The Heat Shock Transcription Factor HsfA Is Essential for Thermotolerance and Regulates Cell Wall Integrity in Aspergillus fumigatus

The deleterious effects of human-induced climate change have long been predicted. However, the imminent emergence and spread of new diseases, including fungal infections through the rise of thermotolerant strains, is still neglected, despite being a potential consequence of global warming. Thermotolerance is a remarkable virulence attribute of the mold Aspergillus fumigatus. Under high-temperature stress, opportunistic fungal pathogens deploy an adaptive mechanism known as heat shock (HS) response controlled by heat shock transcription factors (HSFs). In eukaryotes, HSFs regulate the expression of several heat shock proteins (HSPs), such as the chaperone Hsp90, which is part of the cellular program for heat adaptation and a direct target of HSFs. We recently observed that the perturbation in cell wall integrity (CWI) causes concomitant susceptibility to elevated temperatures in A. fumigatus, although the mechanisms underpinning the HS response and CWI cross talking are not elucidated. Here, we aim at further deciphering the interplay between HS and CWI. Our results show that cell wall ultrastructure is severely modified when A. fumigatus is exposed to HS. We identify the transcription factor HsfA as essential for A. fumigatus viability, thermotolerance, and CWI. Indeed, HS and cell wall stress trigger the coordinated expression of both hsfA and hsp90. Furthermore, the CWI signaling pathway components PkcA and MpkA were shown to be important for HsfA and Hsp90 expression in the A. fumigatus biofilms. Lastly, RNA-sequencing confirmed that hsfA regulates the expression of genes related to the HS response, cell wall biosynthesis and remodeling, and lipid homeostasis. Our studies collectively demonstrate the connection between the HS and the CWI pathway, with HsfA playing a crucial role in this cross-pathway regulation, reinforcing the importance of the cell wall in A. fumigatus thermophily.

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Aspergillus fumigatus Hsp90 interacts with the main components of the cell wall integrity pathway and cooperates in heat shock and cell wall stress adaptation

Cellular Microbiology ◽

10.1111/cmi.13273 ◽

2020 ◽

Author(s):

Marina Campos Rocha ◽

Karine Minari ◽

João Henrique Tadini Marilhano Fabri ◽

Joshua D. Kerkaert ◽

Lisandra Marques Gava ◽

...

Keyword(s):

Cell Wall ◽

Heat Shock ◽

Aspergillus Fumigatus ◽

Wall Stress ◽

Cell Wall Integrity ◽

Stress Adaptation ◽

Cell Wall Stress ◽

Cell Wall Integrity Pathway ◽

Main Components

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Puupehenone, a Marine-Sponge-Derived Sesquiterpene Quinone, Potentiates the Antifungal Drug Caspofungin by Disrupting Hsp90 Activity and the Cell Wall Integrity Pathway

mSphere ◽

10.1128/msphere.00818-19 ◽

2020 ◽

Vol 5 (1) ◽

Cited By ~ 1

Author(s):

Siddharth K. Tripathi ◽

Qin Feng ◽

Li Liu ◽

David E. Levin ◽

Kuldeep K. Roy ◽

...

Keyword(s):

