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Invasive aspergillosis (IA), caused predominantly by Aspergillus fumigatus, is the most common opportunistic mold infection in immunocompromised patients. Resistance of A. fumigatus to triazoles has been increasingly reported, leading to poor outcomes of IA to the front-line azoles. Triazole resistance is in part driven by exposure to agricultural azoles through mechanisms that are poorly understood beyond mutations in ergosterol biosynthetic genes. Priming is defined as a process in which prior exposures to sublethal stressful stimuli, such as antimicrobial drugs, can enhance the ability of pathogens to withstand reexposure to the same or other stressors. Here, we describe, for the first time, triazole priming, where exposure of conidia of three A. fumigatus strains to subinhibitory concentrations of either agricultural (tebuconazole difenoconazole, epoxiconazole) or medical triazoles (voriconazole) increases germination and growth during subsequent reexposure to subinhibitory triazole challenge. We demonstrate that priming in A. fumigatus is class specific to triazoles, is not confined to a particular isolate, and is retained for extended periods in primed dormant conidia, but is not transferred to subsequent generations. Furthermore, azole priming at subinhibitory triazole concentrations increased the frequency of development of stable resistance development at inhibitory triazole exposures. Triazole priming could have far-reaching clinical implications in generating resistance due to the widespread use of agricultural triazoles or breakthrough IA in patients with subtherapeutic serum levels of azoles.

Aspergillus fumigatus is the leading cause of life-threatening invasive mold infections in immunocompromised individuals. This ubiquitous saprophyte possesses several natural attributes allowing it to evade the immune system, including the ability to withstand high toxic Cu concentrations within the phagosomes of macrophages and neutrophils. We previously established that at high levels, Cu binds and activates the A. fumigatus transcription factor AceA, which upregulates the expression of the Cu exporter CrpA to expel excess Cu. Deletion of aceA or crpA result in extreme Cu sensitivity and attenuated virulence. To identify other elements participating in resistance to Cu, we performed a genome-wide analysis of the transcriptome by RNAseq to analyze the AceA-dependent response of A. fumigatus to excess Cu. We deleted key genes whose transcription was strongly upregulated by high Cu, including those encoding homologs of the three Cu chaperones cox17, atx1 and ccs1. Detailed analysis of these genes indicates that in A. fumigatus, cox17 is an essential gene with a possible role in respiration, the atxA gene product participates in reductive iron uptake and ccsA encodes the Cu chaperone activating A. fumigatus Sod1. Interestingly, although the ccsA-null strain was extremely sensitive to high Cu and oxidative stress, it was not attenuated in virulence in a mouse model of invasive pulmonary aspergillosis. Our work provides (i) a detailed view of the genome-wide transcriptional response of A. fumigatus to excess Cu, (ii) identification of the AceA-dependent transcriptome and (iii) analysis of the roles of the three Cu chaperones cox17, atxA and ccsA.

The ubiquitous fungus Aspergillus flavus is notorious for contaminating many important crops and food-stuffs with the carcinogenic mycotoxin, aflatoxin. This fungus is also the second most frequent Aspergillus pathogen after A. fumigatus infecting immunosuppressed patients. In many human fungal pathogens including A. fumigatus, the ability to defend from toxic levels of copper (Cu) is essential in pathogenesis. In A. fumigatus, the Cu-fist DNA binding protein, AceA, and the Cu ATPase transporter, CrpA, play critical roles in Cu defense. Here, we show that A. flavus tolerates higher concentrations of Cu than A. fumigatus and other Aspergillus spp. associated with the presence of two homologs of A. fumigatus CrpA termed CrpA and CrpB. Both crpA and crpB are transcriptionally induced by increasing Cu concentrations via AceA activity. Deletion of crpA or crpB alone did not alter high Cu tolerance, suggesting they are redundant. Deletion of both genes resulted in extreme Cu sensitivity that was greater than that following deletion of the regulatory transcription factor aceA. The ΔcrpAΔcrpB and ΔaceA strains were also sensitive to ROI stress. Compared to wild type, these mutants were impaired in the ability to colonize maize seed treated with Cu fungicide but showed no difference in virulence on non-treated seed. A mouse model of invasive aspergillosis showed ΔcrpAΔcrpB and to a lesser degree ΔaceA to be significantly reduced in virulence, following the greater sensitivity of ΔcrpAΔcrpB to Cu than ΔaceA.

