Opportunistic fungal pathogens are a leading cause of healthcare associated bloodstream infections. Candida yeasts, specifically, cause 80-90% of biofilm-associated invasive fungal infections and mortality rates can approach 50%. Furthermore, the incidence of Candida infections is rising with the increased use of catheter and other device-based interventions. To date, the majority of work related to fungal infections and potential treatments has focused on biofilms and their prevention. However, recent evidence suggests that the dispersion of yeast cells from the biofilm (into the bloodstream) and the persistence of these dispersed cells are perhaps more important virulence factors and represent significant but underutilized treatment targets. Further, existing assays and instrumentation are not amenable to measuring dispersion and phenotyping dispersed cells. Nor do they recapitulate in vivo conditions (e.g. flow, interaction with host cells). Thus, here we will develop a new type of under-oil open microfluidic system (UOMS) to quantify the dispersive capacity of biofilms and assess the phenotype of dispersed cells in Candida mutants and clinical isolates. The UOMS platform is built on the foundation of a newly observed phenomena called Exclusive Liquid Repellency (ELR). ELR provides a unique environment where liquid is completely repelled from a solid surface to eliminate biofouling. Additionally, ELR expands the capabilities of simple open microfluidic devices allowing us to overcome the limitations of current methods and provide a system capable of quantitatively studying fungal dispersion. We will first (Aim 1) develop an under-oil microchannel device to measure the dispersive capacity of Candida biofilms and virulence phenotypes.
Second (Aim 2) we will automate the UOMS and develop a single- cell distribution assay to measure the phenotype of individual dispersed cells. And finally (Aim 3), we will use the UOMS platform to develop dispersion phenotype profiles for Candida mutant libraries and clinical isolates. We will measure the dispersive capacity and phenotype of dispersed cells from hundreds of Candida albicans clinical isolates and mutants available in existing libraries, providing clues to the genetic determinants of dispersion.
Candida yeast species cause 80-90% of invasive fungal infections and up to 50% of patients who develop invasive Candidiasis die. Dispersion from Candida biofilms is a unique aspect of these fungal infections associated with mortality and morbidity yet dispersion remains poorly understood and therapeutically untargeted due to a lack of quantitative in vitro assays. We propose to develop an under-oil microfluidic system (UOMS) that will allow high-throughput measurement of dispersive capacity and dispersed cell phenotype in Candida mutants and clinical isolates, allowing the fundamentals of this important virulence process to be understood and explored as a drug target.
