Iron is essential for a wide range of cellular processes. Here we show that the bZIP-type regulator HapX is indispensable for the transcriptional remodeling required for adaption to iron starvation in the opportunistic fungal pathogen Aspergillus fumigatus. HapX represses iron-dependent and mitochondrial-localized activities including respiration, TCA cycle, amino acid metabolism, iron-sulfur-cluster and heme biosynthesis. In agreement with the impact on mitochondrial metabolism, HapX-deficiency decreases resistance to tetracycline and increases mitochondrial DNA content. Pathways positively affected by HapX include production of the ribotoxin AspF1 and siderophores, which are known virulence determinants. Iron starvation causes a massive remodeling of the amino acid pool and HapX is essential for the coordination of the production of siderophores and their precursor ornithine. Consistent with HapX-function being limited to iron depleted conditions and A. fumigatus facing iron starvation in the host, HapX-deficiency causes significant attenuation of virulence in a murine model of aspergillosis. Taken together, this study demonstrates that HapX-dependent adaption to conditions of iron starvation is crucial for virulence of A. fumigatus.

Sterol regulatory element binding proteins (SREBPs) are a class of basic helix-loop-helix transcription factors that regulate diverse cellular responses in eukaryotes. Adding to the recognized importance of SREBPs in human health, SREBPs in the human fungal pathogens Cryptococcus neoformans and Aspergillus fumigatus are required for fungal virulence and susceptibility to triazole antifungal drugs. To date, the exact mechanism(s) behind the role of SREBP in these observed phenotypes is not clear. Here, we report that A. fumigatus SREBP, SrbA, mediates regulation of iron acquisition in response to hypoxia and low iron conditions. To further define SrbA's role in iron acquisition in relation to previously studied fungal regulators of iron metabolism, SreA and HapX, a series of mutants were generated in the ΔsrbA background. These data suggest that SrbA is activated independently of SreA and HapX in response to iron limitation, but that HapX mRNA induction is partially dependent on SrbA. Intriguingly, exogenous addition of high iron or genetic deletion of sreA in the ΔsrbA background was able to partially rescue the hypoxia growth, triazole drug susceptibility, and decrease in ergosterol content phenotypes of ΔsrbA. Thus, we conclude that the fungal SREBP, SrbA, is critical for coordinating genes involved in iron acquisition and ergosterol biosynthesis under hypoxia and low iron conditions found at sites of human fungal infections. These results support a role for SREBP–mediated iron regulation in fungal virulence, and they lay a foundation for further exploration of SREBP's role in iron homeostasis in other eukaryotes.

Objectives. -The ongoing COVID-19 pandemic has caused approximately 2,350,000 infections worldwide and killed more than 160,000 individuals. In Sainte-Anne Hospital (GHU PARIS Psychiatrie & Neuroscience, Paris, France) we have observed a lower incidence of symptomatic forms of COVID-19 among patients than among our clinical staff. This observation led us to hypothesize that psychotropic drugs could have a prophylactic action against SARS-CoV-2 and protect patients from the symptomatic and virulent forms of this infection, since several of these psychotropic drugs have documented antiviral properties. Chlorpromazine (CPZ), a phenothiazine derivative, is also known for its antiviral activity via the inhibition of clathrin-mediated endocytosis. Recentin vitro studies have reported that CPZ exhibits anti-MERS-CoV and anti-SARS-CoV-1 activity. Methods. -In this context, the ReCoVery study aims to repurpose CPZ, a molecule with an excellent tolerance profile and a very high biodistribution in the saliva, lungs and brain. We hypothesize that CPZ could reduce the unfavorable course of COVID-19 infection among patients requiring respiratory support without the need for ICU care, and that it could also reduce the contagiousness of SARS-CoV-2. For this purpose, we plan a pilot, multicenter, randomized, single blind, controlled, phase III therapeutic trial (standard treatment vs. CPZ + standard treatment).Conclusion. -This repurposing of CPZ for its anti-SARS-CoV-2 activity could offer an alternative, rapid strategy to alleviate infection severity. This repurposing strategy also avoids numerous developmental and experimental steps, and could save precious time to rapidly establish an anti-COVID-19 therapy with well-known, limited and easily managed side effects.

