Publications by authors named "Nir Osherov"

60 Publications

Transcriptional response of to copper and the role of the Cu chaperones.

Virulence 2021 12;12(1):2186-2200

Department of Clinical Microbiology and Immunology, Sackler School of Medicine Ramat-Aviv, Tel-Aviv, Israel.

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 transcription factor AceA, which upregulates the expression of the Cu exporter CrpA to expel excess Cu. Deletion of or 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 to excess Cu. We deleted key genes whose transcription was strongly upregulated by high Cu, including those encoding homologs of the three Cu chaperones and . Detailed analysis of these genes indicates that in is an essential gene with a possible role in respiration, the gene product participates in reductive iron uptake and encodes the Cu chaperone activating Sod1. Interestingly, although the -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 to excess Cu, (ii) identification of the AceA-dependent transcriptome and (iii) analysis of the roles of the three Cu chaperones and
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http://dx.doi.org/10.1080/21505594.2021.1958057DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8425704PMC
December 2021

Point mutation or overexpression of encoding lanosterol 14α-sterol demethylase, leads to triazole resistance.

Antimicrob Agents Chemother 2021 Jul 26:AAC0125221. Epub 2021 Jul 26.

Department of Clinical Microbiology and Immunology, Sackler School of Medicine, Tel-Aviv University, Tel-Aviv, Israel.

is the most common cause of invasive fungal mold infections in immunocompromised individuals. Current antifungal treatment relies heavily on the triazole antifungals which inhibit fungal Erg11/Cyp51 activity and subsequent ergosterol biosynthesis. However, resistance, due primarily to mutation, is rapidly increasing. contains two Cyp51 isoenzymes, Cyp51A and Cyp51B. Overexpression and mutation of Cyp51A is a major cause of triazole resistance in . The role of Cyp51B in generating resistance is unclear. Here we show that overexpression or mutation of results in triazole resistance. We demonstrate that introduction of a G457S Cyp51B mutation identified in a resistant clinical isolate, results in voriconazole resistance in the naïve recipient strain. Our results indicate that mutations in resulting in clinical resistance do exist and should be monitored.
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http://dx.doi.org/10.1128/AAC.01252-21DOI Listing
July 2021

Triazole resistant in an Israeli patient with Chronic Cavitary Pulmonary Aspergillosis due to a novel E306K Substitution in Hmg1.

Antimicrob Agents Chemother 2021 Jul 19:AAC0108921. Epub 2021 Jul 19.

Department of Clinical Microbiology and Immunology, Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel.

Triazole resistance in the pathogenic mold has increased worldwide, posing a growing therapeutic challenge. Recently, mutations in the 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA) reductase gene () have been associated with triazole resistance. Here, we describe a novel E306K triazole resistance-conferring mutation in the HMG-CoA reductase gene from an Israeli patient with chronic cavitary pulmonary aspergillosis (CCPA).
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http://dx.doi.org/10.1128/AAC.01089-21DOI Listing
July 2021

Emergence of W272C Substitution in Hmg1 in a Triazole-Resistant Isolate of Aspergillus fumigatus from a Chinese Patient with Chronic Cavitary Pulmonary Aspergillosis.

Antimicrob Agents Chemother 2021 06 17;65(7):e0026321. Epub 2021 Jun 17.

Department of Dermatology and Venerology, Peking University First Hospital, National Clinical Research Center for Skin and Immune Diseases, Research Center for Medical Mycology, Peking University, Beijing Key Laboratory of Molecular Diagnosis on Dermatoses, Beijing, China.

Recently, mutations in the 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA) reductase gene () have been identified to be associated with triazole resistance in Aspergillus fumigatus. Here, we describe the first case of the G929C mutation in the gene, leading to the W272C amino acid substitution, in a triazole-resistant isolate of A. fumigatus recovered from a chronic cavitary pulmonary aspergillosis patient who failed voriconazole therapy in China.
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http://dx.doi.org/10.1128/AAC.00263-21DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8218700PMC
June 2021

The Essential Thioredoxin Reductase of the Human Pathogenic Mold Is a Promising Antifungal Target.

Front Microbiol 2020 25;11:1383. Epub 2020 Jun 25.

Mikrobiologisches Institut - Klinische Mikrobiologie, Immunologie und Hygiene, Universitätsklinikum Erlangen and Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany.

