The Proteasome Governs Fungal Morphogenesis via Functional Connections with Hsp90 and cAMP-Protein Kinase A Signaling

ABSTRACT Protein homeostasis is critical for proliferation and viability of all organisms. For Candida albicans, protein homeostasis also modulates the transition between yeast and filamentous forms, which is critical for virulence. A key regulator of morphogenesis is the molecular chaperone Hsp90, which mediates proteostasis under physiological and stress conditions. Hsp90 regulates morphogenesis by repressing cyclic AMP-protein kinase A (cAMP-PKA) signaling, such that inhibition of Hsp90 causes filamentation in the absence of an inducing cue. We explored the effect of perturbation of another facet of protein homeostasis and discovered that morphogenesis is also regulated by the proteasome, a large 33-subunit protein complex consisting of a 20S catalytic core and two 19S regulatory particles, which controls degradation of intracellular proteins. We identified a conserved role of the proteasome in morphogenesis as pharmacological inhibition of the proteasome induced filamentation of C. albicans and the related species Candida dubliniensis, Candida tropicalis, Candida krusei, and Candida parapsilosis. For C. albicans, genetic depletion of any of 29 subunits of the 19S or 20S particle induced filamentation. Filaments induced by inhibition of either the proteasome or Hsp90 have shared structural characteristics, such as aberrant nuclear content, and shared genetic dependencies, such as intact cAMP-PKA signaling. Consistent with a functional connection between these facets of protein homeostasis that modulate morphogenesis, we observed that proteasome inhibition results in an accumulation of ubiquitinated proteins that overwhelm Hsp90 function, relieving Hsp90-mediated repression of morphogenesis. Together, our findings provide a mechanism whereby interconnected facets of proteostasis regulate C. albicans morphogenesis. IMPORTANCE Fungi cause life-threatening infections and pose a serious threat to human health as there are very few effective antifungal drugs. Candida albicans is a major human fungal pathogen and cause of morbidity and mortality in immunocompromised individuals. A key trait that enables C. albicans virulence is its ability to transition between yeast and filamentous forms. Understanding the mechanisms regulating this virulence trait can facilitate the development of much-needed, novel therapeutic strategies. A key regulator of morphogenesis is the molecular chaperone Hsp90, which is crucial for proteostasis. Here, we expanded our understanding of how proteostasis regulates fungal morphogenesis and identified the proteasome as a repressor of filamentation in C. albicans and related species. Our work suggests that proteasome inhibition overwhelms Hsp90 function, thereby inducing morphogenesis. This work provides a foundation for understanding the role of the proteasome in fungal virulence and offers potential for targeting the proteasome to disarm fungal pathogens.

Analogous and Diverse Functions of APSES-Type Transcription Factors in the Morphogenesis of the Entomopathogenic Fungus Metarhizium rileyi

ABSTRACT APSES-type transcription factors (TFs) have analogous and diverse functions in the regulation of fungal morphogenesis processes. However, little is known about these functions in microsclerotium formation. In this study, we characterized two orthologous APSES genes (MrStuA and MrXbp) in the entomopathogenic fungus Metarhizium rileyi. Deletion of either MrStuA or MrXbp impaired dimorphic transition, conidiation, fungal virulence, and microsclerotium formation. Compared with the wild-type strain, ΔMrStuA and ΔMrXbp mutants were hypersensitive to thermal and oxidative stress. Furthermore, transcriptome sequencing analysis revealed that MrStuA and MrXbp independently regulate their own distinctive subsets of signaling pathways during dimorphic transition and microsclerotium formation, but they also show an overlapping regulation of genes during these two distinct morphogenesis processes. These results provide a global insight into vital roles of MrStuA and MrXbp in M. rileyi and aid in dissection of the interacting regulatory mechanisms of dimorphism transition and microsclerotium development. IMPORTANCE Transcription factors (TFs) are core components of the signaling pathway and play an important role in transcriptional regulation of gene expression during fungal morphogenesis processes. A prevailing theory suggests an interplay between different TFs regulating microsclerotial differentiation; however, the persisting issue remains that these interplay mechanisms are not clear. Here, we analyzed two members of the APSES-type TFs in Metarhizium rileyi using a gene deletion strategy and transcriptome analysis. Mutants were significantly impaired in microsclerotium formation and dimorphic transition. Transcriptome analysis provided evidence for interacting regulatory mechanisms by the two TFs in microsclerotium formation and dimorphic transition. Furthermore, we investigated their overlapping roles in mediating the expression of genes required for different fungal morphogenesis processes. Characterization of TFs in this study will aid in dissecting the interplay between regulatory mechanisms in fungal morphogenesis processes.

