scholarly journals Fluorescence Polarization Binding Assay for Aspergillus fumigatus Virulence Factor UDP-Galactopyranose Mutase

2011 ◽  
Vol 2011 ◽  
pp. 1-9 ◽  
Author(s):  
Jun Qi ◽  
Michelle Oppenheimer ◽  
Pablo Sobrado
Keyword(s):  
Aspergillus Fumigatus ◽  
Fluorescence Polarization ◽  
Binding Assay ◽  
Pathogenic Fungus ◽  
Affinity Binding ◽  
Good Tolerance ◽  
High Affinity ◽  
Essential Components ◽  
Human Pathogenic Fungus ◽  
Lung Infections

Aspergillus fumigatus is an opportunistic human pathogenic fungus responsible for deadly lung infections in immunocompromised individuals. Galactofuranose (Galf) residues are essential components of the cell wall and play an important role in A. fumigatus virulence. The flavoenzyme UDP-galactopyranose mutase (UGM) catalyzes the isomerization of UDP-galactopyranose to UDP-galactofuranose, the biosynthetic precursor of Galf. Thus, inhibitors of UGM that block the biosynthesis of Galf can lead to novel chemotherapeutics for treating A. fumigatus-related diseases. Here, we describe the synthesis of fluorescently labeled UDP analogs and the development of a fluorescence polarization (FP) binding assay for A. fumigatus UGM (AfUGM). High-affinity binding to AfUGM was only obtained with the chromophore TAMRA, linked to UDP by either 2 or 6 carbons with Kd values of 2.6 ± 0.2 μM and 3.0 ± 0.7 μM, respectively. These values were ~6 times lower than when UDP was linked to fluorescein. The FP assay was validated against several known ligands and displayed an excellent Z′ factor (0.79 ± 0.02) and good tolerance to dimethyl sulfoxide.

scholarly journals The Putative APSES Transcription Factor RgdA Governs Growth, Development, Toxigenesis, and Virulence in Aspergillus fumigatus

mSphere ◽  
2020 ◽  
Vol 5 (6) ◽  
Author(s):  
Sang-Cheol Jun ◽  
Yong-Ho Choi ◽  
Min-Woo Lee ◽  
Jae-Hyuk Yu ◽  
Kwang-Soo Shin
Keyword(s):  
Transcription Factor ◽  
Aspergillus Fumigatus ◽  
Molecular Mechanisms ◽  
Pathogenic Fungus ◽  
Mrna Levels ◽  
Transcript Levels ◽  
Content Type ◽  
Asexual Sporulation ◽  
Human Pathogenic Fungus ◽  
Growth Development

ABSTRACT The APSES transcription factor (TF) in Aspergillus species is known to govern diverse cellular processes, including growth, development, and secondary metabolism. Here, we investigated functions of the rgdA gene (Afu3g13920) encoding a putative APSES TF in the opportunistic human-pathogenic fungus Aspergillus fumigatus. The rgdA deletion resulted in significantly decreased hyphal growth and asexual sporulation. Consistently, transcript levels of the key asexual developmental regulators abaA, brlA, and wetA were decreased in the ΔrgdA mutant compared to those in the wild type (WT). Moreover, ΔrgdA resulted in reduced spore germination rates and elevated transcript levels of genes associated with conidium dormancy. The conidial cell wall hydrophobicity and architecture were changed, and levels of the RodA protein were decreased in the ΔrgdA mutant. Comparative transcriptomic analyses revealed that the ΔrgdA mutant showed higher mRNA levels of gliotoxin (GT)-biosynthetic genes and GT production. While the ΔrgdA mutant exhibited elevated production of GT, ΔrgdA strains showed reduced virulence in the mouse model. In addition, mRNA levels of genes associated with the cyclic AMP (cAMP)-protein kinase A (PKA) signaling pathway and the SakA mitogen-activated protein (MAP) kinase pathway were increased in the ΔrgdA mutant. In summary, RgdA plays multiple roles in governing growth, development, GT production, and virulence which may involve attenuation of PKA and SakA signaling. IMPORTANCE Immunocompromised patients are susceptible to infections with the opportunistic human-pathogenic fungus Aspergillus fumigatus. This fungus causes systemic infections such as invasive aspergillosis (IA), which is one of the most life-threatening fungal diseases. To control this serious disease, it is critical to identify new antifungal drug targets. In fungi, the transcriptional regulatory proteins of the APSES family play crucial roles in controlling various biological processes, including mating, asexual sporulation and dimorphic growth, and virulence traits. This study found that a putative APSES transcription factor, RgdA, regulates normal growth, asexual development, conidium germination, spore wall architecture and hydrophobicity, toxin production, and virulence in A. fumigatus. Better understanding the molecular mechanisms of RgdA in human-pathogenic fungi may reveal a novel antifungal target for future drug development.

