Fungal infections cause more than 1.5 million deaths annually. With an increase in immune-deficient susceptible populations and the emergence of antifungal drug resistance, there is an urgent need for novel strategies to combat these life-threatening infections. Here, we use a combinatorial screening approach to identify an imidazopyrazoindole, NPD827, that synergizes with fluconazole against azole-sensitive and -resistant isolates of Candida albicans. NPD827 interacts with sterols, resulting in profound effects on fungal membrane homeostasis and induction of membrane-associated stress responses. The compound impairs virulence in a Caenorhabditis elegans model of candidiasis, blocks C. albicans filamentation in vitro, and prevents biofilm formation in a rat model of catheter infection by C. albicans. Collectively, this work identifies an imidazopyrazoindole scaffold with a non-protein-targeted mode of action that re-sensitizes the leading human fungal pathogen, C. albicans, to azole antifungals.

For the fungal pathogen Candida albicans, genetic overexpression readily occurs via a diversity of genomic alterations, such as aneuploidy and gain-of-function mutations, with important consequences for host adaptation, virulence, and evolution of antifungal drug resistance. Given the important role of overexpression on C. albicans biology, it is critical to develop and harness tools that enable the analysis of genes expressed at high levels in the fungal cell. Here, we describe the development, optimization, and application of a novel, single-plasmid-based CRISPR activation (CRISPRa) platform for targeted genetic overexpression in C. albicans, which employs a guide RNA to target an activator complex to the promoter region of a gene of interest, thus driving transcriptional expression of that gene. Using this system, we demonstrate the ability of CRISPRa to drive high levels of gene expression in C. albicans, and we assess optimal guide RNA targeting for robust and constitutive overexpression. We further demonstrate the specificity of the system via RNA sequencing. We highlight the application of CRISPRa to overexpress genes involved in pathogenesis and drug susceptibility and contribute towards the identification of novel phenotypes. Consequently, this tool will facilitate a broad range of applications for the study of C. albicans genetic overexpression.

Filamentous bacteriophages (bacterial viruses) are semiflexible proteinous nanofilaments with high aspect ratios for which the surface chemistry can be controlled with atomic precision via genetic engineering. That, in addition to their ability to self-propagate and replicate a nearly monodisperse batch of biologically and chemically identical nanofilaments, makes these bionanofilaments superior to most synthetic nanoparticles and thus a powerful tool in the bioengineers’ toolbox. Furthermore, filamentous phages form liquid crystalline structures at high concentrations; these ordered assemblies create hierarchically ordered macro-, micro-, and nanostructures that, once cross-linked, can form hierarchically ordered hydrogels, hydrated soft material with a variety of physical and chemical properties suitable for biomedical applications (e.g., wound dressings and tissue engineering scaffolds) as well as biosensing, diagnostic assays. We provide a critical review of these hydrogels of filamentous phage, and their physical, mechanical, chemical, and biological properties and current applications, as well as an overview of limitations and challenges and outlook for future applications. In addition, we present a list of design parameters for filamentous phage hydrogels to serve as a guide for the (bio)­engineer and (bio)­chemist interested in utilizing these powerful bionanofilaments for designing smart, bioactive materials and devices.

As widespread antimicrobial resistance threatens to propel the world into a postantibiotic era, there is a pressing need to identify mechanistically distinct antimicrobial agents. This is of particular concern when considering the limited arsenal of drugs available to treat fungal infections, coupled with the emergence of highly drug-resistant fungal pathogens, including Candida auris .