Objectives Over the last 30 years, the number of invasive fungal infections among immunosuppressed patients has increased significantly, while the number of effective systemic antifungal drugs remains low. The aim of this study was to identify and characterize antifungal compounds that inhibit fungus-specific metabolic pathways not conserved in humans. Methods We screened a diverse compound library for antifungal activity in the pathogenic mould Aspergillus fumigatus . We determined the in vitro activity of bromoquinol by MIC determination against a panel of fungi, bacteria and cell lines. The mode of action of bromoquinol was determined by screening an Aspergillus nidulans overexpression genomic library for resistance-conferring genes and by RNAseq analysis in A. fumigatus . In vivo efficacy was tested in Galleria mellonella and murine models of A. fumigatus infection. Results Screening of a diverse chemical library identified three compounds interfering with fungal iron utilization. The most potent, bromoquinol, shows potent wide-spectrum antifungal activity that was blocked in the presence of exogenous iron. Mode-of-action analysis revealed that overexpression of the dba secondary metabolite cluster gene dbaD , encoding a metabolite transporter, confers bromoquinol resistance in A. nidulans , possibly by efflux. RNAseq analysis and subsequent experimental validation revealed that bromoquinol induces oxidative stress and apoptosis in A. fumigatus . Bromoquinol significantly reduced mortality rates of G. mellonella infected with A. fumigatus , but was ineffective in a murine model of infection. Conclusions Bromoquinol is a promising antifungal candidate with a unique mode of action. Its activity is potentiated by iron starvation, as occurs during in vivo growth.

We have found a remarkable capacity for the ubiquitous Gram-negative rod bacterium Serratia marcescens to migrate along and kill the mycelia of zygomycete molds. This migration was restricted to zygomycete molds and several basidiomycete species. No migration was seen on any molds of the phylum Ascomycota. S. marcescens migration did not require fungal viability or surrounding growth medium, as bacteria migrated along aerial hyphae as well. S. marcescens did not exhibit growth tropism toward zygomycete mycelium. Bacterial migration along hyphae proceeded only when the hyphae grew into the bacterial colony. S. marcescens cells initially migrated along the hyphae, forming attached microcolonies that grew and coalesced to generate a biofilm that covered and killed the mycelium. Flagellum-defective strains of S. marcescens were able to migrate along zygomycete hyphae, although they were significantly slower than the wild-type strain and were delayed in fungal killing. Bacterial attachment to the mycelium does not necessitate type 1 fimbrial adhesion, since mutants defective in this adhesin migrated equally well as or faster than the wild-type strain. Killing does not depend on the secretion of S. marcescens chitinases, as mutants in which all three chitinase genes were deleted retained wild-type killing abilities. A better understanding of the mechanisms by which S. marcescens binds to, spreads on, and kills fungal hyphae might serve as an excellent model system for such interactions in general; fungal killing could be employed in agricultural fungal biocontrol. Fil: Hover, Tal. Tel Aviv University; Israel Fil: Maya, Tal. Tel Aviv University; Israel Fil: Ron, Sapir. Tel Aviv University; Israel Fil: Sandovsky, Hani. Tel Aviv University; Israel Fil: Shadkchan, Yana. Tel Aviv University; Israel Fil: Kijner, Nitzan. Tel Aviv University; Israel Fil: Mitiagin, Yulia. Tel Aviv University; Israel Fil: Fichtman, Boris. Bar Ilan University; Israel Fil: Harel, Amnon. Bar Ilan University; Israel Fil: Shanks, Robert M. Q.. University of Pittsburgh Medical Center; Estados Unidos Fil: Bruna, Roberto Emanuel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina Fil: Garcia Vescovi, Eleonora. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina Fil: Osherov, Nir. Tel Aviv University; Israel

Systemic fungal infections are an increasingly prevalent health problem, especially among immunocompromised patients. Antifungal drug development lags far behind in comparison to other types of antimicrobial drugs. Current commercially available antifungals are limited by their insufficient potency, side effects, drug-drug interactions, developing drug-resistance, and narrow formulation options. Here, we report the preparation and evaluation of two novel PEG amide conjugates of amphotericin B (AMB (1)): AB1 (4) and AM2 (5). These compounds are nonlabile, they are prepared in only two and three synthetic steps, respectively, and they show antifungal activity against a wide range of clinical fungal isolates. Their toxicity is significantly lower, and their water solubility is up to 5000-fold higher than that of AMB (1). In vivo efficacy studies in a mouse model of systemic candidiasis showed that AM2 (5) successfully cured all the mice at concentrations above 3.5 mg/kg body weight. In conclusion, these properties make AB1 (4) and AM2 (5) promising candidates for clinical use.

