubated at 37 °C for 24 hours to allow for biofilm development. During this period the spots grow into fully formed "nanobiofilms" that display typical structural and phenotypic characteristics associated with mature C. albicans biofilms (i.e. morphological complexity, 3D architecture and drug resistance). In its final format, a C. albicans biofilm chip is composed of 768 equivalent and spatially distinct biofilms on a single glass slide; and multiple chips can be printed and processed simultaneously. A fluorometric assay with FUN1 viability stain is then used to determine the metabolic activity of the cells within each of the nanobiofilms, the intensity of which is determined using a microarray scanner. This fungal chip is ideally suited for use in true high throughput screening for antifungal drug discovery. Compared to current industry standard (namely the 96-well microtiter plate model of biofilm formation), this fungal biofilm chip has advantages in terms of miniaturization and automation, which combine to cut reagent use and analysis time, minimize or eliminate labor intensive steps, and dramatically reduce assay costs. Such a chip will speed up the drug discovery process by enabling rapid, convenient and inexpensive screening of hundreds-to-thousands of compounds simultaneously.