The identification of cellular targets for antifungal compounds is a cornerstone for the development of novel antimycotics, for which a significant need exists due to increasing numbers of susceptible patients, emerging pathogens, and evolving resistance. For the human pathogenic mold , the causative agent of the opportunistic disease aspergillosis, only a limited number of established targets and corresponding drugs are available. Among several targets that were postulated from a variety of experimental approaches, the conserved thioredoxin reductase (TrxR) activity encoded by the gene was assessed in this study. Its essentiality could be confirmed following a conditional TetOFF promoter replacement strategy. Relevance of the gene product for oxidative stress resistance was revealed and, most importantly, its requirement for full virulence of s in two different models of infection resembling invasive aspergillosis. Our findings complement the idea of targeting the reductase component of the fungal thioredoxin system for antifungal therapy.
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http://dx.doi.org/10.3389/fmicb.2020.01383DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7330004PMC
June 2020

Arginine Auxotrophy Affects Siderophore Biosynthesis and Attenuates Virulence of .

Genes (Basel) 2020 04 15;11(4). Epub 2020 Apr 15.

Institute of Molecular Biology, Biocenter, Medical University of Innsbruck, 6020 Innsbruck, Austria.

is an opportunistic human pathogen mainly infecting immunocompromised patients. The aim of this study was to characterize the role of arginine biosynthesis in virulence of via genetic inactivation of two key arginine biosynthetic enzymes, the bifunctional acetylglutamate synthase/ornithine acetyltransferase (/AFUA_5G08120) and the ornithine carbamoyltransferase (/AFUA_4G07190). Arginine biosynthesis is intimately linked to the biosynthesis of ornithine, a precursor for siderophore production that has previously been shown to be essential for virulence in . ArgJ is of particular interest as it is the only arginine biosynthetic enzyme lacking mammalian homologs. Inactivation of either ArgJ or ArgB resulted in arginine auxotrophy. Lack of ArgJ, which is essential for mitochondrial ornithine biosynthesis, significantly decreased siderophore production during limited arginine supply with glutamine as nitrogen source, but not with arginine as sole nitrogen source. In contrast, siderophore production reached wild-type levels under both growth conditions in ArgB null strains. These data indicate that siderophore biosynthesis is mainly fueled by mitochondrial ornithine production during limited arginine availability, but by cytosolic ornithine production during high arginine availability via cytosolic arginine hydrolysis. Lack of ArgJ or ArgB attenuated virulence of in the insect model and in murine models for invasive aspergillosis, indicating limited arginine availability in the investigated host niches.
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http://dx.doi.org/10.3390/genes11040423DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7231135PMC
April 2020

The Lysine Deacetylase RpdA Is Essential for Virulence in .

Front Microbiol 2019 4;10:2773. Epub 2019 Dec 4.

Institute of Molecular Biology, Biocenter, Medical University of Innsbruck, Innsbruck, Austria.

Current suboptimal treatment options of invasive fungal infections and emerging resistance of the corresponding pathogens urge the need for alternative therapy strategies and require the identification of novel antifungal targets. is the most common airborne opportunistic mold pathogen causing invasive and often fatal disease. Establishing a novel conditional gene expression system, we demonstrate that downregulation of the class 1 lysine deacetylase (KDAC) RpdA leads to avirulence of in a murine model for pulmonary aspergillosis. The promoter used has previously been shown to allow xylose-induced gene expression in different molds. Here, we demonstrate for the first time that this promoter also allows tuning of gene activity by supplying xylose in the drinking water of mice. In the absence of xylose, an strain expressing under control of the promoter, , was avirulent and lung histology showed significantly less fungal growth. With xylose, however, displayed full virulence demonstrating that xylose was taken up by the mouse, transported to the site of fungal infection and caused induction . These results demonstrate that (i) RpdA is a promising target for novel antifungal therapies and (ii) the expression system is a powerful new tool for gene silencing in .
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http://dx.doi.org/10.3389/fmicb.2019.02773DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6905131PMC
December 2019

The leucine biosynthetic pathway is crucial for adaptation to iron starvation and virulence in .

Virulence 2019 12;10(1):925-934

Institute of Molecular Biology, Medical University of Innsbruck, Innsbruck, Austria.