Cell Wall ◽

Heat Shock ◽

Marine Sponge ◽

Fungal Pathogens ◽

Fungal Infections ◽

Pathogen Resistance ◽

Cell Wall Integrity ◽

Combination Therapies ◽

Cell Wall Integrity Pathway ◽

Hsp90 Activity

ABSTRACT The cell wall-targeting echinocandin antifungals, although potent and well tolerated, are inadequate in treating fungal infections due to their narrow spectrum of activity and their propensity to induce pathogen resistance. A promising strategy to overcome these drawbacks is to combine echinocandins with a molecule that improves their activity and also disrupts drug adaptation pathways. In this study, we show that puupehenone (PUUP), a marine-sponge-derived sesquiterpene quinone, potentiates the echinocandin drug caspofungin (CAS) in CAS-resistant fungal pathogens. We have conducted RNA sequencing (RNA-seq) analysis, followed by genetic and molecular studies, to elucidate PUUP’s CAS-potentiating mechanism. We found that the combination of CAS and PUUP blocked the induction of CAS-responding genes required for the adaptation to cell wall stress through the cell wall integrity (CWI) pathway. Further analysis showed that PUUP inhibited the activation of Slt2 (Mpk1), the terminal mitogen-activated protein (MAP) kinase in this pathway. We also found that PUUP induced heat shock response genes and inhibited the activity of heat shock protein 90 (Hsp90). Molecular docking studies predicted that PUUP occupies a binding site on Hsp90 required for the interaction between Hsp90 and its cochaperone Cdc37. Thus, we show that PUUP potentiates CAS activity by a previously undescribed mechanism which involves a disruption of Hsp90 activity and the CWI pathway. Given the requirement of the Hsp90-Cdc37 complex in Slt2 activation, we suggest that inhibitors of this complex would disrupt the CWI pathway and synergize with echinocandins. Therefore, the identification of PUUP’s CAS-potentiating mechanism has important implications in the development of new antifungal combination therapies. IMPORTANCE Fungal infections cause more fatalities worldwide each year than malaria or tuberculosis. Currently available antifungal drugs have various limitations, including host toxicity, narrow spectrum of activity, and pathogen resistance. Combining these drugs with small molecules that can overcome these limitations is a useful strategy for extending their clinical use. We have investigated the molecular mechanism by which a marine-derived compound potentiates the activity of the antifungal echinocandin caspofungin. Our findings revealed a mechanism, different from previously reported caspofungin potentiators, in which potentiation is achieved by the disruption of Hsp90 activity and signaling through the cell wall integrity pathway, processes that play important roles in the adaptation to caspofungin in fungal pathogens. Given the importance of stress adaptation in the development of echinocandin resistance, this work will serve as a starting point in the development of new combination therapies that will likely be more effective and less prone to pathogen resistance.

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The Transcription Factor SomA Synchronously Regulates Biofilm Formation and Cell Wall Homeostasis in Aspergillus fumigatus

mBio ◽

10.1128/mbio.02329-20 ◽

2020 ◽

Vol 11 (6) ◽

Author(s):

Yuan Chen ◽

Francois Le Mauff ◽

Yan Wang ◽

Ruiyang Lu ◽

Donald C. Sheppard ◽

...

Keyword(s):

Cell Wall ◽

Transcription Factor ◽

Aspergillus Fumigatus ◽

Biofilm Formation ◽

Wall Stress ◽

Fungal Cell ◽

Glucan Synthase ◽

Content Type ◽

Cell Wall Stress ◽

Chitin Synthases

ABSTRACT Polysaccharides are key components of both the fungal cell wall and biofilm matrix. Despite having distinct assembly and regulation pathways, matrix exopolysaccharide and cell wall polysaccharides share common substrates and intermediates in their biosynthetic pathways. It is not clear, however, if the biosynthetic pathways governing the production of these polysaccharides are cooperatively regulated. Here, we demonstrate that cell wall stress promotes production of the exopolysaccharide galactosaminogalactan (GAG)-depend biofilm formation in the major fungal pathogen of humans Aspergillus fumigatus and that the transcription factor SomA plays a crucial role in mediating this process. A core set of SomA target genes were identified by transcriptome sequencing and chromatin immunoprecipitation coupled to sequencing (ChIP-Seq). We identified a novel SomA-binding site in the promoter regions of GAG biosynthetic genes agd3 and ega3, as well as its regulators medA and stuA. Strikingly, this SomA-binding site was also found in the upstream regions of genes encoding the cell wall stress sensors, chitin synthases, and β-1,3-glucan synthase. Thus, SomA plays a direct regulation of both GAG and cell wall polysaccharide biosynthesis. Consistent with these findings, SomA is required for the maintenance of normal cell wall architecture and compositions in addition to its function in biofilm development. Moreover, SomA was found to globally regulate glucose uptake and utilization, as well as amino sugar and nucleotide sugar metabolism, which provides precursors for polysaccharide synthesis. Collectively, our work provides insight into fungal adaptive mechanisms in response to cell wall stress where biofilm formation and cell wall homeostasis were synchronously regulated. IMPORTANCE The cell wall is essential for fungal viability and is absent from human hosts; thus, drugs disrupting cell wall biosynthesis have gained more attention. Caspofungin is a member of a new class of clinically approved echinocandin drugs to treat invasive aspergillosis by blocking β-1,3-glucan synthase, thus damaging the fungal cell wall. Here, we demonstrate that caspofungin and other cell wall stressors can induce galactosaminogalactan (GAG)-dependent biofilm formation in the human pathogen Aspergillus fumigatus. We further identified SomA as a master transcription factor playing a dual role in both biofilm formation and cell wall homeostasis. SomA plays this dual role by direct binding to a conserved motif upstream of GAG biosynthetic genes and genes involved in cell wall stress sensors, chitin synthases, and β-1,3-glucan synthase. Collectively, these findings reveal a transcriptional control pathway that integrates biofilm formation and cell wall homeostasis and suggest SomA as an attractive target for antifungal drug development.

