Characterisation of novel-cell-wall LysM-domain proteins LdpA and LdpB from the human pathogenic fungus Aspergillus fumigatus

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.

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

Infection and Immunity ◽

10.1128/iai.01037-07 ◽

2007 ◽

Vol 76 (2) ◽

pp. 820-827 ◽

Cited By ~ 80

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.

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Mitogen-Activated Protein Kinase Cross-Talk Interaction Modulates the Production of Melanins in Aspergillus fumigatus

mBio ◽

10.1128/mbio.00215-19 ◽

2019 ◽

Vol 10 (2) ◽

Cited By ~ 17

Author(s):

Adriana Oliveira Manfiolli ◽

Filipe Silva Siqueira ◽

Thaila Fernanda dos Reis ◽

Patrick Van Dijck ◽

Sanne Schrevens ◽

...

Keyword(s):

Cell Wall ◽

Aspergillus Fumigatus ◽

Cross Talk ◽

Pathogenic Fungus ◽

Antifungal Drugs ◽

Mitogen Activated Protein Kinase ◽

Melanin Production ◽

Content Type ◽

Mitogen Activated Protein ◽

Salvage Pathways

ABSTRACT The pathogenic fungus Aspergillus fumigatus is able to adapt to extremely variable environmental conditions. The A. fumigatus genome contains four genes coding for mitogen-activated protein kinases (MAPKs), which are important regulatory knots involved in diverse cellular responses. From a clinical perspective, MAPK activity has been connected to salvage pathways, which can determine the failure of effective treatment of invasive mycoses using antifungal drugs. Here, we report the characterization of the Saccharomyces cerevisiae Fus3 ortholog in A. fumigatus, designated MpkB. We demonstrate that MpkB is important for conidiation and that its deletion induces a copious increase of dihydroxynaphthalene (DHN)-melanin production. Simultaneous deletion of mpkB and mpkA, the latter related to maintenance of the cell wall integrity, normalized DHN-melanin production. Localization studies revealed that MpkB translocates into the nuclei when A. fumigatus germlings are exposed to caspofungin stress, and this is dependent on the cross-talk interaction with MpkA. Additionally, DHN-melanin formation was also increased after deletion of genes coding for the Gα protein GpaA and for the G protein-coupled receptor GprM. Yeast two-hybrid and coimmunoprecipitation assays confirmed that GpaA and GprM interact, suggesting their role in the MpkB signaling cascade. IMPORTANCE Aspergillus fumigatus is the most important airborne human pathogenic fungus, causing thousands of deaths per year. Its lethality is due to late and often inaccurate diagnosis and the lack of efficient therapeutics. The failure of efficient prophylaxis and therapy is based on the ability of this pathogen to activate numerous salvage pathways that are capable of overcoming the different drug-derived stresses. A major role in the protection of A. fumigatus is played by melanins. Melanins are cell wall-associated macromolecules classified as virulence determinants. The understanding of the various signaling pathways acting in this organism can be used to elucidate the mechanism beyond melanin production and help to identify ideal drug targets.

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Correction for Macdonald et al., “Inducible Cell Fusion Permits Use of Competitive Fitness Profiling in the Human Pathogenic Fungus Aspergillus fumigatus”

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00706-19 ◽

2019 ◽

Vol 63 (6) ◽

Cited By ~ 4

Author(s):

Darel Macdonald ◽

Darren D. Thomson ◽

Anna Johns ◽

Adriana Contreras Valenzuela ◽

Jane M. Gilsenan ◽

...

Keyword(s):

Aspergillus Fumigatus ◽

Cell Fusion ◽

Pathogenic Fungus ◽

Competitive Fitness ◽

Human Pathogenic Fungus

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

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01615-18 ◽

2018 ◽

Vol 63 (1) ◽

Cited By ~ 2

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.

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

Enzyme Research ◽

10.4061/2011/513905 ◽

2011 ◽

Vol 2011 ◽

pp. 1-9 ◽

Cited By ~ 4

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.

