TOR adaptation to iron deficiency in pathogenic fungi

TOR (Target Of Rapamycin) is a highly conserved serinehreonine protein kinase that plays a central role in orchestrating nutrient-sensing networks, thereby ensuring cell survival and growth. On the other hand, iron is essential for virtually every organism, but its excess can be highly toxic. Therefore, organisms have developed tightly regulated mechanisms to maintain the balance between uptake, storage and consumption of iron. Currently, little is known about the link between TOR and iron-regulatory pathways. Because many of the cellular processes controlled by TOR require iron as a cofactor, we propose that iron depletion should inhibit TOR activity, and that this regulation is important for optimal adaption to iron starvation and for virulence. Recent studies suggest a crosstalk between energy- sensing and iron-regulatory pathways in the regulation of TOR activity, and TOR has been implicated in the regulation of iron homeostasis by controlling iron content and uptake. Pathogenic fungi cause devastating yield losses and life-threatening human mycoses. Consequently, there is a strong need for the development of new antifungal drugs. Previous work established that adaptation to iron deficiency is strictly required for fungal virulence on humans and plants. Here we will investigate the role of TOR in adaptation to iron deprivation in the plant pathogen Fusarium oxysporum and the human pathogen Aspergillus fumigatus. Using a combination of complementary genetic, biochemical and phosphoproteomic approaches, we will characterize the impact of iron on TOR activity, and the role of upstream and downstream components of the TOR signaling pathway in adaptation to iron starvation. Phosphoproteomics will be employed to identify new key targets of TOR that are linked to iron regulation. Collectively, the results will establish unprecedented links between TOR signaling, iron homeostasis and fungal pathogenicity and reveal new ways to control fungal infections, both in clinical and agricultural settings.

Pathogenic fungi cause devastating yield losses and life-threatening human mycoses; consequently, there is an urgent need for the development of new antifungal compounds, a process that requires a comprehensive knowledge of the mechanisms underlying pathogenicity. Previous work established that adaptation to iron-depletion is strictly required for virulence of fungi on humans and plants. Since both lack the fungal siderophore-mediated iron uptake system, it represents a promising target for the development of new antifungal therapies. Moreover, we propose that the cellular components regulating iron homeostasis are excellent targets for new antifungals. Although this project was initially conceived to study the role of the protein kinase TOR in the adaptation to iron deficiency in pathogenic fungi, the evolution of the research has led us to investigate not only this, but also other relevant aspects of the regulation of iron homeostasis in the human pathogen Aspergillus fumigatus. Using different readouts, like phospho-quantification of TOR downstream targets, or measuring mRNA levels of different ribosomal proteins, we have shown that the protein kinase TOR remains inactive under iron depletion and is activated once we shift to iron- replete conditions. TOR activation is detectable half an hour after the iron shift, while classical iron responses take place in minutes, if not seconds. We have carried out a complex transcriptome analysis under different iron conditions, with and without rapamycin, that is helping us to distinguish between pure TOR signaling and iron-mediated responses prompting TOR activation. We believe that understanding the functional links between iron metabolism and the TOR signaling pathway will open novel ways to control fungal infections. In another line, we have greatly increased our knowledge about the control of iron homeostasis from a proteomic point of view, in particular on how HapX, the major coordinator for the adaptation to iron deficiency, is regulated in A. fumigatus. We have conducted an in-depth whole proteome analysis with the wild-type strain under different iron conditions with very robust and informative results, and through biochemical techniques, we have shown that HapX half-life is extremely short and that the protein is quickly degraded in shift experiments from iron depleted to iron-replete conditions. Moreover, we have identified, at least partially, the mechanism governing this regulation. In addition, our genetic studies demonstrate that iron also regulates the stability of hapX mRNA and that overexpression of this key iron regulator is highly detrimental. Collectively, our results indicate that HapX is subjected to multiple levels of control in A. fumigatus. Taken together, our results indicate that iron is critical for TOR activation while TORC1 seems to have no influence in the transcriptional response of iron-regulated genes. Moreover, we show that the transcription factor HapX is subjected to multiple levels of control to ensure a balanced regulation of iron homeostasis in A. fumigatus.