scholarly journals The Opportunistic Human Pathogenic Fungus Aspergillus fumigatus Evades the Host Complement System

2007 ◽  
Vol 76 (2) ◽  
pp. 820-827 ◽  
Author(s):  
Judith Behnsen ◽  
Andrea Hartmann ◽  
Jeannette Schmaler ◽  
Alexander Gehrke ◽  
Axel A. Brakhage ◽  
...  
Keyword(s):  
Aspergillus Fumigatus ◽  
Complement System ◽  
Developmental Stages ◽  
Fungal Infections ◽  
Alternative Pathway ◽  
Pathogenic Fungus ◽  
Factor H ◽  
Human Sera ◽  
Human Pathogenic Fungus ◽  
Complement Regulators

ABSTRACT The opportunistic human pathogenic fungus Aspergillus fumigatus causes severe systemic infections and is a major cause of fungal infections in immunocompromised patients. A. fumigatus conidia activate the alternative pathway of the complement system. In order to assess the mechanisms by which A. fumigatus evades the activated complement system, we analyzed the binding of host complement regulators to A. fumigatus. The binding of factor H and factor H-like protein 1 (FHL-1) from human sera to A. fumigatus conidia was shown by adsorption assays and immunostaining. In addition, factor H-related protein 1 (FHR-1) bound to conidia. Adsorption assays with recombinant factor H mutants were used to localize the binding domains. One binding region was identified within N-terminal short consensus repeats (SCRs) 1 to 7 and a second one within C-terminal SCR 20. Plasminogen was identified as the fourth host regulatory molecule that binds to A. fumigatus conidia. In contrast to conidia, other developmental stages of A. fumigatus, like swollen conidia or hyphae, did not bind to factor H, FHR-1, FHL-1, and plasminogen, thus indicating the developmentally regulated expression of A. fumigatus surface ligands. Both factor H and plasminogen maintained regulating activity when they were bound to the conidial surface. Bound factor H acted as a cofactor to the factor I-mediated cleavage of C3b. Plasminogen showed proteolytic activity when activated to plasmin by urokinase-type plasminogen activator. These data show that A. fumigatus conidia bind to complement regulators, and these bound host regulators may contribute to evasion of a host complement attack.

scholarly journals Flotillin-dependent lipid-raft microdomains are required for functional phagolysosomes against fungal infections

2019 ◽  
Author(s):  
Franziska Schmidt ◽  
Andreas Thywißen ◽  
Marie Röcker ◽  
Cristina Cunha ◽  
Zoltán Cseresnyés ◽  
...  
Keyword(s):  
Lipid Rafts ◽  
Lipid Raft ◽  
Fungal Infections ◽  
Pathogenic Fungus ◽  
Cell Transplant ◽  
Hematopoietic Stem ◽  
Essential Components ◽  
Human Pathogenic Fungus ◽  
Lipid Raft Microdomains

SUMMARYLipid rafts form signaling platforms on biological membranes with incompletely characterized role in immune response to infection. Here we report that lipid raft microdomains are essential components of the phagolysosomal membrane of macrophages. Genetic deletion of the lipidraft chaperons flotillin-1 and flotillin-2 demonstrate that the assembly of both major defense complexes vATPase and NADPH oxidase on the phagolysosomal membrane requires lipid rafts. Furthermore, we discovered a new virulence mechanism leading to the dysregulation of lipid-raft formation by melanized wild-type conidia of the important human-pathogenic fungusAspergillus fumigatus. This results in reduced phagolysosomal acidification. Phagolysosomes with ingested melanized conidia contain a reduced amount of free Ca2+ions as compared to phagolysosomes with melanin-free conidia. In agreement with a role of Ca2+for generation of functional lipid rafts, we show that Ca2+-dependent calmodulin activity is required for lipid-raft formation on the phagolysosome. We identified a single nucleotide polymorphism in the humanFLOT1gene that results in heightened susceptibility for invasive aspergillosis in hematopoietic stem-cell transplant recipients. Collectively, flotillin-dependent lipid rafts on the phagolysosomal membrane play an essential role in protective antifungal immunity in humans.

