Abstract
Although three major classes of systemic antifungal agents are clinically available, each is characterized by important limitations. Thus, there has been considerable ongoing effort to develop novel and repurposed agents for the therapy of invasive fungal infections. In an effort to address these needs, we developed a novel high-throughput, multiplexed screening method that utilizes small molecules to probe candidate drug targets in the opportunistic fungal pathogen Candida albicans. This method is amenable to high-throughput automated screening and is based upon detection of changes in GFP levels of individually tagged target proteins. We first selected four GFP-tagged membrane-bound proteins associated with virulence or antifungal drug resistance in C. albicans. We demonstrated proof-of-principle that modulation of fluorescence intensity can be used to assay the expression of specific GFP-tagged target proteins to inhibitors (and inducers), and this change is measurable within the HyperCyt automated flow cytometry sampling system. Next, we generated a multiplex of differentially color-coded C. albicans strains bearing C-terminal GFP-tags of each gene encoding candidate drug targets incubated in the presence of small molecules from the Prestwick Chemical Library in 384-well microtiter plate format. Following incubation, cells were sampled through the HyperCyt system and modulation of protein levels, as indicated by changes in GFP-levels of each strain, was used to identify compounds of interest. The hit rate for both inducers and inhibitors identified in the primary screen did not exceed 1% of the total number of compounds in the small-molecule library that was probed, as would be expected from a robust target-specific, high-throughput screening campaign. Secondary assays for virulence characteristics based on null mutant strains were then used to further validate specificity. In all, this study presents a method for the identification and verification of new antifungal drugs targeted to fungal virulence proteins using C. albicans as a model fungal pathogen.
Highlights
Candida infections are a major cause of morbidity and mortality in a wide range of patient populations
We developed a screening approach to assay the levels of C. albicans candidate drug targets by conjugating the protein of interest with green fluorescent protein (GFP), which can be detected and quantified as the live cell carrying this fusion protein passes through the flow cytometer
We used a series of C. albicans strains in which GFP was fused to the C-termini of the plasma membrane transporters Cdr1p and Ftr1p, the vacuolar membrane ABC transporter Mlt1p, and the plasma membrane protein Sur7p [30,42]
Summary
Candida infections are a major cause of morbidity and mortality in a wide range of patient populations. Despite improvements in antifungal therapy, the high attributable mortality rate due to Candida infections has improved little from two decades ago. Current antifungal agents are limited in the following aspects: (i) available formulations, (ii) accessibility to the site of infection and ability to achieve therapeutic dose, (iii) hepato- and nephro-toxicity, and/or (iv) emergence of antifungal drug resistant strains [4,5]. Usually well-tolerated, are limited by substantial resistance in several important non-albicans Candida species and within C. albicans biofilms. The echinocandins, are costly, and while highly effective against most Candida species, can be ineffective in the presence of point mutations in the FKS1 gene which encodes the glucan synthase drug target [7,8]. Echinocandins have limited central nervous system and urinary penetration, and are of limited to no utility against Cryptococcus species, some other rare opportunistic yeast species, and many invasive mold infections [13,14]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