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Phosphoproteomics of Aspergillus fumigatus Exposed to the Antifungal Drug Caspofungin

mSphere ◽

10.1128/msphere.00365-20 ◽

2020 ◽

Vol 5 (3) ◽

Author(s):

Eliciane Cevolani Mattos ◽

Giuseppe Palmisano ◽

Gustavo H. Goldman

Keyword(s):

Cell Wall ◽

Transcription Factors ◽

Aspergillus Fumigatus ◽

Protein Kinases ◽

Pathogenic Fungus ◽

Fungal Growth ◽

Wild Type ◽

Content Type ◽

Mitogen Activated Protein ◽

High Concentrations

ABSTRACT Aspergillus fumigatus is an opportunistic and allergenic pathogenic fungus, responsible for fungal infections in humans. A. fumigatus infections are usually treated with polyenes, azoles, or echinocandins. Echinocandins, such as caspofungin, can inhibit the biosynthesis of the β-1,3-glucan polysaccharide, affecting the integrity of the cell wall and leading to fungal death. In some A. fumigatus strains, caspofungin treatment at high concentrations induces an increase of fungal growth, a phenomenon called the caspofungin paradoxical effect (CPE). Here, we analyze the proteome and phosphoproteome of the A. fumigatus wild-type strain and of mitogen-activated protein kinase (MAPK) mpkA and sakA null mutant strains during CPE (2 μg/ml caspofungin for 1 h). The wild-type proteome showed 75 proteins and 814 phosphopeptides (corresponding to 520 proteins) altered in abundance in response to caspofungin treatment. The ΔmpkA (ΔmpkA caspofungin/wild-type caspofungin) and ΔsakA (ΔsakA caspofungin/wild-type caspofungin) strains displayed 626 proteins and 1,236 phosphopeptides (corresponding to 703 proteins) and 101 proteins and 1,217 phosphopeptides (corresponding to 645 proteins), respectively, altered in abundance. Functional characterization of the phosphopeptides from the wild-type strain exposed to caspofungin showed enrichment for transcription factors, protein kinases, and cytoskeleton proteins. Proteomic analysis of the ΔmpkA and ΔsakA mutants indicated that control of proteins involved in metabolism, such as in production of secondary metabolites, was highly represented in both mutants. Results of functional categorization of phosphopeptides from both mutants were very similar and showed a high number of proteins with decreased phosphorylation of proteins involved in transcriptional control, DNA/RNA binding, cell cycle control, and DNA processing. This report reveals novel transcription factors involved in caspofungin tolerance. IMPORTANCE Aspergillus fumigatus is an opportunistic human-pathogenic fungus causing allergic reactions or systemic infections, such as invasive pulmonary aspergillosis in immunocompromised patients. Caspofungin is an echinocandin that impacts the construction of the fungal cell wall by inhibiting the biosynthesis of the β-1,3-glucan polysaccharide. Caspofungin is a fungistatic drug and is recommended as a second-line therapy for treatment of aspergillosis. Treatment at high concentrations induces an increase of fungal growth, a phenomenon called the caspofungin paradoxical effect (CPE). Collaboration between the mitogen-activated protein kinases (MAPK) of the cell wall integrity (MapkA) and high-osmolarity glycerol (SakA) pathways is essential for CPE. Here, we investigate the global proteome and phosphoproteome of A. fumigatus wild-type, ΔmpkA, and ΔsakA strains upon CPE. This study showed intense cross talk between the two MAPKs for the CPE and identified novel protein kinases and transcription factors possibly important for CPE. Increased understanding of how the modulation of protein phosphorylation may affect the fungal growth in the presence of caspofungin represents an important step in the development of new strategies and methods to combat the fungus inside the host.

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

Applied Microbiology and Biotechnology ◽

10.1007/s00253-015-6898-1 ◽

2015 ◽

Vol 99 (23) ◽

pp. 10151-10161 ◽

Cited By ~ 30

Author(s):

Derek J. Mattern ◽

Hanno Schoeler ◽

Jakob Weber ◽

Silvia Novohradská ◽

Kaswara Kraibooj ◽

...

Keyword(s):

Aspergillus Fumigatus ◽

Gene Cluster ◽

Pathogenic Fungus ◽

Human Pathogenic Fungus

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Role for Chitin and Chitooligomers in the Capsular Architecture of Cryptococcus neoformans

Eukaryotic Cell ◽

10.1128/ec.00142-09 ◽

2009 ◽

Vol 8 (10) ◽

pp. 1543-1553 ◽

Cited By ~ 38

Author(s):

Fernanda L. Fonseca ◽

Leonardo Nimrichter ◽

Radames J. B. Cordero ◽

Susana Frases ◽

Jessica Rodrigues ◽

...