Evaluation of the Binding to Fixed Platelets of Agonists and Antagonists of ADP-Induced Aggregation

1989 ◽  
Vol 62 (04) ◽  
pp. 1103-1106 ◽  
Author(s):  
Ashok K Agarwal ◽  
Narendra N Tandon ◽  
Nicholas J Greco ◽  
Noel J Cusack ◽  
G A Jamieson
Keyword(s):  
Binding Sites ◽  
Binding Assay ◽  
Relative Magnitude ◽  
Affinity Binding ◽  
Inhibitory Potency ◽  
Competitive Binding Assay ◽  
High Affinity ◽  
High Affinity Site ◽  
Purine Ring ◽  
Induced Aggregation

SummarySteady state binding of eleven different ADP analogues to formaldehyde-fixed platelets has been determined in a competitive binding assay using 3H-ADP. The compounds tested were the inactive analogues L-ADP and L-ATP; the agonists 2-chloroadenosine 5’-diphosphate, adenosine 5’-O-(2-thiodiphosphate)and the diastereoisomeric pair Sp-adenosine 5’-(1-thiodiphosphate) (Sp-ADP-α-S) and Rp-adenosine 5’-(1-thiodiphosphate) (Rp-ADP-α-S); and the antagonists adenosine 5’-O-thiomonophosphate, 2-chloroadenosine 5’-O-thiomonophosphate, 2-chloroadenosine 5’-triphoshate, and the diastereoisomeric pair 5’-(1-thiotriphosphate) (Sp-ATP-α-S) and Rp-adenosine 5’-(1-thiotriphosphate) (Rp-ATP-α-S). All compounds tested competed at the high affinity binding sites for ADP previously identified (Blood 1988; 71: 110-6) but in some cases competition could not be demonstrated at the low affinity sites because of the high nucleotide concentrations required. As a group, C2-substituted analogues bound less strongly (Ki >2 μM) than did the analogues without substituents in the purine ring (Ki <0.7 μM). With the pair of diastereoisomeric agonists Sp-ADP-α-S and Rp-ADP-α-S the Ki values at the high affinity site (210 nM and 560 nM) were of the same relative magnitude and in the same direction as their reported potencies as agonists (Ki 4 μM and 20 μM). With the diastereoisomeric antagonists Sp-ATP-α-S and Rp-ATP-α-S a similar relationship was seen between affinity (17 nM and 156 nM) and inhibitory potency (Ki 4 μM and 20 μM). These results may help to differentiate possible mechanisms in the interaction of ADP with its receptors.

scholarly journals Inducible Cell Fusion Permits Use of Competitive Fitness Profiling in the Human Pathogenic Fungus Aspergillus fumigatus

2018 ◽  
Vol 63 (1) ◽  
Author(s):  
Darel Macdonald ◽  
Darren D. Thomson ◽  
Anna Johns ◽  
Adriana Contreras Valenzuela ◽  
Jane M. Gilsenan ◽  
...  
Keyword(s):  
Aspergillus Fumigatus ◽  
Cell Fusion ◽  
Pathogenic Fungus ◽  
Brefeldin A ◽  
Culture Conditions ◽  
Competitive Fitness ◽  
Content Type ◽  
Genetic Profiling ◽  
Standard Culture ◽  
Human Pathogenic Fungus

ABSTRACT Antifungal agents directed against novel therapeutic targets are required for treating invasive, chronic, and allergic Aspergillus infections. Competitive fitness profiling technologies have been used in a number of bacterial and yeast systems to identify druggable targets; however, the development of similar systems in filamentous fungi is complicated by the fact that they undergo cell fusion and heterokaryosis. Here, we demonstrate that cell fusion in Aspergillus fumigatus under standard culture conditions is not predominately constitutive, as with most ascomycetes, but can be induced by a range of extracellular stressors. Using this knowledge, we have developed a barcode-free genetic profiling system that permits high-throughput parallel determination of strain fitness in a collection of diploid A. fumigatus mutants. We show that heterozygous cyp51A and arf2 null mutants have reduced fitness in the presence of itraconazole and brefeldin A, respectively, and a heterozygous atp17 null mutant is resistant to brefeldin A.