Aspergillus fumigatus is the most common airborne fungal pathogen for humans. In this mold, iron starvation induces production of the siderophore triacetylfusarinine C (TAFC). Here we demonstrate a link between TAFC and ergosterol biosynthetic pathways, which are both critical for virulence and treatment of fungal infections. Consistent with mevalonate being a limiting prerequisite for TAFC biosynthesis, we observed increased expression of 3-hydroxy-3-methyl-glutaryl (HMG)-CoA reductase (Hmg1) under iron starvation, reduced TAFC biosynthesis following lovastatin-mediated Hmg1 inhibition, and increased TAFC biosynthesis following Hmg1 overexpression. We identified enzymes, the acyl-CoA ligase SidI and the enoyl-CoA hydratase SidH, linking biosynthesis of mevalonate and TAFC, deficiency of which under iron starvation impaired TAFC biosynthesis, growth, oxidative stress resistance, and murine virulence. Moreover, inactivation of these enzymes alleviated TAFC-derived biosynthetic demand for mevalonate, as evidenced by increased resistance to lovastatin. Concordant with bilateral demand for mevalonate, iron starvation decreased the ergosterol content and composition, a phenotype that is mitigated in TAFC-lacking mutants.siderophore | isoprenoide | statins

The diagnosis of invasive pulmonary aspergillosis (IPA) is difficult and lacks specificity and sensitivity. In the pathophysiology of Aspergillus fumigatus, iron plays an essential role as a nutrient during infection. A. fumigatus uses a specific and highly efficient iron uptake mechanism based on iron-complexing ferric ion Fe(III) siderophores, which are a requirement for A. fumigatus virulence. We aimed to evaluate the potential of siderophores radiolabeled with 68 Ga, a positron emitter with complexing properties comparable to those of Fe(III), as a radiopharmaceutical for imaging IPA. Methods: 68 Ga radiolabeling of the A. fumigatus siderophores desferri-triacetylfusarinine C (TAFC) and desferriferricrocin (FC) was performed at high specific activity. Stability, protein binding, and log P values were determined. In vitro uptake in A. fumigatus cultures was tested under varying conditions. Biodistribution was studied in healthy noninfected BALB/c mice, and uptake was studied in a model of A. fumigatus infection using immunosuppressed Lewis rats. Results: High-specific-activity 68 Ga labeling could be achieved, and resulting complexes were stable in serum, toward diethylenetriaminepentaacetic acid and Fe(III) challenge. Both siderophores showed hydrophilic properties ( 68 Ga-TAFC, log P 5 22.59; 68 Ga-FC, log P 5 23.17) with low values of protein binding for 68 Ga-TAFC (,2%). Uptake of both siderophores was highly dependent on the mycelial iron load and could be blocked with an excess (10 mM) of siderophore or NaN 3 , indicating specific, energy-dependent uptake. In noninfected mice, 68 Ga-TAFC showed rapid renal excretion and low blood values (1.6 6 0.37 percentage injected dose per gram [%ID/g] at 30 min); in urine only intact 68 Ga-TAFC was detected. In contrast, 68 Ga-FC revealed high retention in blood (16.1 6 1.07 %ID/g at 90 min) and rapid metabolism. In the rat IPA model, lung uptake of 68 Ga-TAFC was dependent on the severity of infection, with less than 0.04 %ID/g in control rats (n 5 5) and 0.29 6 0.11 %ID/g in mildly infected (n 5 3) and 0.95 6 0.37 %ID/g in severely infected (n 5 4) rats. PET showed focal accumulation in infected lung tissue. Conclusion: Both siderophores bound 68 Ga with high affinity, and 68 Ga-TAFC, especially, showed high stability. 68 Ga-TAFC displayed highly selective accumulation by A. fumigatus subspecies in vitro and in vivo. The high and specific uptake by A. fumigatus proves the potential of 68 Ga-labeled siderophores for the specific detection of A. fumigatus during infection. They hold promise as new PET agents for IPA.