Summary: The Fenton-chemistry-generating properties of copper ions are considered a potent phagolysosome defense against pathogenic microbes, yet our understanding of underlying host/microbe dynamics remains unclear. We address this issue in invasive aspergillosis and demonstrate that host and fungal responses inextricably connect copper and reactive oxygen intermediate (ROI) mechanisms. Loss of the copper-binding transcription factor AceA yields an Aspergillus fumigatus strain displaying increased sensitivity to copper and ROI in vitro, increased intracellular copper concentrations, decreased survival in challenge with murine alveolar macrophages (AMΦs), and reduced virulence in a non-neutropenic murine model. ΔaceA survival is remediated by dampening of host ROI (chemically or genetically) or enhancement of copper-exporting activity (CrpA) in A. fumigatus. Our study exposes a complex host/microbe multifactorial interplay that highlights the importance of host immune status and reveals key targetable A. fumigatus counter-defenses. : Wiemann et al. find that Aspergillus fumigatus employs the copper-sensing transcription factor AceA to express the copper exporter CrpA as a defense mechanism against macrophages. Copper and reactive oxygen intermediate attack and defense are inextricably connected on the side of both host and pathogen during infection. Keywords: ATP7A, CrpA, AtfA, CTR1, CGD, ROI, ROS, PHOX, Aspergillus fumigatus, copper

Aspergillus fumigatus is the most common mold species to cause disease in immunocompromised patients. Infection usually begins when its spores (conidia) are inhaled into the airways, where they germinate, forming hyphae that penetrate and destroy the lungs and disseminate to other organs, leading to high mortality. The ability of hyphae to penetrate the pulmonary epithelium is a key step in the infectious process. A. fumigatus produces extracellular proteases that are thought to enhance penetration by degrading host structural barriers. This study explores the role of the A. fumigatus transcription factor XprG in controlling secreted proteolytic activity and fungal virulence. We deleted xprG, alone and in combination with prtT, a transcription factor previously shown to regulate extracellular proteolysis. xprG deletion resulted in abnormal conidiogenesis and formation of lighter colored, more fragile conidia and a moderate reduction in the ability of culture filtrates (CFs) to degrade substrate proteins. Deletion of both xprG and prtT resulted in an additive reduction, generating a mutant strain producing CF with almost no ability to degrade substrate proteins. Detailed proteomic analysis identified numerous secreted proteases regulated by XprG and PrtT, alone and in combination. Interestingly, proteomics also identified reduced levels of secreted cell wall modifying enzymes (glucanases, chitinases) and allergens following deletion of these genes, suggesting they target additional cellular processes. Surprisingly, despite the major alteration in the secretome of the xprG/prtT null mutant, including two to fivefold reductions in the level of 24 proteases, 18 glucanases, 6 chitinases, and 19 allergens, it retained wild-type virulence in murine systemic and pulmonary models of infection. This study highlights the extreme adaptability of A. fumigatus during infection based on extensive gene redundancy.

Fungal cell-wall proteins containing the conserved fungal CFEM domain have been implicated in host-pathogen interactions and virulence. To determine the role of these proteins in the mold pathogen Aspergillus fumigatus, we deleted the entire family of three CFEM-containing genes (CfmA-C), singly and in all combinations. We found an additive increase in the susceptibility of the single, double and triple ΔCfm mutants towards the chitin/β-glucan-microfibril destabilizing compounds Congo Red (CR) and Calcofluor White (CFW), indicating that the A. fumigatus CFEM proteins are involved in stabilizing the cell wall. No defects in growth or germination were observed, indicating that CFEM proteins do not have an essential role in the morphogenesis of A. fumigatus. Unlike in Candida albicans, the A. fumigatus CFEM proteins were not implicated in heme uptake or biofilm formation. The ΔTriple-Cfm deletion strain did not exhibit altered virulence in either insect or murine models of infection, suggesting that cell-wall proteins containing the conserved fungal CFEM domain are not a significant virulence factor in A. fumigatus.

