Fabio Gsaller

The opportunistic mold Aspergillus fumigatus , a WHO-priority fungal pathogen, causes life-threatening invasive infections in immunocompromised patients. A major antifungal drug target is the ergosterol biosynthesis pathway, primarily regulated by the transcription factors SrbA and AtrR. Recently, we identified RttA as an additional regulator of sterol homeostasis that mediates the activation of the sterol C24-methyltransferase-encoding gene erg6 . Here, we demonstrate that overexpression of rttA leads to elevated production of the secondary metabolite fumicycline via induction of fccR , coding for the principal transcriptional activator of fumicycline biosynthesis. Lack of fccR blocked activation of fumicycline biosynthetic genes ( fccA-fccE ) during rttA overexpression, highlighting the indispensable role of FccR for activation of this cluster. In contrast, rttA inactivation did not abolish fumicycline production during fccR overexpression or during co-cultivation with Streptomyces rapamycinicus . In addition to fccA-fccE , our findings reveal a crucial role of FccR for the upregulation of a gene located adjacent to the fumicycline biosynthesis cluster, here termed fccF . Deletion of fccF severely diminished the conversion of fumicycline B into fumicycline C, uncovering FccF as fumicycline B O-acetyltransferase. Intriguingly, during fccR overexpression and resulting fumicycline overproduction, inactivation of fccF caused growth defects, likely due to precursor toxicity. Together, these findings broaden our understanding of fungal secondary metabolite regulation and reveal a novel role of RttA in the activation of fccR encoding the cluster-specific regulator that activates fumicycline production, including FccF, the terminal acetyltransferase in the pathway. IMPORTANCE: Aspergillus fumigatus is a major opportunistic fungal pathogen that causes severe infections in immunocompromised individuals and represents a significant global health threat. Understanding the regulatory networks controlling fungal metabolism is important for identifying potential therapeutic vulnerabilities. Here, we uncover a previously unrecognized role for the sterol homeostasis regulator RttA in activating fumicycline production through induction of the pathway-specific transcription factor FccR. We further show that FccR controls expression of an additional biosynthetic gene, fccF , and identify FccF as the terminal O-acetyltransferase required for conversion of fumicycline B to fumicycline C. Disruption of this final biosynthetic step impairs fungal growth during metabolite overproduction, indicating that accumulation of fumicycline B or related intermediates can negatively affect fungal growth. Together, these findings expand our understanding of fungal secondary metabolite regulation and provide new insights into the organization and function of the fumicycline biosynthetic pathway.

The increasing prevalence of triazole resistance among Aspergillus fumigatus clinical isolates threatens the efficacy of this front-line treatment, highlighting the urgent need for novel therapeutic targets. In this study, we characterized three putative lanosterol synthase (also known as oxidosqualene cyclase) paralogs and identified Erg7A as the dominant enzyme. Although erg7B provided functional redundancy when overexpressed, deletion of erg7A alone created a significant ergosterol biosynthetic blockade. erg7A deficiency led to the accumulation of upstream substrates (squalene and 2,3-oxidosqualene) as well as downstream sterol intermediates, including lanosterol and eburicol. This sterol perturbation conferred triazole hypersusceptibility to a laboratory strain and restored susceptibility in resistant clinical isolates harboring cyp51A or hmg1 mutations. We further validated these findings using Ro 48-8071, a specific oxidosqualene cyclase inhibitor, and found that Ro 48-8071 exhibited potent synergy with the long-tailed triazoles, itraconazole, and posaconazole. Our findings demonstrate that targeting Erg7 sensitizes A. fumigatus to triazoles, offering a potential combination therapy against resistant isolates. IMPORTANCE: Aspergillus fumigatus is a mold pathogen and the major etiologic agent of invasive aspergillosis. Treatment of invasive aspergillosis is currently limited to only three classes of antifungals, the use of which is hampered by toxicity and rising antifungal resistance rates. The front-line therapeutic class, the triazoles, functions through the inhibition of ergosterol biosynthesis, and the majority of clinical triazole resistance is caused by target gene mutations impacting the sterol demethylase Cyp51. In this study, we differentiate the biological roles of three putative A. fumigatus lanosterol synthase paralogs as enzymes functioning upstream of the triazole target. We provide strong genetic and pharmacological evidence that inhibition of this upstream enzymatic step in the ergosterol biosynthetic pathway is synergistic with triazoles and can resensitize resistant clinical isolates. The development of lanosterol synthase inhibitors with fungus-specific activity is expected to provide new stand-alone and/or combination therapies to improve outcomes of invasive aspergillosis.