Identification of the antiphagocytic trypacidin gene cluster in the human-pathogenic fungus Aspergillus fumigatus

2015 ◽  
Vol 99 (23) ◽  
pp. 10151-10161 ◽  
Author(s):  
Derek J. Mattern ◽  
Hanno Schoeler ◽  
Jakob Weber ◽  
Silvia Novohradská ◽  
Kaswara Kraibooj ◽  
...  
Keyword(s):  
Aspergillus Fumigatus ◽  
Gene Cluster ◽  
Pathogenic Fungus ◽  
Human Pathogenic Fungus

scholarly journals Conidial Melanin of the Human-Pathogenic Fungus Aspergillus fumigatus Disrupts Cell Autonomous Defenses in Amoebae

mBio ◽  
2020 ◽  
Vol 11 (3) ◽  
Author(s):  
Iuliia Ferling ◽  
Joe Dan Dunn ◽  
Alexander Ferling ◽  
Thierry Soldati ◽  
Falk Hillmann
Keyword(s):  
Aspergillus Fumigatus ◽  
Dictyostelium Discoideum ◽  
Immune Cells ◽  
Pathogenic Fungus ◽  
Innate Immune ◽  
Innate Immune Cells ◽  
Membrane Repair ◽  
Protective Function ◽  
Content Type ◽  
Human Pathogenic Fungus

ABSTRACT The human-pathogenic fungus Aspergillus fumigatus is a ubiquitous saprophyte that causes fatal lung infections in immunocompromised individuals. Following inhalation, conidia are ingested by innate immune cells and can arrest phagolysosome maturation. How this virulence trait could have been selected for in natural environments is unknown. Here, we found that surface exposure of the green pigment 1,8-dihydroxynaphthalene-(DHN)-melanin can protect conidia from phagocytic uptake and intracellular killing by the fungivorous amoeba Protostelium aurantium and delays its exocytosis from the nonfungivorous species Dictyostelium discoideum. To elucidate the antiphagocytic properties of the surface pigment, we followed the antagonistic interactions of A. fumigatus conidia with the amoebae in real time. For both amoebae, conidia covered with DHN-melanin were internalized at far lower rates than were seen with conidia lacking the pigment, despite high rates of initial attachment to nonkilling D. discoideum. When ingested by D. discoideum, the formation of nascent phagosomes was followed by transient acidification of phagolysosomes, their subsequent neutralization, and, finally, exocytosis of the conidia. While the cycle was completed in less than 1 h for unpigmented conidia, the process was significantly prolonged for conidia covered with DHN-melanin, leading to an extended intracellular residence time. At later stages of this cellular infection, pigmented conidia induced enhanced damage to phagolysosomes and infected amoebae failed to recruit the ESCRT (endosomal sorting complex required for transport) membrane repair machinery or the canonical autophagy pathway to defend against the pathogen, thus promoting prolonged intracellular persistence in the host cell and the establishment of a germination niche in this environmental phagocyte. IMPORTANCE Infections with Aspergillus fumigatus are usually acquired by an inhalation of spores from environmental sources. How spores of a saprophytic fungus have acquired abilities to withstand and escape the phagocytic attacks of innate immune cells is not understood. The fungal surface pigment dihydroxynaphtalene-melanin has been shown to be a crucial factor for the delay in phagosome maturation. Here, we show that this pigment also has a protective function against environmental phagocytes. Pigmented conidia escaped uptake and killing by the fungus-eating amoeba Protostelium aurantium. When ingested by the nonfungivorous phagocyte Dictyostelium discoideum, the pigment attenuated the launch of cell autonomous defenses against the fungal invader, such as membrane repair and autophagy, leading to prolonged intracellular retention. Membrane damage and cytoplasmic leakage may result in an influx of nutrients and thus may further promote intracellular germination of the fungus, indicating that A. fumigatus has acquired some of the basic properties of intracellular pathogens.

Sign in / Sign up

Export Citation Format

Share Document