Candida auris is an emerging fungal pathogen and a serious global health threat as the majority of clinical isolates display elevated resistance to currently available antifungal drugs. Despite the increased prevalence of C. auris infections, the mechanisms governing drug resistance remain largely elusive. In diverse fungi, the evolution of drug resistance is enabled by the essential molecular chaperone Hsp90, which stabilizes key regulators of cellular responses to drug-induced stress. Hsp90 also orchestrates temperature-dependent morphogenesis in Candida albicans, a key virulence trait. However, the role of Hsp90 in the pathobiology of C. auris remains unknown. In order to study regulatory functions of Hsp90 in C. auris, we placed HSP90 under the control of a doxycycline-repressible promoter to enable transcriptional repression. We found that Hsp90 is essential for growth in C. auris and that it enables tolerance of clinical isolates with respect to the azoles, which inhibit biosynthesis of the membrane sterol ergosterol. High-level azole resistance was independent of Hsp90 but dependent on the ABC transporter CDR1, deletion of which resulted in abrogated resistance. Strikingly, we discovered that C. auris undergoes a morphogenetic transition from yeast to filamentous growth in response to HSP90 depletion or cell cycle arrest but not in response to other cues that induce C. albicans filamentation. Finally, we observed that this developmental transition is associated with global transcriptional changes, including the induction of cell wall-related genes. Overall, this report provides a novel insight into mechanisms of drug tolerance and resistance in C. auris and describes a developmental transition in response to perturbation of a core regulator of protein homeostasis. IMPORTANCE Fungal pathogens pose a serious threat to public health. Candida auris is an emerging fungal pathogen that is often resistant to commonly used antifungal drugs. However, the mechanisms governing drug resistance and virulence in this organism remain largely unexplored. In this study, we adapted a conditional expression system to modulate the transcription of an essential gene, HSP90, which regulates antifungal resistance and virulence in diverse fungal pathogens. We showed that Hsp90 is essential for growth in C. auris and is important for tolerance of the clinically important azole antifungals, which block ergosterol biosynthesis. Further, we established that the Cdr1 efflux transporter regulates azole resistance. Finally, we discovered that C. auris transitions from yeast to filamentous growth in response to Hsp90 inhibition, accompanied by global transcriptional remodeling. Overall, this work provides a novel insight into mechanisms regulating azole resistance in C. auris and uncovers a distinct developmental program regulated by Hsp90.

Aspergillus fumigatus is an opportunistic fungal pathogen that secretes an array of immune-modulatory molecules, including secondary metabolites (SMs), which contribute to enhancing fungal fitness and growth within the mammalian host. Gliotoxin (GT) is a SM that interferes with the function and recruitment of innate immune cells, which are essential for eliminating A . fumigatus during invasive infections. We identified a C6 Zn cluster-type transcription factor (TF), subsequently named RglT, important for A . fumigatus oxidative stress resistance, GT biosynthesis and self-protection. RglT regulates the expression of several gli genes of the GT biosynthetic gene cluster, including the oxidoreductase-encoding gene gliT , by directly binding to their respective promoter regions. Subsequently, RglT was shown to be important for virulence in a chemotherapeutic murine model of invasive pulmonary aspergillosis (IPA). Homologues of RglT and GliT are present in eurotiomycete and sordariomycete fungi, including the non-GT-producing fungus A . nidulans , where a conservation of function was described. Phylogenetically informed model testing led to an evolutionary scenario in which the GliT-based resistance mechanism is ancestral and RglT-mediated regulation of GliT occurred subsequently. In conclusion, this work describes the function of a previously uncharacterised TF in oxidative stress resistance, GT biosynthesis and self-protection in both GT-producing and non-producing Aspergillus species.