Major mechanisms of azole resistance in Aspergillus fumigatus involve overexpression of the azole target protein encoded by cyp51A . The elevated expression originates from the duplication of an enhancer element in its promoter, serving as a binding platform for AtrR and SrbA, two central transcription factors that orchestrate the activation of ergosterol biosynthesis genes and azole resistance. Alongside AtrR and SrbA, several other transcription factors were shown to be crucial to mediate azole tolerance. Here, we unveil RttA as a further protein involved in the regulation of ergosterol biosynthesis and azole resistance in A. fumigatus . Because the gene was wrongly annotated, its encoded protein remained a protein with unclear function. Based on mapped RNA-seq reads, the coding sequence was corrected, yielding a DNA-binding protein comprising a Zn ₂ Cys ₆ binuclear zinc cluster. Domain analysis and structural comparisons implied similarity between RttA and Neurospora crassa NcSR and yeast Upc2, both involved in sterol regulation and azole resistance. Through deletion and overexpression of rttA , we confirm its role in azole resistance. Transcriptional profiling of atrR , srbA , and rttA deletion mutants revealed that rttA expression depends on both AtrR and SrbA. In addition, our analyses uncovered a positive regulatory role for RttA in the expression of efflux pump-encoding genes and sterol homeostasis through activation of erg6 expression. In agreement, the lack of rttA led to a substantial accumulation of the Erg6 substrate lanosterol. Collectively, this work elucidates RttA as a transcription factor in the clinically important fungal pathogen A. fumigatus involved in the regulation of ergosterol biosynthesis and azole tolerance. IMPORTANCE: Azole antifungals are frontline treatments against Aspergillus fumigatus , a major cause of life-threatening fungal infections. Resistance to azoles is a growing concern, often linked to increased expression of cyp51A , which encodes the azole target enzyme. This upregulation depends on the transcription factors AtrR and SrbA, key activators of ergosterol biosynthesis genes. Here, we identify and characterize a previously misannotated gene, rttA , encoding a Zn₂Cys₆ transcription factor structurally related to Upc2 and NcSR, sterol regulators in yeast and Neurospora crassa . Functional analyses, including gene deletion, overexpression, and transcriptomics, show that RttA promotes azole resistance and regulates sterol homeostasis by activating erg6 , encoding sterol C24-methyltransferase. Loss of rttA leads to lanosterol accumulation, indicating disrupted ergosterol biosynthesis. Moreover, rttA expression depends on both AtrR and SrbA, placing RttA within their regulatory network. Our findings offer new insight into sterol regulation and antifungal resistance in A. fumigatus , highlighting RttA as a novel regulator.

Background: The discovery of CRISPR/Cas9 and its subsequent accessibility in daily research initiated a new era in genome editing. This game-changing genetic instrument enabled a vast array of challenging applications requiring site-specific genome engineering as well as applications involving the equipment of cells with additional genetic traits. Despite the undisputed benefits of this technology, for facile and efficient selection of successfully manipulated cells selectable markers remain indispensable. Over the past years endogenous counter-selectable markers have come into focus in antifungal research enabling site-directed integration of multiple genes into the genome of the human mold pathogen Aspergillus fumigatus. However, gene cassettes had to be transformed in a consecutive manner keeping multigene integrations laborious and time-consuming. Results: In this work, we coupled the use of CRISPR/Cas9 with endogenous counter-selectable markers to achieve the simultaneous integration of multiple expression cassettes. The three markers used in this work included the herein employed azgA and the previously identified fcyB and cntA, responsible for 8-azaguanine, 5-fluorocytosine and 5-fluorouridine uptake, respectively. Exploiting their role in uptake of different selective agents, a triple selective transformation procedure and genomic integration of three expression cassettes in A. fumigatus was successfully accomplished. In addition to three distinct cellular reporters, we introduced strain-specific fluorescent reporters into four isolates displaying different levels of antifungal azole resistance to subsequently visualize and monitor their growth patterns in the same growth environment. Conclusions: The technology described in this study significantly streamlines the genetic manipulation process, reducing both time and labor associated with sequential transformations. By enabling the introduction of multiple genetic traits in a single transformation event, this strategy provides a flexible and efficient platform for a wide range of applications. As such, it enhances the potential for rapid and effective multigene integration, advancing the field of genetic engineering in fungi.

