Molecular Mechanisms of Pathogenicity of 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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Transcriptional Regulation of Chitin Synthases by Calcineurin Controls Paradoxical Growth of Aspergillus fumigatus in Response to Caspofungin

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00854-09 ◽

2010 ◽

Vol 54 (4) ◽

pp. 1555-1563 ◽

Cited By ~ 103

Author(s):

Jarrod R. Fortwendel ◽

Praveen R. Juvvadi ◽

B. Zachary Perfect ◽

Luise E. Rogg ◽

John R. Perfect ◽

...

Keyword(s):

Aspergillus Fumigatus ◽

Molecular Mechanisms ◽

Laboratory Strain ◽

Chitin Synthase ◽

High Dose ◽

Paradoxical Effect ◽

Paradoxical Response ◽

Paradoxical Growth ◽

High Concentrations ◽

Calcineurin Signaling

ABSTRACT Attenuated activity of echinocandin antifungals at high concentrations, known as the “paradoxical effect,” is a well-established phenomenon in Candida albicans and Aspergillus fumigatus. In the yeast C. albicans, upregulation of chitin biosynthesis via the protein kinase C (PKC), high-osmolarity glycerol response (HOG), and Ca2+/calcineurin signaling pathways is an important cell wall stress response that permits growth in the presence of high concentrations of echinocandins. However, nothing is known of the molecular mechanisms regulating the mold A. fumigatus and its paradoxical response to echinocandins. Here, we show that the laboratory strain of A. fumigatus and five of seven clinical A. fumigatus isolates tested display various magnitudes of paradoxical growth in response to caspofungin. Interestingly, none of the eight strains showed paradoxical growth in the presence of micafungin or anidulafungin. Treatment of the ΔcnaA and ΔcrzA strains, harboring gene deletions of the calcineurin A subunit and the calcineurin-dependent transcription factor, respectively, with high concentrations of caspofungin revealed that the A. fumigatus paradoxical effect is calcineurin pathway dependent. Exploring a molecular role for CnaA in the compensatory chitin biosynthetic response, we found that caspofungin treatment resulted in increased chitin synthase gene expression, leading to a calcineurin-dependent increase in chitin synthase activity. Taken together, our data suggest a mechanistic role for A. fumigatus calcineurin signaling in the chitin biosynthetic response observed during paradoxical growth in the presence of high-dose caspofungin treatment.

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Insights in the molecular mechanisms of an azole stress adapted laboratory-generated Aspergillus fumigatus strain

Medical Mycology ◽

10.1093/mmy/myaa118 ◽

2021 ◽

Author(s):

Marion Aruanno ◽

Samantha Gozel ◽

Isabelle Mouyna ◽

Josie E Parker ◽

Daniel Bachmann ◽

...

Keyword(s):

Transcription Factor ◽

Aspergillus Fumigatus ◽

Molecular Mechanisms ◽

Drug Transporters ◽

Major Facilitator Superfamily ◽

Ergosterol Biosynthesis ◽

Azole Resistance ◽

Drug Transporter

Abstract Aspergillus fumigatus is the main cause of invasive aspergillosis, for which azole drugs are the first-line therapy. Emergence of pan-azole resistance among A. fumigatus is concerning and has been mainly attributed to mutations in the target gene (cyp51A). However, azole resistance may also result from other mutations (hmg1, hapE) or other adaptive mechanisms. We performed microevolution experiment exposing an A. fumigatus azole-susceptible strain (Ku80) to sub-minimal inhibitory concentration of voriconazole to analyze emergence of azole resistance. We obtained a strain with pan-azole resistance (Ku80R), which was partially reversible after drug relief, and without mutations in cyp51A, hmg1, and hapE. Transcriptomic analyses revealed overexpression of the transcription factor asg1, several ATP-binding cassette (ABC) and major facilitator superfamily transporters and genes of the ergosterol biosynthesis pathway in Ku80R. Sterol analysis showed a significant decrease of the ergosterol mass under voriconazole exposure in Ku80, but not in Ku80R. However, the proportion of the sterol compounds was similar between both strains. To further assess the role of transporters, we used the ABC transporter inhibitor milbemycine oxime (MLB). MLB inhibited transporter activity in both Ku80 and Ku80R and demonstrated some potentiating effect on azole activity. Criteria for synergism were reached for MLB and posaconazole against Ku80. Finally, deletion of asg1 revealed some role of this transcription factor in controlling drug transporter expression, but had no impact on azole susceptibility. This work provides further insight in mechanisms of azole stress adaptation and suggests that drug transporters inhibition may represent a novel therapeutic target. Lay Summary A pan-azole-resistant strain was generated in vitro, in which drug transporter overexpression was a major trait. Analyses suggested a role of the transporter inhibitor milbemycin oxime in inhibiting drug transporters and potentiating azole activity.

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Resistance profiling of Aspergillus fumigatus to olorofim indicates absence of intrinsic resistance and unveils the molecular mechanisms of acquired olorofim resistance

10.1101/2021.11.16.468917 ◽

2021 ◽

Author(s):

Jochem Berend Buil ◽

Jason D Oliver ◽

Derek Law ◽

Tim J. H. Baltussen ◽

Jan Zoll ◽

...

