The negative cofactor 2 complex is a key regulator of drug resistance in Aspergillus fumigatus

Furukawa, Tatsuhiko; Rhijn, Norman van; Fraczek, Marcin G.; Gsaller, Fabio; Davies, Emma; Carr, P.D.; Gago, Sara; Fortune‐Grant, Rachael; Rahman, Sanzidur; Gilsenan, Jane Mabey; Houlder, Emma L; Kowalski, Caitlin H.; Raj, Shriya; Paul, Sanjoy; Cook, Peter C.; Parker, Josie E.; Kelly, S. L.; Cramer, Robert A.; Latgé, Jean‐Paul; Moye‐Rowley, W. Scott; Bignell, Elaine; Bowyer, Paul

doi:10.1038/s41467-019-14191-1

Cited by 106 publications

(171 citation statements)

References 84 publications

(111 reference statements)

Supporting

Mentioning

170

Contrasting

Unclassified

Order By: Relevance

“…To assess if any other TF plays a role in CPE, a library of 484 A. fumigatus TF null mutants (25) was screened for sensitivity to high concentrations of caspofungin (16 g/ml). At this concentration, the CPE is induced in the CEA17 parental strain (22,23).…”

Section: Eleven Transcriptional Factors Govern Caspofungin Paradoxicamentioning

confidence: 99%

Aspergillus fumigatus Transcription Factors Involved in the Caspofungin Paradoxical Effect

Valero

Colabardini

Chiaratto

et al. 2020

mBio

View full text Add to dashboard Cite

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.

show abstract

Section: Eleven Transcriptional Factors Govern Caspofungin Paradoxicamentioning

confidence: 99%

Aspergillus fumigatus Transcription Factors Involved in the Caspofungin Paradoxical Effect

Valero

Colabardini

Chiaratto

et al. 2020

mBio

View full text Add to dashboard Cite

show abstract

“…The transcription factor knockout mutant collection was generated in the A. fumigatus strain A1160 (a derivative of CEA17, ΔKu80 pyrG + ) according to Furukawa et al [68]. Gene knockout cassettes were generated using a fusion PCR approach.…”

Section: Generation Validation and Screening Of Transcription Factomentioning

confidence: 99%

Aspergillus fumigatus calcium-responsive transcription factors regulate cell wall architecture promoting stress tolerance, virulence and caspofungin resistance

et al. 2019

Self Cite

View full text Add to dashboard Cite

Aspergillus fumigatus causes invasive aspergillosis, the most common life-threatening fungal disease of immuno-compromised humans. The treatment of disseminated infections with antifungal drugs, including echinocandin cell wall biosynthesis inhibitors, is increasingly challenging due to the rise of drug-resistant pathogens. The fungal calcium responsive calcineurin-CrzA pathway influences cell morphology, cell wall composition, virulence, and echinocandin resistance. A screen of 395 A. fumigatus transcription factor mutants identified nine transcription factors important to calcium stress tolerance, including CrzA and ZipD. Here, comparative transcriptomics revealed CrzA and ZipD regulated the expression of shared and unique gene networks, suggesting they participate in both converged and distinct stress response mechanisms. CrzA and ZipD additively promoted calcium stress tolerance. However, ZipD also regulated cell wall organization, osmotic stress tolerance and echinocandin resistance. The absence of ZipD in A. fumigatus caused a significant virulence reduction in immunodeficient and immunocompetent mice. The ΔzipD mutant displayed altered cell wall organization and composition, while being more susceptible to macrophage killing and eliciting an increased pro-inflammatory cytokine response. A higher number of neutrophils, macrophages and activated macrophages were found in ΔzipD infected mice lungs. Collectively, this shows that ZipD-mediated regulation of the fungal cell wall PLOS Genetics | https://doi.contributes to the evasion of pro-inflammatory responses and tolerance of echinocandin antifungals, and in turn promoting virulence and complicating treatment options. Author summaryA. fumigatus is the main ethiological agent of one of the most important life-threatening fungal diseases in immuno-compromised humans, invasive aspergillosis. Treatment commonly involves echinocandin antifungal drugs that inhibit cell wall β-1,3-glucan polysaccharide biosynthesis. Calcium is an important messenger for many signaling pathways regulating the fungal response to stress and antifungal drugs. The calcium responsive calcineurin phosphatase influences the localization and activity of the CrzA transcription factor (TF), regulating the activation of several stress responses and cell wall modifications. For many years, CrzA has been recognized as the sole calcium/calcineurin-dependent TF. Here, we identify nine TFs involved in the calcium/calcineurin metabolism, including a novel A. fumigatus calcium/calcineurin dependent TF named ZipD. Transcriptional profiling of the response of A. fumigatus wild-type, plus the ΔcrzA and ΔzipD mutant, strains shows CrzA and ZipD to have shared and unique functions. ZipD was found to be important for not only calcium metabolism, but also for the cell wall organization, osmotic stress and echinocandin tolerance. During host infection, ZipD plays an important role in modulating fungal cell walls, promoting evasion of the host pro-inflammatory immune responses and virulence. Our work emphasize...