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 transfer RNA (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 liquid chromatography-tandem mass spectrometry 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 Aspergillus nidulans Major Facilitator Superfamily transporter nmeA as specific CPC-client genes in A. fumigatus. Overexpression of Mod5-target tRNATyrGΨA in the Δmod5 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.

Fluorescent proteins (FPs) are indispensable tools used for molecular imaging, single-cell dynamics, imaging in infection models, and more. However, next-generation FPs have yet to be characterized in Aspergillus . Here, we characterize 18 FPs in the pathogenic filamentous fungus Aspergillus fumigatus spanning the visible light spectrum. We report on in vivo FP brightness in hyphal and spore morphotypes and show how a fluoropyrimidine-based selection system can be used to iteratively introduce four distinct FPs enabling the simultaneous visualization of the cell membrane, mitochondria, peroxisomes, and vacuoles. Using this strain, we describe and compare the dynamic responses of organelles to stresses induced by voriconazole, amphotericin B, and the novel antifungal drugs olorofim and manogepix. The expansion to the fluorescent genetic toolbox will overcome boundaries in research applications that involve fluorescence imaging in filamentous fungi.

Olorofim, the first member of the novel class of antifungal drugs, the orotomides, shows promising anti- Aspergillus activity and is currently in phase III clinical development. Using high-throughput microscopy, we monitored olorofim’s antifungal potential at sub-minimum inhibitory concentration (MIC) levels with a focus on early-stage growth. Unlike voriconazole, olorofim showed significant growth inhibitory activities against three main pathogenic Aspergillus species, Aspergillus fumigatus , Aspergillus flavus , and Aspergillus niger , at concentrations >100,000-fold below its MIC. IMPORTANCE: Among antifungal compounds in clinical development for systemic disease, the orotomide olorofim is one of only two that target a completely new mechanism of action. Olorofim is highly potent against pathogenic Aspergillus species including cryptic species that frequently show increased resistance to current agents. In this study, our primary focus was on evaluating in detail the inhibitory activity of voriconazole and olorofim against different pathogenic Aspergillus species employing high-throughput microscopy. Compared to standardized, less-sensitive visual assessment-based methods, microscopy-assisted growth monitoring allowed us to detect sub-MIC drug concentration ranges with significant inhibitory activity at early-stage growth. This revealed that olorofim exerts growth inhibition at concentrations that are several magnitudes below those of voriconazole.

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 tRNA ^Tyr _GΨA in the Δ 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 i ⁶ A 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.

Azole antifungals remain the “gold standard” therapy for invasive aspergillosis. The world-wide emergence of isolates resistant to this drug class, however, developed into a steadily increasing threat to human health over the past years. In Aspergillus fumigatus , major mechanisms of resistance involve increased expression of cyp51A encoding one of two isoenzymes targeted by azoles. Yet, the level of resistance caused by cyp51A upregulation , driven by either clinically relevant tandem repeat mutations within its promoter or the use of high expressing heterologous promoters, is limited. Cytochrome P450 enzymes such as Cyp51A rely on redox partners that provide electrons for their activity. A. fumigatus harbors several genes encoding putative candidate proteins including two paralogous cytochrome P450 reductases, CprA and CprB, and the cytochrome b ₅ CybE. In this work, we investigated the contribution of each cprA , cprB, and cybE overexpression to cyp51A -mediated resistance to different medical and agricultural azoles. Using the bidirectional promoter PxylP , we conditionally expressed these genes in combination with cyp51A , revealing cprA as the main limiting factor. Similar to this approach, we overexpressed cprA in an azole-resistant background strain carrying a cyp51A allele with TR34 in its promoter, which led to a further increase in its resistance. Employing sterol measurements, we demonstrate an enhanced eburicol turnover during upregulation of either cprA or cyp51A , which was even more pronounced during their simultaneous overexpression. In summary, our work suggests that mutations leading to increased Cyp51A activity through increased electron supply could be key factors that elevate azole resistance.