Aspergillus fumigatus is the leading cause of pulmonary fungal diseases. Azoles have been used for many years as the main antifungal agents to treat and prevent invasive aspergillosis. However, in the last 10 years there have been several reports of azole resistance in A. fumigatus and new strategies are needed to combat invasive aspergillosis. Caspofungin is effective against other human-pathogenic fungal species, but it is fungistatic only against A. fumigatus. Resistance to caspofungin in A. fumigatus has been linked to mutations in the fksA gene that encodes the target enzyme of the drug β-1,3-glucan synthase. However, tolerance of high caspofungin concentrations, a phenomenon known as the caspofungin paradoxical effect (CPE), is also important for subsequent adaptation and drug resistance evolution. Here, we identified and characterized the transcription factors involved in the response to CPE by screening an A. fumigatus library of 484 null transcription factors (TFs) in CPE drug concentrations. We identified 11 TFs that had reduced CPE and that encoded proteins involved in the basal modulation of the RNA polymerase II initiation sites, calcium metabolism, and cell wall remodeling. One of these TFs, FhdA, was important for mitochondrial respiratory function and iron metabolism. The ΔfhdA mutant showed decreased growth when exposed to Congo red or to high temperature. Transcriptome sequencing (RNA-seq) analysis and further experimental validation indicated that the ΔfhdA mutant showed diminished respiratory capacity, probably affecting several pathways related to the caspofungin tolerance and resistance. Our results provide the foundation to understand signaling pathways that are important for caspofungin tolerance and resistance. IMPORTANCE Aspergillus fumigatus, one of the most important human-pathogenic fungal species, is able to cause aspergillosis, a heterogeneous group of diseases that presents a wide range of clinical manifestations. Invasive pulmonary aspergillosis is the most serious pathology in terms of patient outcome and treatment, with a high mortality rate ranging from 50% to 95% primarily affecting immunocompromised patients. Azoles have been used for many years as the main antifungal agents to treat and prevent invasive aspergillosis. However, there were several reports of evolution of clinical azole resistance in the last decade. Caspofungin, a noncompetitive β-1,3-glucan synthase inhibitor, has been used against A. fumigatus, but it is fungistatic and is recommended as second-line therapy for invasive aspergillosis. More information about caspofungin tolerance and resistance is necessary in order to refine antifungal strategies that target the fungal cell wall. Here, we screened a transcription factor (TF) deletion library for TFs that can mediate caspofungin tolerance and resistance. We have identified 11 TFs that are important for caspofungin sensitivity and/or for the caspofungin paradoxical effect (CPE). These TFs encode proteins involved in the basal modulation of the RNA polymerase II initiation sites, calcium metabolism or cell wall remodeling, and mitochondrial respiratory function. The study of those genes regulated by TFs identified in this work will provide a better understanding of the signaling pathways that are important for caspofungin tolerance and resistance.

In teleosts, the onset of puberty in females is marked by the appearance of the first wave of pre-vitellogenic (PV) follicles from the pool of primary growth (PG) follicles (follicle activation) in the ovary during sexual maturation. To understand the mechanisms underlying follicle activation and therefore puberty onset, we undertook this transcriptomic study to investigate gene expression profiles in the event. Our analysis revealed a total of 2,027 up-regulated and 859 down-regulated genes during the PG-PV transition. Gene Ontology (GO) analysis showed that in addition to basic cellular functions such as gene transcription, cell differentiation, and cell migration, other biological processes such as steroidogenesis, cell signaling and angiogenesis were also enriched in up-regulated genes; by comparison, some processes were down-regulated including piRNA metabolism, gene silencing and proteolysis. Further Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis identified a variety of signaling pathways that might play pivotal roles in PG-PV transition, including MAPK, TGF-β, Hedgehog, FoxO, VEGF, Jak-STAT, and phosphatidylinositol signaling pathways. Other pathways of particular interest included endocytosis and glycosaminoglycan biosynthesis. We also analyzed expression changes of genes expressed in different compartments viz. oocytes and follicle cells. Interestingly, most oocyte-specific genes remained unchanged in expression during follicle activation whereas a great number of genes specifically expressed in the follicle cells showed significant changes in expression. Overall, this study reported a comprehensive analysis for genes, biological processes and pathways involved in follicle activation, which also marks female puberty onset in the zebrafish when occurring for the first time in sexual maturation.