Keyword(s):

Cell Wall ◽

Cryptococcus Neoformans ◽

Pathogenic Fungus ◽

Monosaccharide Composition ◽

Altered Expression ◽

Functional Activities ◽

Differential Reactivity ◽

Human Pathogenic Fungus

ABSTRACT Molecules composed of β-1,4-linked N-acetylglucosamine (GlcNAc) and deacetylated glucosamine units play key roles as surface constituents of the human pathogenic fungus Cryptococcus neoformans. GlcNAc is the monomeric unit of chitin and chitooligomers, which participate in the connection of capsular polysaccharides to the cryptococcal cell wall. In the present study, we evaluated the role of GlcNAc-containing structures in the assembly of the cryptococcal capsule. The in vivo expression of chitooligomers in C. neoformans varied depending on the infected tissue, as inferred from the differential reactivity of yeast forms to the wheat germ agglutinin (WGA) in infected brain and lungs of rats. Chromatographic and dynamic light-scattering analyses demonstrated that glucuronoxylomannan (GXM), the major cryptococcal capsular component, interacts with chitin and chitooligomers. When added to C. neoformans cultures, chitooligomers formed soluble complexes with GXM and interfered in capsular assembly, as manifested by aberrant capsules with defective connections with the cell wall and no reactivity with a monoclonal antibody to GXM. Cultivation of C. neoformans in the presence of an inhibitor of glucosamine 6-phosphate synthase resulted in altered expression of cell wall chitin. These cells formed capsules that were loosely connected to the cryptococcal wall and contained fibers with decreased diameters and altered monosaccharide composition. These results contribute to our understanding of the role played by chitin and chitooligosaccharides on the cryptococcal capsular structure, broadening the functional activities attributed to GlcNAc-containing structures in this biological system.

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Biosynthesis of β-(1→5)-Galactofuranosyl Chains of Fungal-Type and O-Mannose-Type Galactomannans within the Invasive Pathogen Aspergillus fumigatus

10.1101/814756 ◽

2019 ◽

Author(s):

Yuria Chihara ◽

Yutaka Tanaka ◽

Minoru Izumi ◽

Daisuke Hagiwara ◽

Akira Watanabe ◽

...

Keyword(s):

Cell Wall ◽

Cell Surface ◽

Aspergillus Fumigatus ◽

Pathogenic Fungus ◽

Cell Surface Hydrophobicity ◽

Surface Hydrophobicity ◽

Wall Structure ◽

Fungal Cell ◽

Animal Pathogens

ABSTRACTThe pathogenic fungus Aspergillus fumigatus contains galactomannans localized on the surface layer of its cell walls, which are involved in various biological processes. Galactomannans comprise α-(1→2)-/α-(1→6)-mannan and β-(1→5)-/β-(1→6)-galactofuranosyl chains. We previously revealed that GfsA is a β-galactofuranoside β-(1→5)-galactofuranosyltransferase involved in the biosynthesis of β-(1→5)-galactofuranosyl chains. Here, we clarified the entire biosynthesis of β-(1→5)-galactofuranosyl chains in A. fumigatgus. Two paralogs exist within A. fumigatus: GfsB and GfsC. We show that GfsB and GfsC, in addition to GfsA, are β-galactofuranoside β-(1→5)-galactofuranosyltransferases by biochemical and genetic analyses. GfsA, GfsB, and GfsC can synthesize β-(1→5)-galactofuranosyl oligomers up to lengths of 7, 3, and 5 galactofuranoses within an established in vitro highly efficient assay of galactofuranosyltransferase activity. Structural analyses of galactomannans extracted from the strains ΔgfsB, ΔgfsC, ΔgfsAC, and ΔgfsABC revealed that GfsA and GfsC synthesized all β-(1→5)-galactofuranosyl residues of fungal-type and O-mannose-type galactomannans, and GfsB exhibited limited function in A. fumigatus. The loss of β-(1→5)-galactofuranosyl residues decreased the hyphal growth rate and conidia formation ability as well as increased the abnormal hyphal branching structure and cell surface hydrophobicity, but this loss is dispensable for sensitivity to antifungal agents and virulence toward immune-compromised mice.IMPORTANCEβ-(1→5)-galactofuranosyl residues are widely distributed in the subphylum Pezisomycotina of the phylum Ascomycota. Pezizomycotina includes many plant and animal pathogens. Although the structure of β-(1→5)-galactofuranosyl residues of galactomannans in filamentous fungi was discovered long ago, it remains unclear which enzyme is responsible for biosynthesis of this glycan. Fungal cell wall formation processes are complicated, and information concerning glycosyltransferases is essential for their understanding. In this study, we show that GfsA and GfsC are responsible for the biosynthesis of all β-(1→5)-galactofuranosyl residues of fungal-type and O-mannose-type galactomannans. The data presented here indicates that β-(1→5)-galactofuranosyl residues are involved in cell growth, conidiation, polarity, and cell surface hydrophobicity. Our new understanding of β-(1→5)-galactofuranosyl residue biosynthesis provides important novel insights into the formation of the complex cell wall structure and the virulence of the subphylum Pezisomycotina.

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