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TRNA hypomodification facilitates 5-fluorocytosine resistance via cross-pathway control system activation in Aspergillus fumigatus.

ABSTRACT: Increasing antifungal drug resistance is a major concern associated with human fungal pathogens like Aspergillus fumigatus. Genetic mutation and epimutation mechanisms clearly drive resistance, yet the epitranscriptome remains relatively untested. Here, deletion of the A. fumigatus tRNA-modifying isopentenyl transferase ortholog, Mod5, led to altered stress response and unexpected resistance against the antifungal drug 5-fluorocytosine (5-FC). After confirming the canonical isopentenylation activity of Mod5 by LC-MS/MS and Nano-tRNAseq, we performed simultaneous profiling of transcriptomes and proteomes to reveal a comparable overall response to 5-FC stress; however, a premature activation of cross-pathway control (CPC) genes in the knockout was further increased after antifungal treatment. We identified several orthologues of the A. nidulans Major Facilitator Superfamily (MFS) transporter nmeA as specific CPC-client genes in A. fumigatus. Overexpression of Mod5-target tRNATyrGΨA in the ∆mod5 strain rescued select phenotypes but failed to reverse 5-FC resistance, whereas deletion of nmeA largely, but incompletely, reverted the resistance phenotype, implying additional relevant exporters. In conclusion, 5-FC resistance in the absence of Mod5 and i6A likely originates from multifaceted transcriptional and translational changes that skew the fungus towards premature CPC-dependent activation of antifungal toxic-intermediate exporter nmeA, offering a potential mechanism reliant on RNA modification to facilitate transient antifungal resistance.

ORGANISM(S): Aspergillus fumigatus

PROVIDER: GSE276346 | GEO | 2024/10/16

REPOSITORIES: GEO

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Mod5 mediates a molecular trade-off between transcriptional and translational regulation and antifungal resistance.

Project description:Increasing antifungal drug resistance is a major concern associated with human fungal infections. Genetic mutation and epimutation mechanisms are clearly linked to resistance in some fungi, but few studies have investigated RNA modifications (the epitranscriptome) as a mediator of resistance. Here, deletion of the Aspergillus fumigatus tRNA-modifying isopentenyl transferase ortholog, Mod5, led to altered growth and stress response, but also unexpected resistance against the antifungal drug 5-fluorocytosine (5-FC). Simultaneous profiling of the transcriptome and proteome of 5-FC-stressed wild-type and ∆mod5 strains revealed a similar general adaptation to stress; however, the knockout displayed elevated expression of cross-pathway control (CPC) genes. Quantification of CPC transcription factor cpcA and client gene argB showed premature CPC activation in ∆mod5, which was further increased upon treatment with 5-FC. By associating codon usage patterns with proteomics abundances, we observed the number of tRNATyrGΨA-decoded codons to negatively correlate with protein abundance in the knockout, indicative of modification-tunable transcripts. Overexpression of tRNATyrGΨA in the ∆mod5 strain rescued select phenotypes but, surprisingly, failed to reverse 5-FC resistance. Investigation of the purported tRNA gene-mediated silencing function of Mod5 uncovered a negative correlation between tRNA proximity and gene induction under normal growth, suggesting that tRNA gene-mediated silencing is active in A. fumigatus. In conclusion, 5-FC resistance in the absence of bifunctional Mod5 likely originates from multifaceted transcriptional and translational changes that skew the fungus towards starvation responses over optimal growth, potentially offering a mechanism reliant on RNA modification and tRNA gene-mediated silencing capable of facilitating transient antifungal resistance.

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Project description:Increasing antifungal drug resistance is a major concern associated with human fungal infections. Genetic mutation and epimutation mechanisms are clearly linked to resistance in some fungi, but few studies have investigated RNA modifications (the epitranscriptome) as a mediator of resistance. Here, deletion of the Aspergillus fumigatus tRNA-modifying isopentenyl transferase ortholog, Mod5, led to altered growth and stress response, but also unexpected resistance against the antifungal drug 5-fluorocytosine (5-FC). Simultaneous profiling of the transcriptome and proteome of 5-FC-stressed wild-type and ∆mod5 strains revealed a similar general adaptation to stress; however, the knockout displayed elevated expression of cross-pathway control (CPC) genes. Quantification of CPC transcription factor cpcA and client gene argB showed premature CPC activation in ∆mod5, which was further increased upon treatment with 5-FC. By associating codon usage patterns with proteomics abundances, we observed the number of tRNATyrGΨA-decoded codons to negatively correlate with protein abundance in the knockout, indicative of modification-tuneable transcripts. Subsequent overexpression of tRNATyrGΨA in the ∆mod5 strain rescued select phenotypes but, surprisingly, failed to reverse 5-FC resistance. Investigation of the purported tRNA gene-mediated silencing function of Mod5 uncovered a negative correlation between tRNA proximity and gene induction under normal growth, suggesting that tRNA gene-mediated silencing is active in A. fumigatus. In conclusion, 5-FC resistance in the absence of the bifunctional Mod5 protein likely originates from multifaceted transcriptional and translational changes that skew the fungus towards starvation responses over optimal growth, potentially offering a mechanism reliant on RNA modification and tRNA gene-mediated silencing capable of facilitating transient antifungal resistance.

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