In contrast to mammalia, fungi are able to synthesize the branched-chain amino acid leucine . Recently, the transcription factor LeuB has been shown to cross-regulate leucine biosynthesis, nitrogen metabolism and iron homeostasis in , the most common human mold pathogen. Moreover, the leucine biosynthetic pathway intermediate α-isopropylmalate (α-IPM) has previously been shown to posttranslationally activate LeuB homologs in and . Here, we demonstrate that in inactivation of both leucine biosynthetic enzymes α-IPM synthase (LeuC), which disrupts α-IPM synthesis, and α-IPM isomerase (LeuA), which causes cellular α-IPM accumulation, results in leucine auxotrophy. However, compared to lack of LeuA, lack of LeuC resulted in increased leucine dependence, a growth defect during iron starvation and decreased expression of LeuB-regulated genes including genes involved in iron acquisition. Lack of either LeuA or LeuC decreased virulence in an insect infection model, and inactivation of LeuC rendered avirulent in a pulmonary aspergillosis mouse model. Taken together, we demonstrate that the lack of two leucine biosynthetic enzymes, LeuA and LeuC, results in significant phenotypic consequences indicating that the regulator LeuB is activated by α-IPM in and that the leucine biosynthetic pathway is an attractive target for the development of antifungal drugs.
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http://dx.doi.org/10.1080/21505594.2019.1682760DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6844326PMC
December 2019

Copper Utilization, Regulation, and Acquisition by .

Int J Mol Sci 2019 Apr 23;20(8). Epub 2019 Apr 23.

Department of Clinical Microbiology and Immunology, Sackler School of Medicine, Tel-Aviv University, Ramat-Aviv 69978, Tel-Aviv, Israel.

Copper is an essential micronutrient for the opportunistic human pathogen, . Maintaining copper homeostasis is critical for survival and pathogenesis. Copper-responsive transcription factors, AceA and MacA, coordinate a complex network responsible for responding to copper in the environment and determining which response is necessary to maintain homeostasis. For example, uses copper exporters to mitigate the toxic effects of copper while simultaneously encoding copper importers and small molecules to ensure proper supply of the metal for copper-dependent processes such a nitrogen acquisition and respiration. Small molecules called isocyanides recently found to be produced by may bind copper and partake in copper homeostasis similarly to isocyanide copper chelators in bacteria. Considering that the host uses copper as a microbial toxin and copper availability fluctuates in various environmental niches, understanding how maintains copper homeostasis will give insights into mechanisms that facilitate the development of invasive aspergillosis and its survival in nature.
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http://dx.doi.org/10.3390/ijms20081980DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6514546PMC
April 2019

Siroheme Is Essential for Assimilation of Nitrate and Sulfate as Well as Detoxification of Nitric Oxide but Dispensable for Murine Virulence of .

Front Microbiol 2018 12;9:2615. Epub 2018 Nov 12.

Division of Molecular Biology, Biocenter, Medical University of Innsbruck, Innsbruck, Austria.

The saprophytic mold is the most common airborne fungal pathogen causing severe invasive fungal infections in immunocompromised patients. Siroheme is a heme-like prosthetic group used by plants and microorganisms for sulfate and nitrate assimilation but is absent in higher eukaryotes. Here, we investigated the role of siroheme in by deletion of the gene encoding the bifunctional dehydrogenase/ferrochelatase enzyme Met8. Met8-deficiency resulted in the inability to utilize sulfate and nitrate as sulfur and nitrogen sources, respectively. These results match previous data demonstrating that siroheme is an essential cofactor for nitrite and sulfite reductases. Moreover, Met8-deficiency caused significantly decreased resistance against nitric oxide (NO) underlining the importance of nitrite reductase in NO detoxification. Met8-deficiency did not affect virulence in murine models for invasive aspergillosis indicating that neither NO-detoxification nor assimilation of sulfate and nitrate play major roles in virulence in this host. Interestingly, Met8-deficiency resulted in mild virulence attenuation in the infection model revealing differences in interaction of with and mouse.
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http://dx.doi.org/10.3389/fmicb.2018.02615DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6240589PMC
November 2018

Riboflavin and pantothenic acid biosynthesis are crucial for iron homeostasis and virulence in the pathogenic mold Aspergillus fumigatus.

