Azole antifungals inhibit the fungal ergosterol biosynthesis pathway, resulting in either growth inhibition or killing of the pathogen, depending on the species. Here we report that azoles have an initial growth-inhibitory (fungistatic) activity against the pathogen Aspergillus fumigatus that can be separated from the succeeding fungicidal effects. At a later stage, the cell wall salvage system is induced. This correlates with successive cell integrity loss and death of hyphal compartments. Time-lapse fluorescence microscopy reveals excessive synthesis of cell wall carbohydrates at defined spots along the hyphae, leading to formation of membrane invaginations and eventually rupture of the plasma membrane. Inhibition of β-1,3-glucan synthesis reduces the formation of cell wall carbohydrate patches and delays cell integrity failure and fungal death. We propose that azole antifungals exert their fungicidal activity by triggering synthesis of cell wall carbohydrate patches that penetrate the plasma membrane, thereby killing the fungus. The elucidated mechanism may be potentially exploited as a novel approach for azole susceptibility testing.

In Escherichia coli or Salmonella enterica, the stress-associated mammalian hormones epinephrine (E) and norepinephrine (NE) trigger a signaling cascade by interacting with the QseC sensor protein. Here we show that Vibrio cholerae, the causative agent of cholera, exhibits a specific response to E and NE. These catecholates (0.1 mM) enhanced the growth and swimming motility of V. cholerae strain O395 on soft agar in a medium containing calf serum, which simulated the environment within the host. During growth, the hormones were converted to degradation products, including adrenochrome formed by autooxidation with O 2 or superoxide. In E. coli, the QseC sensor kinase, which detects the autoinducer AI-3, also senses E or NE. The genome of V. cholerae O395 comprises an open reading frame coding for a putative protein with 29% identity to E. coli QseC. Quantitative reverse transcriptase PCR (qRT-PCR) experiments revealed increased transcript levels of the qseC-like gene and of pomB, a gene encoding a structural component of the flagellar motor complex, under the influence of E or NE. Phentolamine blocks the response of E. coli QseC to E or NE. A V. cholerae mutant devoid of the qseC-like gene retained the phentolamine-sensitive motility in the presence of E, whereas NE-stimulated motility was no longer inhibited by phentolamine. Our study demonstrates that V. cholerae senses the stress hormones E and NE. A sensor related to the histidine kinase QseC from E. coli is identified and is proposed to participate in the sensing of NE. IMPORTANCEVibrio cholerae is a Gram-negative bacterium that may cause cholera, a severe illness with high mortality due to acute dehydration caused by diarrhea and vomiting. Pathogenic V. cholerae strains possess virulence factors like the cholera toxin (CTX) and the toxin-coregulated pilus (TCP) produced in response to signals provided by the host. In pathogenic enterobacteria, the stressassociated hormones epinephrine (E) and norepinephrine (NE) of the human host act as signal molecules for the production of virulence factors and promote bacterial growth by the sequestration of iron from the host. Here we show that V. cholerae, like some enterobacteria, benefits from these stress hormones and possesses a sensor to recognize them. Stress enhances the susceptibility of a mammalian host to infection by bacteria. The stress-associated mammalian hormones epinephrine (E) and norepinephrine (NE), for example, support the growth (1-3) and motility (4, 5) of the enterobacteria Escherichia coli, Salmonella enterica serovar Typhimurium, and Klebsiella pneumoniae (6), of Pseudomonas aeruginosa (7,8), and of some Vibrio species (9) in a serum-based bacteriostatic medium. As E and NE share structural similarities to the bacterial catechol siderophores-a benzene ring with two adjacent hydroxyl groups-it was suggested that these stress hormones may be involved in the binding and uptake of iron by bacteria, thus promoting growth or motility (1-5). Recently, the mechanism by which catecholamine hormo...

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.

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