Live Monitoring and Analysis of Fungal Growth, Viability, and Mycelial Morphology Using the IncuCyte NeuroTrack Processing Module

ABSTRACTEfficient live-imaging methods are pivotal to understand fungal morphogenesis, especially as it relates to interactions with host immune cells and mechanisms of antifungal drugs. Due to the notable similarities in growth patterns of neuronal cells and mycelial networks, we sought to repurpose the NeuroTrack (NT) processing module of the IncuCyte time-lapse microscopy system as a tool to quantify mycelial growth and branching of pathogenic fungi. We showed the robustness of NT analysis to studyCandida albicansand five different molds and confirmed established characteristics of mycelial growth kinetics. We also documented high intra- and interassay reproducibility of the NT module for a spectrum of spore inocula and culture periods. Using GFP-expressingAspergillus fumigatusandRhizopus arrhizus, the feasibility of fluorescence-based NT analysis was validated. In addition, we performed proof-of-concept experiments of NT analysis for several translational applications such as studying the morphogenesis of a filamentation-defectiveC. albicansmutant, the effects of different classes of antifungals (polyenes, azoles, and echinocandins), and coculture with host immune cells. High accuracy was found, even at high immune cell-to-fungus ratios or in the presence of fungal debris. For antifungal efficacy studies, addition of a cytotoxicity dye further refined IncuCyte-based analysis, facilitating real-time determination of fungistatic and fungicidal activity in a single assay. Complementing conventional MIC-based assays, NT analysis is an appealing method to study fungal morphogenesis and viability in the context of antifungal compound screening and evaluation of novel immune therapeutics.IMPORTANCEPathogenic fungi remain a major cause of infectious complications in immunocompromised patients. Microscopic techniques are crucial for our understanding of fungal biology, host-pathogen interaction, and the pleiotropic effects of antifungal drugs on fungal cell growth and morphogenesis. Taking advantage of the morphological similarities of neuronal cell networks and mycelial growth patterns, we employed the IncuCyte time-lapse microscopy system and its NeuroTrack image analysis software package to study growth and branching of a variety of pathogenic yeasts and molds. Using optimized image processing definitions, we validated IncuCyte NeuroTrack analysis as a reliable and efficient tool for translational applications such as antifungal efficacy evaluation and coculture with host immune effector cells. Hence, the IncuCyte system and its NeuroTrack module provide an appealing platform for efficientin vitrostudies of antifungal compounds and immunotherapeutic strategies in medical mycology.

New sulfone and sulfanyl derivatives with antifungal activity

Introduction: Increasing occurrence of fungal infections raises the need to develop novel antifungal agents. In this context, an inhibition of morphological switch may provide an alternative approach to find compounds with a potential to control the Candida albicans infections. Methods: A series of 17 sulfone and sulfanyl derivatives was synthesized and evaluated for activity against the C. albicans wild type (SC5314, ATCC) and SAP-deficient mutant strains using the broth microdilution method M27-A3. Afterwards, phase-contrast microscopy was applied to evaluate the inhibition of fungal morphogenesis under the influence of randomly selected active compounds: 1, 5 and 6. Results: By in vitro susceptibility testing of C. albicans, we identified the effective antifungal agents displaying moderate-to-good activity. Newly synthesized sulfanyl and sulfone derivatives strongly inhibited the C. albicans morphogenetic transition under the hyphae inducing conditions. Conclusions: The leading compound 6 has the potential to be used as a base structure in antifungal drug development, however further structural optimalization and toxicity studies are required.