Many natural broad-spectrum cationic antimicrobial peptides (AMPs) possess a general mode of action that is dependent on lipophilicity and charge. Modulating the lipophilicity of AMPs by the addition of a fatty acid has been an effective strategy to increase the lytic activity and can further broaden the spectrum of AMPs. However, lipophilic modifications that narrow the spectrum of activity and exclusively direct peptides to fungi are less common. Here, we show that short peptide sequences can be targeted to fungi with structured lipophilic biomolecules, such as vitamin E and cholesterol. The conjugates were active against Aspergillus fumigatus , Cryptococcus neoformans , and Candida albicans but not against bacteria and were observed to cause membrane perturbation by transmission electron microscopy and in membrane permeability studies. However, for C. albicans , selected compounds were effective without the perturbation of the cell membrane, and synergism was seen with a vitamin E conjugate and amphotericin B. Moreover, in combination with β-cyclodextrin, antibacterial activity emerged in selected compounds. Biocompatibility for selected active compounds was tested in vitro and in vivo using toxicity assays on erythrocytes, macrophages, and mice. In vitro cytotoxicity experiments led to selective toxicity ratios (50% lethal concentration/MIC) of up to 64 for highly active antifungal compounds, and no in vivo murine toxicity was seen. Taken together, these results highlight the importance of the conjugated lipophilic structure and suggest that the modulation of other biologically relevant peptides with hydrophobic moieties, such as cholesterol and vitamin E, generate compounds with unique bioactivity.

The Aspergillus nidulans genes cetA (AN3079.2) and calA (AN7619.2) encode a novel class of fungal thaumatin-like proteins of unknown function. Deletion of cetA does not result in an observable phenotype [Greenstein, S., Shadkchan, Y., Jadoun, J., Sharon, C., Markovich, S., Osherov, N., 2006. Analysis of the Aspergillus nidulans thaumatin-like cetA gene and evidence for transcriptional repression of pyr4 expression in the cetA-disrupted strain. Fungal Genet. Biol. 43, 42–53]. We prepared knockout calA and calA/cetA A. nidulans strains. The calA mutants were phenotypically identical to the wild-type. In contrast, the cetA/calA double mutant showed a synthetic lethal phenotype suggesting that the two genes affect a single function or pathway: most of its conidia were completely inhibited in germination. Many collapsed and underwent lysis. A few showed abnormal germination characterized by short swollen hyphae and abnormal hyphal branching. Nongerminated conidia contained a single condensed nucleus suggesting a block in early germination. This is the first functional analysis of the novel cetA/calA family of thaumatin-like genes and their role in A. nidulans conidial germination. We show that CETA and CALA are secreted proteins that together play an essential role in early conidial germination.

The activity of antimicrobial peptides (AMPs) that contain a large proportion of histidine residues (pK(a) ∼ 6) depends on the physiological pH environment. Advantages of these AMPs include high activity in slightly acidic areas of the human body and relatively low toxicity in other areas. Also, many AMPs are highly active in a multivalent form, but this often increases toxicity. Here we designed pH dependent amphiphilic compounds consisting of multiple ultrashort histidine lipopeptides on a triazacyclophane scaffold, which showed high activity toward Aspergillus fumigatus and Cryptococcus neoformans at acidic pH, yet remained nontoxic. In vivo, treatment with a myristic acid conjugated trivalent histidine-histidine dipeptide resulted in 55% survival of mice (n = 9) in an otherwise lethal murine lung Aspergillus infection model. Fungal burden was assessed and showed completely sterile lungs in 80% of the mice (n = 5). At pH 5.5 and 7.5, differing peptide-membrane interactions and peptide nanostructures were observed. This study underscores the potential of unique AMPs to become the next generation of clinical antimicrobial therapy.

cspA (for cell surface protein A) encodes a repeat-rich glycophosphatidylinositol (GPI)-anchored cell wall protein (CWP) in the pathogenic fungus Aspergillus fumigatus . The number of repeats in cspA varies among isolates, and this trait is used for typing closely related strains of A. fumigatus . We have previously shown that deletion of cspA is associated with rapid conidial germination and reduced adhesion of dormant conidia. Here we show that cspA can be extracted with hydrofluoric acid (HF) from the cell wall, suggesting that it is a GPI-anchored CWP. The cspA -encoded CWP is unmasked during conidial germination and is surface expressed during hyphal growth. Deletion of cspA results in weakening of the conidial cell wall, whereas its overexpression increases conidial resistance to cell wall-degrading enzymes and inhibits conidial germination. Double mutant analysis indicates that cspA functionally interacts with the cell wall protein-encoding genes ECM33 and GEL2 . Deletion of cspA together with ECM33 or GEL2 results in strongly reduced conidial adhesion, increased disorganization of the conidial cell wall, and exposure of the underlying layers of chitin and β-glucan. This is correlated with increasing susceptibility of the Δ cspA , Δ ECM33 , and Δ cspA Δ ECM33 mutants to conidial phagocytosis and killing by human macrophages and hyphal damage induced by neutrophils. However, these strains did not exhibit altered virulence in mice with infected lungs. Collectively, these results suggest a role for cspA in maintaining the strength and integrity of the cell wall.