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Azole resistance of the fungal pathogen Aspergillus fumigatus is an emerging problem. To identify novel mechanisms that could mediate azole resistance in A. fumigatus, we analyzed the transcriptome of a mitochondrial fission/fusion mutant that exhibits increased azole tolerance. Approximately 12% of the annotated genes are differentially regulated in this strain. This comprises upregulation of Cyp51A, the azole target structure, upregulation of ATP-binding cassette (ABC) superfamily and major facilitator superfamily (MFS) transporters and differential regulation of transcription factors. To study their impact on azole tolerance, conditional mutants were constructed of seven ABC transporters and 17 transcription factors. Under repressed conditions, growth rates and azole susceptibility of the mutants were similar to wild type. Under induced conditions, several transcription factor mutants showed growth phenotypes. In addition, four ABC transporter mutants and seven transcription factor mutants exhibited altered azole susceptibility. However, deletion of individual identified ABC transporters and transcription factors did not affect the increased azole tolerance of the fission/fusion mutant. Our results revealed the ability of multiple ABC transporters and transcription factors to modulate the azole susceptibility of A. fumigatus and support a model where mitochondrial dysfunctions trigger a drug resistance network that mediates azole tolerance of this mold.
Super-resolution microscopy has evolved as a powerful method for subdiffraction-resolution fluorescence imaging of cells and cellular organelles, but requires sophisticated and expensive installations. Expansion microscopy (ExM), which is based on the physical expansion of the cellular structure of interest, provides a cheap alternative to bypass the diffraction limit and enable super-resolution imaging on a conventional fluorescence microscope. While ExM has shown impressive results for the magnified visualization of proteins and RNAs in cells and tissues, it has not yet been applied in fungi, mainly due to their complex cell wall. Here we developed a method that enables reliable isotropic expansion of ascomycetes and basidiomycetes upon treatment with cell wall degrading enzymes. Confocal laser scanning microscopy (CLSM) and structured illumination microscopy (SIM) images of 4.5-fold expanded sporidia of Ustilago maydis expressing fluorescent fungal rhodopsins and hyphae of Fusarium oxysporum or Aspergillus fumigatus expressing either histone H1-mCherry together with Lifeact-sGFP or mRFP targeted to mitochondria, revealed details of subcellular structures with an estimated spatial resolution of around 30 nm. ExM is thus well suited for cell biology studies in fungi on conventional fluorescence microscopes.
Echinocandin antifungals represent one of the most important drug classes for the treatment of invasive fungal infections. The mode of action of the echinocandins relies on inhibition of the β-1,3-glucan synthase, an enzyme essentially required for the synthesis of the major fungal cell wall carbohydrate β-1,3-glucan. Depending on the species, echinocandins may exert fungicidal or fungistatic activity. Apparently independent of this differential activity, a surprising in vitro phenomenon called the “paradoxical effect” can be observed. The paradoxical effect is characterized by the ability of certain fungal isolates to reconstitute growth in the presence of higher echinocandin concentrations, while being fully susceptible at lower concentrations. The nature of the paradoxical effect is not fully understood and has been the focus of multiple studies in the last two decades. Here we concisely review the current literature and propose an updated model for the paradoxical effect, taking into account recent advances in the field.