Overcoming Fungal Echinocandin Resistance through Inhibition of the Non-essential Stress Kinase Yck2.

New strategies are urgently needed to counter the threat to human health posed by drug-resistant fungi. To explore an as-yet unexploited target space for antifungals, we screened a library of protein kinase inhibitors for the ability to reverse resistance of the most common human fungal pathogen, Candida albicans, to caspofungin, a widely used antifungal. This screen identified multiple 2,3-aryl-pyrazolopyridine scaffold compounds capable of restoring caspofungin sensitivity. Using chemical genomic, biochemical, and structural approaches, we established the target for our most potent compound as Yck2, a casein kinase 1 family member. Combination of this compound with caspofungin eradicated drug-resistant C. albicans infection while sparing co-cultured human cells. In mice, genetic depletion of YCK2 caused an ∼3-log10 decline in fungal burden in a model of systemic caspofungin-resistant C. albicans infection. Structural insights and our tool compound's profile in culture support targeting the Yck2 kinase function as a broadly active antifungal strategy.

Functional Genomic Screening Reveals Core Modulators of Echinocandin Stress Responses in Candida albicans.

Candida albicans is a leading cause of death due to fungal infection. Treatment of systemic candidiasis often relies on echinocandins, which disrupt cell wall synthesis. Resistance is readily acquired via mutations in the drug target gene, FKS1. Both basal tolerance and resistance to echinocandins require cellular stress responses. We performed a systematic analysis of 3,030 C. albicans mutants to define circuitry governing cellular responses to echinocandins. We identified 16 genes for which deletion or transcriptional repression enhanced echinocandin susceptibility, including components of the Pkc1-MAPK signaling cascade. We discovered that the molecular chaperone Hsp90 is required for the stability of Pkc1 and Bck1, establishing key mechanisms through which Hsp90 mediates echinocandin resistance. We also discovered that perturbation of the CCT chaperonin complex causes enhanced echinocandin sensitivity, altered cell wall architecture, and aberrant septin localization. Thus, we provide insights into the mechanisms by which cellular chaperones enable crucial responses to echinocandin-induced stress.

Molecular Evolution of Antifungal Drug Resistance.

The fungal pathogens Candida albicans, Cryptococcus neoformans, and Aspergillus fumigatus have transitioned from a rare curiosity to a leading cause of human mortality. The management of infections caused by these organisms is intimately dependent on the efficacy of antifungal agents; however, fungi that are resistant to these treatments are regularly isolated in the clinic, impeding our ability to control infections. Given the significant impact fungal pathogens have on human health, it is imperative to understand the molecular mechanisms that govern antifungal drug resistance. This review describes our current knowledge of the mechanisms by which antifungal drug resistance evolves in experimental populations and clinical settings. We explore current antifungal treatment options and discuss promising strategies to impede the evolution of drug resistance. By tackling antifungal drug resistance as an evolutionary problem, there is potential to improve the utility of current treatments and accelerate the development of novel therapeutic strategies.

ProP-ProP and ProP-phospholipid interactions determine the subcellular distribution of osmosensing transporter ProP in Escherichia coli.

Osmosensing transporter ProP protects bacteria from osmotically induced dehydration by mediating the uptake of zwitterionic osmolytes. ProP activity is a sigmoidal function of the osmolality. ProP orthologues share an extended, cytoplasmic C-terminal domain. Orthologues with and without a C-terminal, α-helical coiled-coil domain respond similarly to the osmolality. ProP concentrates at the poles and septa of Escherichia coli cells in a cardiolipin (CL)-dependent manner. The roles of phospholipids and the C-terminal domain in subcellular localization of ProP were explored. Liposome association of peptides representing the C-terminal domains of ProP orthologues and variants in vitro was compared with subcellular localization of the corresponding orthologues and variants in vivo. In the absence of coiled-coil formation, the C-terminal domain bound liposomes and ProP concentrated at the cell poles in a CL-independent manner. The presence of the coiled-coil replaced those phenomena with CL-dependent binding and localization. The effects of amino acid replacements on lipid association of the C-terminal peptide fully recapitulated their effects on the subcellular localization of ProP. These data suggest that polar localization of ProP results from association of its C-terminal domain with the anionic lipid-enriched membrane at the cell poles. The coiled-coil domain present on only some orthologues renders that phenomenon CL-dependent.