A Novel Actin Binding Drug with In Vivo Efficacy.

Occidiofungin is produced by the soil bacterium Burkolderia contaminans MS14 and is structurally similar or identical to the burkholdines, xylocandins, and cepacidines. This study identified the primary cellular target of occidiofungin, which was determined to be actin. The modification of occidiofungin with a functional alkyne group enabled affinity purification assays and localization studies in yeast. Occidiofungin has a subtle effect on actin dynamics that triggers apoptotic cell death. We demonstrate the highly specific localization of occidiofungin to cellular regions rich in actin in yeast and the binding of occidiofungin to purified actin in vitro Furthermore, a disruption of actin-mediated cellular processes, such as endocytosis, nuclear segregation, and hyphal formation, was observed. All of these processes require the formation of stable actin cables, which are disrupted following the addition of a subinhibitory concentration of occidiofungin. We were also able to demonstrate the effectiveness of occidiofungin in treating a vulvovaginal yeast infection in a murine model. The results of this study are important for the development of an efficacious novel class of actin binding drugs that may fill the existing gap in treatment options for fungal infections or different types of cancer.

A Human IRE1 Inhibitor Blocks the Unfolded Protein Response in the Pathogenic Fungus Aspergillus fumigatus and Suggests Noncanonical Functions within the Pathway.

The unfolded protein response (UPR) is a signaling network that maintains homeostasis of the endoplasmic reticulum (ER). In the human-pathogenic fungus Aspergillus fumigatus, the UPR is initiated by activation of an endoribonuclease (RNase) domain in the ER transmembrane stress sensor IreA, which splices the downstream mRNA hacAu into its active form, hacAi, encoding the master transcriptional regulator of the pathway. Small-molecule inhibitors against IRE1, the human ortholog of IreA, have been developed for anticancer therapy, but their effects on the fungal UPR are unexplored. Here, we demonstrate that the IRE1 RNase inhibitor 4µ8C prevented A. fumigatus from increasing the levels of hacAi mRNA, thereby blocking induction of downstream UPR target gene expression. Treatment with 4µ8C had minimal effects on growth in minimal medium but severely impaired growth on a collagen substrate that requires high levels of hydrolytic enzyme secretion, mirroring the phenotype of other fungal UPR mutants. 4µ8C also increased sensitivity to carvacrol, a natural compound that disrupts ER integrity in fungi, and hygromycin B, which correlated with reduced expression of glycosylation-related genes. Interestingly, treatment with 4µ8C was unable to induce all of the phenotypes attributed to the loss of the canonical UPR in a ΔhacA mutant but showed remarkable similarity to the phenotype of an RNase-deficient IreA mutant that is also unable to generate the hacAi mRNA. These results establish proof of principle that pharmacological inhibition of the canonical UPR pathway is feasible in A. fumigatus and support a noncanonical role for the hacAu mRNA in ER stress response.IMPORTANCE The unfolded protein response (UPR) is a signaling pathway that maintains endoplasmic reticulum (ER) homeostasis, with functions that overlap virulence mechanisms in the human-pathogenic mold Aspergillus fumigatus The canonical pathway centers on HacA, its master transcriptional regulator. Translation of this protein requires the removal of an unconventional intron from the cytoplasmic mRNA of the hacA gene, which is achieved by an RNase domain located in the ER-transmembrane stress sensor IreA. Here, we show that targeting this RNase activity with a small-molecule inhibitor effectively blocked UPR activation, resulting in effects that mirror the consequences of genetic deletion of the RNase domain. However, these phenotypes were surprisingly narrow in scope relative to those associated with a complete deletion of the hacA gene. These findings expand the understanding of UPR signaling in this species by supporting the existence of noncanonical functions for the unspliced hacA mRNA in ER stress response.