Virulence 2018 ;9(1):1036-1049

a Division of Molecular Biology, Biocenter , Medical University of Innsbruck , Innsbruck , Austria.

Background: Aspergillus fumigatus is the most prevalent airborne fungal pathogen, causing invasive fungal infections mainly in immunosuppressed individuals. Death rates from invasive aspergillosis remain high because of limited treatment options and increasing antifungal resistance. The aim of this study was to identify key fungal-specific genes participating in vitamin B biosynthesis in A. fumigatus. Because these genes are absent in humans they can serve as possible novel targets for antifungal drug development.

Methods: By sequence homology we identified, deleted and analysed four key A. fumigatus genes (riboB, panA, pyroA, thiB) involved respectively in the biosynthesis of riboflavin (vitamin B2), pantothenic acid (vitamin B5), pyridoxine (vitamin B6) and thiamine (vitamin B1).

Results: Deletion of riboB, panA, pyroA or thiB resulted in respective vitamin auxotrophy. Lack of riboflavin and pantothenic acid biosynthesis perturbed many cellular processes including iron homeostasis. Virulence in murine pulmonary and systemic models of infection was severely attenuated following deletion of riboB and panA, strongly reduced after pyroA deletion and weakly attenuated after thiB deletion.

Conclusions: This study reveals the biosynthetic pathways of the vitamins riboflavin and pantothenic acid as attractive targets for novel antifungal therapy. Moreover, the virulence studies with auxotrophic mutants serve to identify the availability of nutrients to pathogens in host niches.

Abbreviations: BPS: bathophenanthrolinedisulfonate; BSA: bovine serum albumin; CFU: colony forming unit; -Fe: iron starvation; +Fe: iron sufficiency; hFe: high iron; NRPSs: nonribosomal peptide synthetases; PKSs: polyketide synthaseses; wt: wild type.
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http://dx.doi.org/10.1080/21505594.2018.1482181DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6068542PMC
October 2018

Contribution of ATPase copper transporters in animal but not plant virulence of the crossover pathogen Aspergillus flavus.

Virulence 2018 ;9(1):1273-1286

b Department of Medical Microbiology and Immunology , University of Wisconsin , Madison , WI , USA.

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.
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http://dx.doi.org/10.1080/21505594.2018.1496774DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6177249PMC
April 2019

Vitamin Biosynthesis as an Antifungal Target.

J Fungi (Basel) 2018 Jun 17;4(2). Epub 2018 Jun 17.

Department of Clinical Microbiology and Immunology, Sackler School of Medicine, Tel-Aviv University, Ramat-Aviv, Tel-Aviv 69978, Israel.

The large increase in the population of immunosuppressed patients, coupled with the limited efficacy of existing antifungals and rising resistance toward them, have dramatically highlighted the need to develop novel drugs for the treatment of invasive fungal infections. An attractive possibility is the identification of possible drug targets within essential fungal metabolic pathways not shared with humans. Here, we review the vitamin biosynthetic pathways (vitamins A⁻E, K) as candidates for the development of antifungals. We present a set of ranking criteria that identify the vitamin B2 (riboflavin), B5 (pantothenic acid), and B9 (folate) biosynthesis pathways as being particularly rich in new antifungal targets. We propose that recent scientific advances in the fields of drug design and fungal genomics have developed sufficiently to merit a renewed look at these pathways as promising sources for the development of novel classes of antifungals.
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http://dx.doi.org/10.3390/jof4020072DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6023522PMC
June 2018

Anti-Aspergillus Activities of the Respiratory Epithelium in Health and Disease.

J Fungi (Basel) 2018 Jan 8;4(1). Epub 2018 Jan 8.

Manchester Fungal Infection Group, Faculty of Biology, Medicine and Health, University of Manchester, Manchester M13 9NT, UK.

