Modular Inducible Multigene Expression System for Filamentous Fungi

Baldin, Clara; Kühbacher, Alexander; Merschak, Petra; Wagener, Johannes; Gsaller, Fabio

doi:10.1128/spectrum.03670-22

Cited by 6 publications

(3 citation statements)

References 71 publications

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“…When comparing effects of the ergosterol biosynthesis pathway inhibitors, the hmg1 pHspA mutants displayed resistance to the triazole antifungals but not to terbinafine (Fig. 5E and F ), coinciding with data from a separate recent report 31 . This was true for all mold-active triazoles (Supplementary Fig.…”

Section: Resultssupporting

confidence: 86%

A secondary mechanism of action for triazole antifungals in Aspergillus fumigatus mediated by hmg1

Rybak,

Xie,

Martin-Vicente

et al. 2024

Nat Commun

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Triazole antifungals function as ergosterol biosynthesis inhibitors and are frontline therapy for invasive fungal infections, such as invasive aspergillosis. The primary mechanism of action of triazoles is through the specific inhibition of a cytochrome P450 14-α-sterol demethylase enzyme, Cyp51A/B, resulting in depletion of cellular ergosterol. Here, we uncover a clinically relevant secondary mechanism of action for triazoles within the ergosterol biosynthesis pathway. We provide evidence that triazole-mediated inhibition of Cyp51A/B activity generates sterol intermediate perturbations that are likely decoded by the sterol sensing functions of HMG-CoA reductase and Insulin-Induced Gene orthologs as increased pathway activity. This, in turn, results in negative feedback regulation of HMG-CoA reductase, the rate-limiting step of sterol biosynthesis. We also provide evidence that HMG-CoA reductase sterol sensing domain mutations previously identified as generating resistance in clinical isolates of Aspergillus fumigatus partially disrupt this triazole-induced feedback. Therefore, our data point to a secondary mechanism of action for the triazoles: induction of HMG-CoA reductase negative feedback for downregulation of ergosterol biosynthesis pathway activity. Abrogation of this feedback through acquired mutations in the HMG-CoA reductase sterol sensing domain diminishes triazole antifungal activity against fungal pathogens and underpins HMG-CoA reductase-mediated resistance.

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Section: Resultssupporting

confidence: 86%

A secondary mechanism of action for triazole antifungals in Aspergillus fumigatus mediated by hmg1

Rybak,

Xie,

Martin-Vicente

et al. 2024

Nat Commun

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“…Consequently, the growth pattern of bol3 Venus was similar to that of Δbol3 , but the growth defect was less severe, which can most likely be explained by the basal PxylP activity in bol3 Venus . As PxylP shows low basal activity [ 31 , 34 ], these data indicate that low bol3 expression already impacts the growth pattern. The Δbol3 strain displayed a similar growth defect also on complex medium (electronic supplementary material, figure S5), revealing that the growth defect cannot be cured by supplementation with metabolites present in yeast extract or peptone such as amino acids or vitamins.…”

Section: Resultsmentioning

confidence: 99%

“…This is most likely caused by the basal PxylP activity. As PxylP shows very low basal activity [ 31 , 34 ], these data indicate that low bol3 expression already affects the growth pattern, or in other words, that only low Bol3c amounts are required for its cellular function. Interestingly, overexpression of Bol3m owing to high PxylP induction ( bol3 M41L-Venus with 1% xylose; figure 2 ) or exchange of the Kozak sequence ( bol3 xylK-M41L-Venus with 0.1% xylose; figure 2 ) cured the growth defect caused by lack of Bol3c ( bol3 M41L-Venus with 0.1% xylose; figure 2 ).…”

Section: Discussionmentioning

confidence: 99%

The cytosolic form of dual localized BolA family protein Bol3 is important for adaptation to iron starvation in Aspergillus fumigatus

Oberegger,

Misslinger,

Faserl

et al. 2024

Open Biol.

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Aspergillus fumigatus is the predominant mould pathogen for humans. Adaption to host-imposed iron limitation has previously been demonstrated to be essential for its virulence. [2Fe–2S] clusters are crucial as cofactors of several metabolic pathways and mediate cytosolic/nuclear iron sensing in fungi including A. fumigatus . [2Fe–2S] cluster trafficking has been shown to involve BolA family proteins in both mitochondria and the cytosol/nucleus. Interestingly, both A. fumigatus homologues, termed Bol1 and Bol3, possess mitochondrial targeting sequences, suggesting the lack of cytosolic/nuclear versions. Here, we show by the combination of mutational, proteomic and fluorescence microscopic analyses that expression of the Bol3 encoding gene leads to dual localization of gene products to mitochondria and the cytosol/nucleus via alternative translation initiation downstream of the mitochondrial targeting sequence, which appears to be highly conserved in various Aspergillus species. Lack of either mitochondrial Bol1 or Bol3 was phenotypically inconspicuous while lack of cytosolic/nuclear Bol3 impaired growth during iron limitation but not iron sensing which indicates a particular importance of [2Fe–2S] cluster trafficking during iron limitation. Remarkably, cytosolic/nuclear Bol3 differs from the mitochondrial version only by N-terminal acetylation, a finding that was only possible by mutational hypothesis testing.

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