Iron is an essential metal for virtually all organisms. Iron acquisition is well characterized for various organisms, whereas intracellular iron distribution is poorly understood. In contrast to bacteria, plants, and animals, most fungi lack ferritin-mediated iron storage but possess an intracellular siderophore shown to be involved in iron storage. Here we demonstrate that deficiency in the intracellular siderophore ferricrocin causes iron starvation in conidia of Aspergillus fumigatus, demonstrating that ferricrocin is also involved in intra-and transcellular iron distribution. Thus, ferricrocin represents the first intracellular iron transporter identified in any organism.Virtually all organisms require iron as an indispensable cofactor for various metabolic processes, including electron transport and redox reactions. Excess or incorrect storage of iron, however, is toxic, as this metal has the capacity to reinforce the production of reactive oxygen species. Therefore, organisms have evolved precisely regulated iron acquisition systems, which are well characterized in numerous prokaryotes and eukaryotes. In contrast, mechanisms for intracellular distribution of acquired iron are poorly understood. We investigated these mechanisms in Aspergillus fumigatus, a typical saprophytic ascomycete, which has become the most common airborne fungal pathogen of humans, causing life-threatening invasive disease especially in immunocompromised patients (16). A. fumigatus employs four siderophores (low-molecularmass ferric iron chelators) for maintenance of iron homeostasis (5): it excretes two siderophores for solubilization and uptake of iron, and it accumulates two structurally different siderophores, ferricrocin (FC) and its hydroxylated derivative hydroxy-FC, within hyphae and conidia, respectively. Both intracellular siderophores are believed to be involved in intracellular iron storage. The siderophore system became a matter of particular interest as it represents an attractive target for antifungal therapy due to its requirement for virulence of A. fumigatus and its lack in mammalian hosts (12, 13). Recently, extra-and intracellular siderophores have also been implicated in the phytopathogenicity of various ascomycetes (6, 11). Here we demonstrate that intracellular siderophores are also involved in the intracellular long-distance distribution of iron.Extracellular siderophores are utilized by most fungi and bacteria and some plants, whereas intracellular siderophores are found exclusively in fungi, which in contrast to bacteria, plants, and animals lack ferritin-mediated iron storage (5). The function of intracellular siderophores has been studied in the most detail with Aspergillus nidulans and A. fumigatus (5). Several lines of evidence support a role for FC and hydroxy-FC in iron storage in these fungi. (i) FC accumulation increases under conditions of intracellular iron excess (4, 9, 14); (ii) FC deficiency reduces the iron content of conidia by 34% and 76% in A. nidulans and A. fumigatus, respectively (4, 13); ...

DCs express intrinsic cellular defense mechanisms to specifically inhibit HIV-1 replication. Thus, DCs are productively infected only at very low levels with HIV-1, and this non-permissiveness of DCs is suggested to go along with viral evasion. We now illustrate that complement-opsonized HIV-1 (HIV-C) efficiently bypasses SAMHD1 restriction and productively infects DCs including BDCA-1 DCs. Efficient DC infection by HIV-C was also observed using single-cycle HIV-C, and correlated with a remarkable elevated SAMHD1 T592 phosphorylation but not SAMHD1 degradation. If SAMHD1 phosphorylation was blocked using a CDK2-inhibitor HIV-C-induced DC infection was also significantly abrogated. Additionally, we found a higher maturation and co-stimulatory potential, aberrant type I interferon expression and signaling as well as a stronger induction of cellular immune responses in HIV-C-treated DCs. Collectively, our data highlight a novel protective mechanism mediated by complement opsonization of HIV to effectively promote DC immune functions, which might be in the future exploited to tackle HIV infection.

Purpose68Ga-triacetylfusarinine C (68Ga-TAFC) and 68Ga-ferrioxamine E (68Ga-FOXE) showed excellent targeting properties in Aspergillus fumigatus rat infection model. Here, we report on the comparison of specificity towards different microorganisms and human lung cancer cells (H1299).ProceduresThe in vitro uptake of 68Ga-TAFC and 68Ga-FOXE was studied in various fungal, bacterial and yeast cultures as well as in H1299 cells. The in vivo imaging was studied in fungal and bacterial rat infection and inflammation models.Results68Ga-TAFC and 68Ga-FOXE showed rapid uptake in A. fumigatus cultures, significantly lower in other fungal species and almost no uptake in other microorganisms and H1299 cells, except for 68Ga-FOXE in Staphylococcus aureus. 68Ga-TAFC and 68Ga-FOXE revealed rapid uptake in the lungs of A. fumigatus-infected rats, low accumulation in sterile inflammation and no uptake in bacterial abscess.ConclusionsWe have shown that 68Ga-FOXE and 68Ga-TAFC have high uptake in A. fumigatus both in vitro and in vivo. 68Ga-TAFC showed higher specificity, while 68Ga-FOXE showed higher sensitivity.