Respiratory epithelia fulfil multiple roles beyond that of gaseous exchange, also acting as primary custodians of lung sterility and inflammatory homeostasis. Inhaled fungal spores pose a continual antigenic, and potentially pathogenic, challenge to lung integrity against which the human respiratory mucosa has developed various tolerance and defence strategies. However, respiratory disease and immune dysfunction frequently render the human lung susceptible to fungal diseases, the most common of which are the aspergilloses, a group of syndromes caused by inhaled spores of . Inhaled spores enter into a multiplicity of interactions with respiratory epithelia, the mechanistic bases of which are only just becoming recognized as important drivers of disease, as well as possible therapeutic targets. In this mini-review we examine current understanding of -epithelial interactions and, based upon the very latest developments in the field, we explore two apparently opposing schools of thought which view epithelial uptake of spores as either a curative or disease-exacerbating event.
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http://dx.doi.org/10.3390/jof4010008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5872311PMC
January 2018

Phenotypic and Proteomic Analysis of the Δ, Δ and Δ/Δ Protease-Deficient Mutants.

Front Microbiol 2017 12;8:2490. Epub 2017 Dec 12.

Aspergillus and Antifungal Research Laboratory, Department of Clinical Microbiology and Immunology, Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel.

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. produces extracellular proteases that are thought to enhance penetration by degrading host structural barriers. This study explores the role of the transcription factor XprG in controlling secreted proteolytic activity and fungal virulence. We deleted , alone and in combination with , a transcription factor previously shown to regulate extracellular proteolysis. 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 and 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 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 during infection based on extensive gene redundancy.
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http://dx.doi.org/10.3389/fmicb.2017.02490DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5732999PMC
December 2017

The quinoline bromoquinol exhibits broad-spectrum antifungal activity and induces oxidative stress and apoptosis in Aspergillus fumigatus.

J Antimicrob Chemother 2017 08;72(8):2263-2272

Department of Clinical Microbiology and Immunology, Sackler School of Medicine, Tel-Aviv University, Tel-Aviv, Israel.

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.
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http://dx.doi.org/10.1093/jac/dkx117DOI Listing
August 2017

Aspergillus fumigatus Copper Export Machinery and Reactive Oxygen Intermediate Defense Counter Host Copper-Mediated Oxidative Antimicrobial Offense.

Cell Rep 2017 05;19(5):1008-1021

Department of Medical Microbiology and Immunology, University of Wisconsin, Madison, WI 53706, USA; Department of Bacteriology, University of Wisconsin, Madison, WI 53706, USA. Electronic address:

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.
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http://dx.doi.org/10.1016/j.celrep.2017.04.019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5512462PMC
May 2017

NK Cell Recognition of Candida glabrata through Binding of NKp46 and NCR1 to Fungal Ligands Epa1, Epa6, and Epa7.

Cell Host Microbe 2016 Oct;20(4):527-534

The Lautenberg Center for General and Tumor Immunology, The BioMedical Research Institute Israel Canada of the Faculty of Medicine (IMRIC), The Hebrew University Hadassah Medical School, 91120 Jerusalem, Israel. Electronic address:

Natural killer (NK) cells form an important arm of the innate immune system and function to combat a wide range of invading pathogens, ranging from viruses to bacteria. However, the means by which NK cells accomplish recognition of pathogens with a limited repertoire of receptors remain largely unknown. In the current study, we describe the recognition of an emerging fungal pathogen, Candida glabrata, by the human NK cytotoxic receptor NKp46 and its mouse ortholog, NCR1. Using NCR1 knockout mice, we observed that this receptor-mediated recognition was crucial for controlling C. glabrata infection in vitro and in vivo. Finally, we delineated the fungal ligands to be the C. glabrata adhesins Epa1, Epa6, and Epa7 and demonstrated that clearance of systemic C. glabrata infections in vivo depends on their recognition by NCR1. As NKp46 and NCR1 have been previously shown to bind viral adhesion receptors, we speculate that NKp46/NCR1 may be a novel type of pattern recognition receptor.
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http://dx.doi.org/10.1016/j.chom.2016.09.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5492882PMC
October 2016

The anti-Aspergillus drug pipeline: Is the glass half full or empty?

Med Mycol 2017 Jan 25;55(1):118-124. Epub 2016 Aug 25.