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Deciphering cell wall integrity signalling in Aspergillus fumigatus: identification and functional characterization of cell wall stress sensors and relevant Rho GTPases

Molecular Microbiology ◽

10.1111/j.1365-2958.2011.07946.x ◽

2012 ◽

Vol 83 (3) ◽

pp. 506-519 ◽

Cited By ~ 71

Author(s):

Karl Dichtl ◽

Christoph Helmschrott ◽

Franziska Dirr ◽

Johannes Wagener

Keyword(s):

Cell Wall ◽

Aspergillus Fumigatus ◽

Rho Gtpases ◽

Functional Characterization ◽

Wall Stress ◽

Cell Wall Integrity ◽

Cell Wall Stress ◽

Stress Sensors

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Heat Shock Protein 90 Is Required for Conidiation and Cell Wall Integrity in Aspergillus fumigatus

Eukaryotic Cell ◽

10.1128/ec.00032-12 ◽

2012 ◽

Vol 11 (11) ◽

pp. 1324-1332 ◽

Cited By ~ 84

Author(s):

Frédéric Lamoth ◽

Praveen R. Juvvadi ◽

Jarrod R. Fortwendel ◽

William J. Steinbach

Keyword(s):

Cell Wall ◽

Heat Shock ◽

Heat Shock Protein ◽

Aspergillus Fumigatus ◽

Cellular Localization ◽

Fluorescent Protein ◽

Heat Shock Protein 90 ◽

Cell Wall Integrity ◽

Compensatory Mechanisms ◽

Content Type

ABSTRACTHeat shock protein 90 (Hsp90) is a eukaryotic molecular chaperone. Its involvement in the resistance ofCandida albicansto azole and echinocandin antifungals is well established. However, little is known about Hsp90's function in the filamentous fungal pathogenAspergillus fumigatus. We investigated the role of Hsp90 inA. fumigatusby genetic repression and examined its cellular localization under various stress conditions. Failure to generate a deletion strain ofhsp90suggested that it is essential. Genetic repression of Hsp90 was achieved by an inducible nitrogen-dependent promoter (pniiA-Hsp90) and led to decreased spore viability, decreased hyphal growth, and severe defects in germination and conidiation concomitant with the downregulation of the conidiation-specific transcription factorsbrlA,wetA, andabaA. Hsp90 repression potentiated the effect of cell wall inhibitors affecting the β-glucan structure of the cell wall (caspofungin, Congo red) and of the calcineurin inhibitor FK506, supporting a role in regulating cell wall integrity pathways. Moreover, compromising Hsp90 abolished the paradoxical effect of caspofungin. Pharmacological inhibition of Hsp90 by geldanamycin and its derivatives (17-AAG and 17-DMAG) resulted in similar effects. C-terminal green fluorescent protein (GFP) tagging of Hsp90 revealed mainly cytosolic distribution under standard growth conditions. However, treatment with caspofungin resulted in Hsp90 accumulation at the cell wall and at sites of septum formation, further highlighting its role in cell wall stress compensatory mechanisms. Targeting Hsp90 with fungal-specific inhibitors to cripple stress response compensatory pathways represents an attractive new antifungal strategy.

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Saccharomyces cerevisiae Heat Shock Transcription Factor Regulates Cell Wall Remodeling in Response to Heat Shock

Eukaryotic Cell ◽

10.1128/ec.4.6.1050-1056.2005 ◽

2005 ◽

Vol 4 (6) ◽

pp. 1050-1056 ◽

Cited By ~ 46

Author(s):

Hiromi Imazu ◽

Hiroshi Sakurai

Keyword(s):

