Publications by authors named "Yana Shadkchan"

19 Publications

  • Page 1 of 1

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

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

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

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

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

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

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

Trivalent ultrashort lipopeptides are potent pH dependent antifungal agents.

J Med Chem 2012 Feb 1;55(3):1296-302. Epub 2012 Feb 1.

Department of Biological Chemistry, The Weizmann Institute of Science, Ullman Building, Rehovot 76100, Israel.

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.
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http://dx.doi.org/10.1021/jm2014474DOI Listing
February 2012

Ultrashort peptide bioconjugates are exclusively antifungal agents and synergize with cyclodextrin and amphotericin B.

Antimicrob Agents Chemother 2012 Jan 17;56(1):1-9. Epub 2011 Oct 17.

Department of Biological Chemistry, The Weizmann Institute of Science, Rehovot 76100, Israel.

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.
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http://dx.doi.org/10.1128/AAC.00468-11DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3256016PMC
January 2012

PrtT-regulated proteins secreted by Aspergillus fumigatus activate MAPK signaling in exposed A549 lung cells leading to necrotic cell death.

PLoS One 2011 Mar 11;6(3):e17509. Epub 2011 Mar 11.

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

Aspergillus fumigatus is the most commonly encountered mold pathogen of humans, predominantly infecting the respiratory system. Colonization and penetration of the lung alveolar epithelium is a key but poorly understood step in the infection process. This study focused on identifying the transcriptional and cell-signaling responses activated in A549 alveolar carcinoma cells incubated in the presence of A. fumigatus wild-type and ΔPrtT protease-deficient germinating conidia and culture filtrates (CF). Microarray analysis of exposed A549 cells identified distinct classes of genes whose expression is altered in the presence of germinating conidia and CF and suggested the involvement of both NFkB and MAPK signaling pathways in mediating the cellular response. Phosphoprotein analysis of A549 cells confirmed that JNK and ERK1/2 are phosphorylated in response to CF from wild-type A. fumigatus and not phosphorylated in response to CF from the ΔPrtT protease-deficient strain. Inhibition of JNK or ERK1/2 kinase activity substantially decreased CF-induced cell damage, including cell peeling, actin-cytoskeleton damage, and reduction in metabolic activity and necrotic death. These results suggest that inhibition of MAPK-mediated host responses to treatment with A. fumigatus CF decreases cellular damage, a finding with possible clinical implications.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0017509PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3055868PMC
March 2011

The Aspergillus fumigatus cspA gene encoding a repeat-rich cell wall protein is important for normal conidial cell wall architecture and interaction with host cells.

Eukaryot Cell 2010 Sep 23;9(9):1403-15. Epub 2010 Jul 23.

Department of Human Microbiology, Sackler School of Medicine, Tel Aviv University, Ramat Aviv 69978, Tel Aviv, Israel.

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 beta-glucan. This is correlated with increasing susceptibility of the DeltacspA, DeltaECM33, and DeltacspA DeltaECM33 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.
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http://dx.doi.org/10.1128/EC.00126-10DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2937338PMC
September 2010

The Aspergillus nidulans cetA and calA genes are involved in conidial germination and cell wall morphogenesis.

Fungal Genet Biol 2008 Mar 20;45(3):232-42. Epub 2007 Jul 20.

Department of Human Microbiology, Sackler School of Medicine, Tel-Aviv University, Ramat-Aviv 69978, Tel-Aviv, Israel.

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.
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http://dx.doi.org/10.1016/j.fgb.2007.07.005DOI Listing
March 2008
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