The Essential Thioredoxin Reductase of the Human Pathogenic Mold Aspergillus fumigatus Is a Promising Antifungal Target

Abstract

The identification of cellular targets for antifungal compounds is a cornerstone for the development of novel antimycotics, for which a significant need exists due to increasing numbers of susceptible patients, emerging pathogens, and evolving resistance. For the human pathogenic mold Aspergillus fumigatus, the causative agent of the opportunistic disease aspergillosis, only a limited number of established targets and corresponding drugs are available. Among several targets that were postulated from a variety of experimental approaches, the conserved thioredoxin reductase (TrxR) activity encoded by the trxR gene was assessed in this study. Its essentiality could be confirmed following a conditional TetOFF promoter replacement strategy. Relevance of the trxR gene product for oxidative stress resistance was revealed and, most importantly, its requirement for full virulence of A. fumigatus in two different models of infection resembling invasive aspergillosis. Our findings complement the idea of targeting the reductase component of the fungal thioredoxin system for antifungal therapy.

Keywords: TetOFF; aspergillosis; conditional promoter replacement; essentiality; thioredoxin.

FIGURE 1

Generation of a conditional promoter replacement (CPR) strain to modulate trxR expression validates essentiality of the thioredoxin reductase-encoding gene for A. fumigatus. (A) Schematic outline of the CPR procedure employing the tetOFF:trxR module of pSK655. (B) Steady state trxR transcript levels in the wild-type strain AfS35 and its tetOFF:trxR derivative AfS227 illustrate a significant down-regulation of trxR transcription for the latter in the presence of doxycycline (+Dox). (C) Growth of AfS227 on minimal (left panel) and rich Sabouraud (right panel) culture medium is strongly inhibited by doxycycline, thereby validating essentiality of the trxR gene.

FIGURE 2

Growth impairment by TrxR deficiency is not rescued substantially by supplementation of glutathione or other organic sources of sulfur like cysteine or methionine. Shown are sporulating mycelia documented after three days of growth on culture medium supplemented with 20 mM glutathione (GSH) or 5 mM cysteine or methionine in the presence or absence of doxycycline.

FIGURE 3

The trxR gene product contributes to oxidative stress resistance of A. fumigatus. (A) Growth inhibition by H₂O₂ correlates with increasing concentrations of doxycycline as monitored by halo formation in agar diffusion tests (left panel, plate assays; right panel, quantification from three replicates). (B) Transcription of trxR in the wild-type isolate AfS35 is not significantly influenced by oxidative stress conditions as triggered by 5 mM H₂O₂, whereas trxR transcript levels increase upon prolonged incubation of AfS227 in the presence of the oxidative stressor. All samples were generated from mycelia grown in the absence of doxycycline to exclude interference with trxR transcription in the TetOFF strain. (C) Oxidative stress resistance is influenced by metronidazole in a trxR-dependent manner, as deduced from a positive correlation of H₂O₂ sensitivity with increasing metronidazole concentrations for the conditional tetOFF:trxR expression strain AfS227.

FIGURE 4

Pathogenicity of A. fumigatus is influenced by trxR transcription. (A) Infections of larvae of the greater wax moth Galleria mellonella (n = 20 for every cohort) with the CPR strain AfS227 reveal a highly significant (p = 0.0071) attenuation of virulence in the presence of doxycycline. (B) In a murine infection model of pulmonary invasive aspergillosis, animals fed with doxycycline show increased survival after infection with the conditional tetOFF:trxR expression strain (n = 10 for every cohort).