RB1 mutational inactivation is a cancer driver in various types of cancer including lung cancer, making it an important target for therapeutic exploitation. We performed chemical and genetic vulnerability screens in RB1-isogenic lung cancer pair and herein report that aurora kinase A (AURKA) inhibition is synthetic lethal in RB1-deficient lung cancer. Mechanistically, RB1−/− cells show unbalanced microtubule dynamics through E2F-mediated upregulation of the microtubule destabilizer stathmin and are hypersensitive to agents targeting microtubule stability. Inhibition of AURKA activity activates stathmin function via reduced phosphorylation and facilitates microtubule destabilization in RB1−/− cells, heavily impacting the bipolar spindle formation and inducing mitotic cell death selectively in RB1−/− cells. This study shows that stathmin-mediated disruption of microtubule dynamics is critical to induce synthetic lethality in RB1-deficient cancer and suggests that upstream factors regulating microtubule dynamics, such as AURKA, can be potential therapeutic targets in RB1-deficient cancer.

Aspergillus fumigatus is the main causative agent of invasive pulmonary aspergillosis (IPA), a severe disease that affects immunosuppressed patients worldwide. The fungistatic drug caspofungin is the second line of therapy against IPA but has increasingly been used against clinical strains that are resistant to azoles, the first line antifungal therapy. In high concentrations, caspofungin induces a tolerance phenotype with partial reestablishment of fungal growth called caspofungin paradoxical effect (CPE), resulting from a change in the composition of the cell wall. An increasing number of studies has shown that different isolates of A. fumigatus exhibit phenotypic heterogeneity, including heterogeneity in their CPE response. To gain insights into the underlying molecular mechanisms of CPE response heterogeneity, we analyzed the transcriptomes of two A. fumigatus reference strains, Af293 and CEA17, exposed to low and high caspofungin concentrations. We found that there is a core transcriptional response that involves genes related to cell wall remodeling processes, mitochondrial function, transmembrane transport, and amino acid and ergosterol metabolism, and a variable response related to secondary metabolite (SM) biosynthesis and iron homeostasis. Specifically, we show here that the overexpression of a SM pathway that works as an iron chelator extinguishes the CPE in both backgrounds, whereas iron depletion is detrimental for the CPE in Af293 but not in CEA17. We next investigated the function of the transcription factor CrzA, whose deletion was previously shown to result in heterogeneity in the CPE response of the Af293 and CEA17 strains. We found that CrzA constitutively binds to and modulates the expression of several genes related to processes involved in caspofungin tolerance, and that crzA deletion differentially impacts the SM production and growth of Af293 and CEA17. As opposed to the ΔcrzACEA17 mutant, the ΔcrzA Af293 mutant fails to activate cell wall remodeling genes upon caspofungin exposure, which most likely severely affects its macrostructure and extinguishes its CPE. This work describes how heterogeneity in the response to an antifungal agent between A. fumigatus strains stems from heterogeneity in the function of a transcription factor and its downstream target genes.

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Single-cell whole-exome sequencing (scWES) is a powerful approach for deciphering intratumor heterogeneity and identifying cancer drivers. So far, however, simultaneous analysis of single nucleotide variants (SNVs) and copy number variations (CNVs) of a single cell has been challenging. By analyzing SNVs and CNVs simultaneously in bulk and single cells of premalignant tissues and tumors from mouse and human BRCA1-associated breast cancers, we discover an evolution process through which the tumors initiate from cells with SNVs affecting driver genes in the premalignant stage and malignantly progress later via CNVs acquired in chromosome regions with cancer driver genes. These events occur randomly and hit many putative cancer drivers besides p53 to generate unique genetic and pathological features for each tumor. Upon this, we finally identify a tumor metastasis suppressor Plekha5, whose deficiency promotes cancer metastasis to the liver and/or lung.