Aspergillus fumigatus Transcription Factors Involved in the Caspofungin Paradoxical Effect

Abstract

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.

The Quiet and Underappreciated Rise of Drug-Resistant Invasive Fungal Pathogens

“…The inhibition of growth at MIC involves a relocalization of β-1,3-d-glucan synthase complex (Fks1p and Rho) from cell wall to vacuole, while continuous exposure to high MIC after 48 h results in returning of this complex to cell wall, which results in normal growth [38]. New lines of studies have found that a transcription factor, FhdA, plays an important role in PGE, which is involved in iron metabolism and mitochondrial respiratory function [39]. Importantly, addition of farnesol can block PGE in A. fumigatus when exposed to caspofungin [38].…”

Direct Visualization of Fungal Burden in Filamentous Fungus-Infected Silkworms

“…We observed that for TFKO mutants with strongly attenuated ( ∆hapB ), attenuated ( ∆rfeC and ∆ AFUB_014000) or unchanged virulence ( ∆nctA ) in mouse infections, our silkworm model could faithfully predict these phenotypes, even though various mouse models had been used for individual A. fumigatus mutants [ 40 , 41 ]. However, the other four TFKO mutants showed slightly attenuated virulence compared to the WT strain in our silkworm model, which in this case could not reflect the mutant virulence in mouse infections [ 42 , 43 ]. In addition, we also compared seven A. fumigatus TFKO mutants that had been previously generated from other WT progenitors and had been validated in various infection models ( Table S2 ).…”

Signaling Pathways Governing the Caspofungin Paradoxical Effect in Aspergillus fumigatus

“…By conducting more detailed phenotype analysis in some of these TF mutant strains, Valero and colleagues (10) nicely demonstrated that NctC and CbfA are also essential for optimal growth under various stress conditions. Unexpectedly, both CBF/NF-Y TFs were also shown to act as negative regulators of the biosynthesis of secondary metabolites belonging to the nonribosomal peptide family (NRPs) such as fumiquinazolines A, C, and F; pyripyropene A; tryprostatin A; and fumitremorgin C. Last but not least, the nctC null mutant was shown to be avirulent in a neutropenic mouse model.…”

“…In a preliminary work, Goldman and colleagues demonstrated the role of the basic leucine zipper ZipD transcription factor (TF) in regulating the calcium-calcineurin pathway that governs CPE in A. fumigatus ( 8 ). To gain further insights into the regulation of the CPE, and considering that this phenomenon could result from the concerted effect of many gene regulators, these authors took the opportunity of a 484-TF null mutant deletion library to address this question ( 9 , 10 ).…”

“…1 ). Importantly, all but two were demonstrated to be important not only for CPE but also for caspofungin basal susceptibility ( 10 ).…”