Saccharomyces Cerevisiae ◽

Cell Wall ◽

Transcription Factor ◽

Heat Shock ◽

Protein Kinase ◽

Elevated Temperatures ◽

Heat Shock Transcription Factor ◽

Mitogen Activated Protein Kinase ◽

Temperature Sensitive ◽

Cell Wall Remodeling

ABSTRACT The heat shock transcription factor Hsf1 of the yeast Saccharomyces cerevisiae regulates expression of genes encoding heat shock proteins and a variety of other proteins as well. To better understand the cellular roles of Hsf1, we screened multicopy suppressor genes of a temperature-sensitive hsf1 mutation. The RIM15 gene, encoding a protein kinase that is negatively regulated by the cyclic AMP-dependent protein kinase, was identified as a suppressor, but Rim15-regulated stress-responsive transcription factors, such as Msn2, Msn4, and Gis1, were unable to rescue the temperature-sensitive growth phenotype of the hsf1 mutant. Another class of suppressors encoded cell wall stress sensors, Wsc1, Wsc2, and Mid2, and the GDP/GTP exchange factor Rom2 that interacts with these cell wall sensors. Activation of a protein kinase, Pkc1, which is induced by these cell wall sensor proteins upon heat shock, but not activation of the Pkc1-regulated mitogen-activated protein kinase cascade, was necessary for the hsf1 suppression. Like Wsc-Pkc1 pathway mutants, hsf1 cells exhibited an osmotic remedial cell lysis phenotype at elevated temperatures. Several of the other suppressors were found to encode proteins functioning in cell wall organization. These results suggest that Hsf1 in concert with Pkc1 regulates cell wall remodeling in response to heat shock.

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The Aspergillus niger MADS-box transcription factor RlmA is required for cell wall reinforcement in response to cell wall stress

Molecular Microbiology ◽

10.1111/j.1365-2958.2005.04827.x ◽

2005 ◽

Vol 58 (1) ◽

pp. 305-319 ◽

Cited By ~ 59

Author(s):

Robbert A. Damveld ◽

Mark Arentshorst ◽

Angelique Franken ◽

Patricia A. VanKuyk ◽

Frans M. Klis ◽

...

Keyword(s):

Cell Wall ◽

Transcription Factor ◽

Aspergillus Niger ◽

Wall Stress ◽

Mads Box ◽

Cell Wall Stress ◽

Cell Wall Reinforcement

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Aspergillus nidulans Protein O-Mannosyltransferases Play Roles in Cell Wall Integrity and Developmental Patterning

Eukaryotic Cell ◽

10.1128/ec.00040-09 ◽

2009 ◽

Vol 8 (10) ◽

pp. 1475-1485 ◽

Cited By ~ 33

Author(s):

Thanyanuch Kriangkripipat ◽

Michelle Momany

Keyword(s):

Cell Wall ◽

Aspergillus Nidulans ◽

Double Mutant ◽

Elevated Temperatures ◽

Cell Wall Integrity ◽

Wild Type ◽

Spatial Cues ◽

Developmental Patterning ◽

In The Wild ◽

Increased Sensitivity

ABSTRACT Protein O-mannosyltransferases (Pmts) initiate O-mannosyl glycan biosynthesis from Ser and Thr residues of target proteins. Fungal Pmts are divided into three subfamilies, Pmt1, -2, and -4. Aspergillus nidulans possesses a single representative of each Pmt subfamily, pmtA (subfamily 2), pmtB (subfamily 1), and pmtC (subfamily 4). In this work, we show that single Δpmt mutants are viable and have unique phenotypes and that the ΔpmtA ΔpmtB double mutant is the only viable double mutant. This makes A. nidulans the first fungus in which all members of individual Pmt subfamilies can be deleted without loss of viability. At elevated temperatures, all A. nidulans Δpmt mutants show cell wall-associated defects and increased sensitivity to cell wall-perturbing agents. The Δpmt mutants also show defects in developmental patterning. Germ tube emergence is early in ΔpmtA and more frequent in ΔpmtC mutants than in the wild type. In ΔpmtB mutants, intrahyphal hyphae develop. All Δpmt mutants show distinct conidiophore defects. The ΔpmtA strain has swollen vesicles and conidiogenous cells, the ΔpmtB strain has swollen conidiophore stalks, and the ΔpmtC strain has dramatically elongated conidiophore stalks. We also show that AN5660, an ortholog of Saccharomyces cerevisiae Wsc1p, is modified by PmtA and PmtC. The Δpmt phenotypes at elevated temperatures, increased sensitivity to cell wall-perturbing agents and restoration to wild-type growth with osmoticum suggest that A. nidulans Pmts modify proteins in the cell wall integrity pathway. The altered developmental patterns in Δpmt mutants suggest that A. nidulans Pmts modify proteins that serve as spatial cues.

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