Department of Infectious Diseases, Infection Control and Employee Health, University of Texas MD Anderson Cancer Center, Houston, TX, USA

Aspergillosis has emerged as important human mycoses, in view of the ever expanding population at risk. The emergence of resistance to the most commonly used drugs for aspergillosis, the azoles, the mediocre activity, and frequent toxicity of the current antifungal armamentarium, support the need for development of novel antifungals for treatment of this disease. In this minireview, we describe recent efforts by small drug companies and University research labs to develop novel therapies for invasive aspergillus infections. We specifically discuss four small-molecule antifungals (T-2307, E1210/APX001, ASP2397, and F901318) with novel modes-of-action, which are currently entering phase I clinical trials. In addition, we provide a nonexhaustive discussion of some interesting, yet early developments in the quest for improved therapeutic strategies such as (i) novel formulations of amphotericin B including AMB nanoparticle suspensions and AMB-arabinogalactan or AMB-PEG conjugates that show low toxicity and high efficacy in preclinical animal models, (ii) repurposed drugs that synergize with existing antifungals (clozafimine, trichostatin A, MGCD290, geldanamycin, tacrolimus, cyclosporin), (iii) natural products (psoriasin, humidimycin), and (iv) immunotherapy using adoptive transfer of activated immune cells with antifungal activity. We argue that despite the plethora of candidates, the extremely low success rates of drug development leading to clinically useful drugs reinforces the need for continued clinical reliance on mainstream antifungals and their improved derivatives.
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http://dx.doi.org/10.1093/mmy/myw060DOI Listing
January 2017

Correction: Modulation of Host Angiogenesis as a Microbial Survival Strategy and Therapeutic Target.

PLoS Pathog 2016 Aug 17;12(8):e1005838. Epub 2016 Aug 17.

[This corrects the article DOI: 10.1371/journal.ppat.1005479.].
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http://dx.doi.org/10.1371/journal.ppat.1005838DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4988771PMC
August 2016

Systematic Identification of Anti-Fungal Drug Targets by a Metabolic Network Approach.

Front Mol Biosci 2016 17;3:22. Epub 2016 Jun 17.

Department of Bioinformatics, Biocenter, University of Würzburg Würzburg, Germany.

New antimycotic drugs are challenging to find, as potential target proteins may have close human orthologs. We here focus on identifying metabolic targets that are critical for fungal growth and have minimal similarity to targets among human proteins. We compare and combine here: (I) direct metabolic network modeling using elementary mode analysis and flux estimates approximations using expression data, (II) targeting metabolic genes by transcriptome analysis of condition-specific highly expressed enzymes, and (III) analysis of enzyme structure, enzyme interconnectedness ("hubs"), and identification of pathogen-specific enzymes using orthology relations. We have identified 64 targets including metabolic enzymes involved in vitamin synthesis, lipid, and amino acid biosynthesis including 18 targets validated from the literature, two validated and five currently examined in own genetic experiments, and 38 further promising novel target proteins which are non-orthologous to human proteins, involved in metabolism and are highly ranked drug targets from these pipelines.
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http://dx.doi.org/10.3389/fmolb.2016.00022DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4911368PMC
July 2016

Modulation of Host Angiogenesis as a Microbial Survival Strategy and Therapeutic Target.

PLoS Pathog 2016 Apr 14;12(4):e1005479. Epub 2016 Apr 14.

Infectious Disease Unit, Tel-Aviv Sourasky Medical Center, Tel-Aviv, Israel.

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http://dx.doi.org/10.1371/journal.ppat.1005479DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4831739PMC
April 2016

Identification and Characterization of a Novel Aspergillus fumigatus Rhomboid Family Putative Protease, RbdA, Involved in Hypoxia Sensing and Virulence.

Infect Immun 2016 06 24;84(6):1866-1878. Epub 2016 May 24.

Department of Clinical Microbiology and Immunology, Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel

Aspergillus fumigatus is the most common pathogenic mold infecting humans and a significant cause of morbidity and mortality in immunocompromised patients. In invasive pulmonary aspergillosis, A. fumigatus spores are inhaled into the lungs, undergoing germination and invasive hyphal growth. The fungus occludes and disrupts the blood vessels, leading to hypoxia and eventual tissue necrosis. The ability of this mold to adapt to hypoxia is regulated in part by the sterol regulatory element binding protein (SREBP) SrbA and the DscA to DscD Golgi E3 ligase complex critical for SREBP activation by proteolytic cleavage. Loss of the genes encoding these proteins results in avirulence. To identify novel regulators of hypoxia sensing, we screened the Neurospora crassa gene deletion library under hypoxia and identified a novel rhomboid family protease essential for hypoxic growth. Deletion of the A. fumigatus rhomboid homolog rbdA resulted in an inability to grow under hypoxia, hypersensitivity to CoCl2, nikkomycin Z, fluconazole, and ferrozine, abnormal swollen tip morphology, and transcriptional dysregulation-accurately phenocopying deletion of srbA. In vivo, rbdA deletion resulted in increased sensitivity to phagocytic killing, a reduced inflammatory Th1 and Th17 response, and strongly attenuated virulence. Phenotypic rescue of the ΔrbdA mutant was achieved by expression and nuclear localization of the N terminus of SrbA, including its HLH domain, further indicating that RbdA and SrbA act in the same signaling pathway. In summary, we have identified RbdA, a novel putative rhomboid family protease in A. fumigatus that mediates hypoxia adaptation and fungal virulence and that is likely linked to SrbA cleavage and activation.
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http://dx.doi.org/10.1128/IAI.00011-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4907145PMC
June 2016

Mechanisms of Bacterial (Serratia marcescens) Attachment to, Migration along, and Killing of Fungal Hyphae.

Appl Environ Microbiol 2016 May 18;82(9):2585-94. Epub 2016 Apr 18.

Department of Clinical Microbiology and Immunology, Sackler School of Medicine, Tel-Aviv University, Tel-Aviv, Israel

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.
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http://dx.doi.org/10.1128/AEM.04070-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4836416PMC
May 2016

Novel Water-Soluble Amphotericin B-PEG Conjugates with Low Toxicity and Potent in Vivo Efficacy.

J Med Chem 2016 Feb 27;59(3):1197-206. Epub 2016 Jan 27.

Schulich Faculty of Chemistry, Israel Institute of Technology , 3200008 Technion-City, Haifa, 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.
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http://dx.doi.org/10.1021/acs.jmedchem.5b01862DOI Listing
February 2016

Identification and characterization of haemofungin, a novel antifungal compound that inhibits the final step of haem biosynthesis.

J Antimicrob Chemother 2016 Apr 7;71(4):946-52. Epub 2016 Jan 7.

Department of Clinical Microbiology and Immunology, Sackler School of Medicine, Tel-Aviv University, Tel-Aviv, Israel

Objectives: During recent decades, the number of invasive fungal infections among immunosuppressed patients has increased significantly, whereas the number of effective systemic antifungal drugs remains low and unsatisfactory. The aim of this study was to characterize a novel antifungal compound, CW-8/haemofungin, which we previously identified in a screen for compounds affecting fungal cell wall integrity.

Methods: The in vitro characteristics of haemofungin were investigated by MIC evaluation against a panel of pathogenic and non-pathogenic fungi, bacteria and mammalian cells in culture. Haemofungin mode-of-action studies were performed by screening an Aspergillus nidulans overexpression genomic library for resistance-conferring plasmids and biochemical validation of the target. In vivo efficacy was tested in the Galleria mellonella and Drosophila melanogaster insect models of infection.

Results: We demonstrate that haemofungin causes swelling and lysis of growing fungal cells. It inhibits the growth of pathogenic Aspergillus, Candida, Fusarium and Rhizopus isolates at micromolar concentrations, while only weakly affecting the growth of mammalian cell lines. Genetic and biochemical analyses in A. nidulans and Aspergillus fumigatus indicate that haemofungin primarily inhibits ferrochelatase (HemH), the last enzyme in the haem biosynthetic pathway. Haemofungin was non-toxic and significantly reduced mortality rates of G. mellonella and D. melanogaster infected with A. fumigatus and Rhizopus oryzae, respectively.

Conclusions: Further development and in vivo validation of haemofungin is warranted.
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http://dx.doi.org/10.1093/jac/dkv446DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5757792PMC
April 2016

Identification and characterization of a novel family of selective antifungal compounds (CANBEFs) that interfere with fungal protein synthesis.

Antimicrob Agents Chemother 2015 Sep 6;59(9):5631-40. Epub 2015 Jul 6.