show abstract

“…A screen for transcription factor deletion strains sensitive to sorbic acid identifies warA. To find genes associated with weak-acid resistance, an Aspergillus fumigatus transcription factor deletant collection (30) was screened for sorbic acid sensitivity. Aspergillus fumigatus is not commonly associated with food spoilage, and it displays relatively high sensitivity to weak acids such as sorbic acid (14).…”

Section: Resultsmentioning

confidence: 99%

“…Strains and media. The Aspergillus fumigatus transcription factor deletant library, derived from wild-type strain MFIG001, was constructed by homologous recombination using gene replacement cassettes and transformation methodologies described previously (30,41). Studies in Aspergillus niger were performed in the A. niger N402 background (referred to as the A. niger WT throughout) and an A. niger ΔcdcA mutant strain (14).…”

Section: Methodsmentioning

confidence: 99%

Weak Acid Resistance A (WarA), a Novel Transcription Factor Required for Regulation of Weak-Acid Resistance and Spore-Spore Heterogeneity in Aspergillus niger

Geoghegan

Stratford

Bromley

et al. 2020

mSphere

View full text Add to dashboard Cite

Propionic, sorbic, and benzoic acids are organic weak acids that are widely used as food preservatives, where they play a critical role in preventing microbial growth. In this study, we uncovered new mechanisms of weak-acid resistance in molds. By screening a library of 401 transcription factor deletion strains in Aspergillus fumigatus for sorbic acid hypersensitivity, a previously uncharacterized transcription factor was identified and named weak acid resistance A (WarA). The orthologous gene in the spoilage mold Aspergillus niger was identified and deleted. WarA was required for resistance to a range of weak acids, including sorbic, propionic, and benzoic acids. A transcriptomic analysis was performed to characterize genes regulated by WarA during sorbic acid treatment in A. niger. Several genes were significantly upregulated in the wild type compared with a ΔwarA mutant, including genes encoding putative weak-acid detoxification enzymes and transporter proteins. Among these was An14g03570, a putative ABC-type transporter which we found to be required for weak-acid resistance in A. niger. We also show that An14g03570 is a functional homologue of the Saccharomyces cerevisiae protein Pdr12p and we therefore name it PdrA. Last, resistance to sorbic acid was found to be highly heterogeneous within genetically uniform populations of ungerminated A. niger conidia, and we demonstrate that pdrA is a determinant of this heteroresistance. This study has identified novel mechanisms of weak-acid resistance in A. niger which could help inform and improve future food spoilage prevention strategies. IMPORTANCE Weak acids are widely used as food preservatives, as they are very effective at preventing the growth of most species of bacteria and fungi. However, some species of molds can survive and grow in the concentrations of weak acid employed in food and drink products, thereby causing spoilage with resultant risks for food security and health. Current knowledge of weak-acid resistance mechanisms in these fungi is limited, especially in comparison to that in yeasts. We characterized gene functions in the spoilage mold species Aspergillus niger which are important for survival and growth in the presence of weak-acid preservatives. Such identification of weak-acid resistance mechanisms in spoilage molds will help in the design of new strategies to reduce food spoilage in the future.

show abstract

The negative cofactor 2 complex is a key regulator of drug resistance in Aspergillus fumigatus

Cited by 106 publications

References 84 publications

Aspergillus fumigatus Transcription Factors Involved in the Caspofungin Paradoxical Effect

Aspergillus fumigatus Transcription Factors Involved in the Caspofungin Paradoxical Effect

Aspergillus fumigatus calcium-responsive transcription factors regulate cell wall architecture promoting stress tolerance, virulence and caspofungin resistance

Weak Acid Resistance A (WarA), a Novel Transcription Factor Required for Regulation of Weak-Acid Resistance and Spore-Spore Heterogeneity in Aspergillus niger

Contact Info

Product

Resources

About