Department of Clinical Microbiology and Immunology, Sackler School of Medicine, Tel-Aviv University, Tel-Aviv, Israel

Invasive mycotic infections have become more common during recent decades, posing an increasing threat to public health. However, despite the growing needs, treatments for invasive fungal infections remain unsatisfactory and are limited to a small number of antifungals. The aim of this study was to identify novel fungal cell wall inhibitors from a library of small chemical compounds using a conditional protein kinase C (PKC)-expressing strain of Aspergillus nidulans sensitive to cell wall-active agents. Eight "hit" compounds affecting cell wall integrity were identified from a screen of 35,000 small chemical compounds. Five shared a common basic molecular structure of 4-chloro-6-arylamino-7-nitro-benzofurazane (CANBEF). The most potent compound, CANBEF-24, was characterized further and was shown to inhibit the growth of pathogenic Aspergillus, Candida, Fusarium, and Rhizopus isolates at micromolar concentrations but not to affect the growth of mammalian cell lines. CANBEF-24 demonstrated strong synergy in combination with caspofungin, an antifungal that inhibits cell wall biosynthesis. Genetic and biochemical analyses with Aspergillus nidulans and Saccharomyces cerevisiae indicated that CANBEFs selectively inhibit fungal rRNA maturation and protein synthesis, suggesting that their effect on the cell wall is indirect. CANBEFs were nontoxic in insect (Galleria mellonella, Drosophila melanogaster) and mouse models of fungal infection. Preliminary evidence showing no therapeutic benefit in these models suggests that further cycles of optimization are needed for the development of this novel class of compounds for systemic use.
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http://dx.doi.org/10.1128/AAC.00850-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4538511PMC
September 2015

Identification of hypoxia-inducible target genes of Aspergillus fumigatus by transcriptome analysis reveals cellular respiration as an important contributor to hypoxic survival.

Eukaryot Cell 2014 Sep 1;13(9):1241-53. Epub 2014 Aug 1.

Department of Molecular and Applied Microbiology, the Leibniz Institute for Natural Product Research and Infection Biology-Hans Knöll Institute (HKI), Jena, Germany Department of Microbiology and Molecular Biology, Institute of Microbiology, Friedrich Schiller University Jena, Jena, Germany Integrated Research Treatment-Center, Center for Sepsis Control and Care (CSCC), University Hospital Jena, Germany

Aspergillus fumigatus is an opportunistic, airborne pathogen that causes invasive aspergillosis in immunocompromised patients. During the infection process, A. fumigatus is challenged by hypoxic microenvironments occurring in inflammatory, necrotic tissue. To gain further insights into the adaptation mechanism, A. fumigatus was cultivated in an oxygen-controlled chemostat under hypoxic and normoxic conditions. Transcriptome analysis revealed a significant increase in transcripts associated with cell wall polysaccharide metabolism, amino acid and metal ion transport, nitrogen metabolism, and glycolysis. A concomitant reduction in transcript levels was observed with cellular trafficking and G-protein-coupled signaling. To learn more about the functional roles of hypoxia-induced transcripts, we deleted A. fumigatus genes putatively involved in reactive nitrogen species detoxification (fhpA), NAD(+) regeneration (frdA and osmA), nitrogen metabolism (niaD and niiA), and respiration (rcfB). We show that the nitric oxygen (NO)-detoxifying flavohemoprotein gene fhpA is strongly induced by hypoxia independent of the nitrogen source but is dispensable for hypoxic survival. By deleting the nitrate reductase gene niaD, the nitrite reductase gene niiA, and the two fumarate reductase genes frdA and osmA, we found that alternative electron acceptors, such as nitrate and fumarate, do not have a significant impact on growth of A. fumigatus during hypoxia, but functional mitochondrial respiratory chain complexes are essential under these conditions. Inhibition studies indicated that primarily complexes III and IV play a crucial role in the hypoxic growth of A. fumigatus.
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http://dx.doi.org/10.1128/EC.00084-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4187615PMC
September 2014

The three Aspergillus fumigatus CFEM-domain GPI-anchored proteins (CfmA-C) affect cell-wall stability but do not play a role in fungal virulence.

Fungal Genet Biol 2014 Feb 20;63:55-64. Epub 2013 Dec 20.

Department of Clinical Microbiology and Immunology, Sackler School of Medicine, Ramat-Aviv, Tel-Aviv, Israel. Electronic address:

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.
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http://dx.doi.org/10.1016/j.fgb.2013.12.005DOI Listing
February 2014
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