Keyword(s):

Growth Rate ◽

Aspergillus Fumigatus ◽

Molecular Mechanisms ◽

Dihydroorotate Dehydrogenase ◽

Intrinsic Resistance ◽

Resistance Development ◽

Resistant Phenotype ◽

Pyre Gene

Olorofim (F901318) is a new antifungal currently under clinical development that shows both in vitro and in vivo activity against a number of filamentous fungi including Aspergillus fumigatus. In this study we screened A. fumigatus isolates for intrinsic olorofim-resistant A. fumigatus and evaluated the ability of A. fumigatus to acquire an olorofim-resistant phenotype. No intrinsic resistance was found in 975 clinical A. fumigatus isolates. However, we found that isolates with increased olorofim MICs (> 8 mg/L) could be selected using a high number of conidia and olorofim exposure under laboratory conditions. Assessment of the frequency of acquired olorofim resistance development of A. fumigatus was shown to be higher than for voriconazole but lower than for itraconazole. Sequencing the PyrE gene of isogenic isolates with olorofim MICs of >8 mg/L identified various amino acid substitutions with a hotspot at locus G119. Olorofim was shown to have reduced affinity to mutated target protein dihydroorotate dehydrogenase (DHODH) and the effect of these mutations were proven by introducing the mutations directly in A. fumigatus. We then investigated whether G119 mutations were associated with a fitness cost in A. fumigatus. These experiments showed a small but significant reduction in growth rate for strains with a G119V substitution, while strains with a G119C substitution did not exhibit a reduction in growth rate. These in vitro findings were confirmed in an in vivo pathogenicity model.

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The Protein Kinase A-Dependent Phosphoproteome of the Human Pathogen Aspergillus fumigatus Reveals Diverse Virulence-Associated Kinase Targets

mBio ◽

10.1128/mbio.02880-20 ◽

2020 ◽

Vol 11 (6) ◽

pp. e02880-20

Author(s):

E. Keats Shwab ◽

Praveen R. Juvvadi ◽

Greg Waitt ◽

Shareef Shaheen ◽

John Allen ◽

...

Keyword(s):

Infectious Disease ◽

Protein Kinase ◽

Aspergillus Fumigatus ◽

Protein Kinase A ◽

Molecular Mechanisms ◽

Targeted Mutagenesis ◽

Human Pathogen ◽

Content Type ◽

Kinase A ◽

Microbial Disease

ABSTRACTProtein kinase A (PKA) signaling plays a critical role in the growth and development of all eukaryotic microbes. However, few direct targets have been characterized in any organism. The fungus Aspergillus fumigatus is a leading infectious cause of death in immunocompromised patients, but the specific molecular mechanisms responsible for its pathogenesis are poorly understood. We used this important pathogen as a platform for a comprehensive and multifaceted interrogation of both the PKA-dependent whole proteome and phosphoproteome in order to elucidate the mechanisms through which PKA signaling regulates invasive microbial disease. Employing advanced quantitative whole-proteomic and phosphoproteomic approaches with two complementary phosphopeptide enrichment strategies, coupled to an independent PKA interactome analysis, we defined distinct PKA-regulated pathways and identified novel direct PKA targets contributing to pathogenesis. We discovered three previously uncharacterized virulence-associated PKA effectors, including an autophagy-related protein, Atg24; a CCAAT-binding transcriptional regulator, HapB; and a CCR4-NOT complex-associated ubiquitin ligase, Not4. Targeted mutagenesis, combined with in vitro kinase assays, multiple murine infection models, structural modeling, and molecular dynamics simulations, was employed to characterize the roles of these new PKA targets in growth, environmental and antimicrobial stress responses, and pathogenesis in a mammalian system. We also elucidated the molecular mechanisms of PKA regulation for these effectors by defining the functionality of phosphorylation at specific PKA target sites. We have comprehensively characterized the PKA-dependent phosphoproteome and validated PKA targets as direct regulators of infectious disease for the first time in any pathogen, providing new insights into PKA signaling and control over microbial pathogenesis.IMPORTANCE PKA is essential for the virulence of eukaryotic human pathogens. Understanding PKA signaling mechanisms is therefore fundamental to deciphering pathogenesis and developing novel therapies. Despite its ubiquitous necessity, specific PKA effectors underlying microbial disease remain unknown. To address this fundamental knowledge gap, we examined the whole-proteomic and phosphoproteomic impacts of PKA on the deadly fungal pathogen Aspergillus fumigatus to uncover novel PKA targets controlling growth and virulence. We also defined the functional consequences of specific posttranslational modifications of these target proteins to characterize the molecular mechanisms of pathogenic effector regulation by PKA. This study constitutes the most comprehensive analysis of the PKA-dependent phosphoproteome of any human pathogen and proposes new and complex roles played by PKA signaling networks in governing infectious disease.

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Gliotoxin Suppresses Macrophage Immune Function by Subverting Phosphatidylinositol 3,4,5-Trisphosphate Homeostasis

mBio ◽

10.1128/mbio.02242-15 ◽

2016 ◽

Vol 7 (2) ◽

Cited By ~ 29

Author(s):

Daniel Schlam ◽

Johnathan Canton ◽

Marvin Carreño ◽

Hannah Kopinski ◽

Spencer A. Freeman ◽

...

Keyword(s):

Aspergillus Fumigatus ◽

Molecular Mechanisms ◽

Fungal Spores ◽

Therapeutic Interventions ◽

Actin Dynamics ◽

Phosphoinositide Metabolism ◽

Pulmonary Tissue ◽

Phagocytic Capacity ◽

Mechanisms Of Toxicity ◽

Healthy Humans

ABSTRACT Aspergillus fumigatus , an opportunistic fungal pathogen, spreads in the environment by releasing numerous conidia that are capable of reaching the small alveolar airways of mammalian hosts. In otherwise healthy individuals, macrophages are responsible for rapidly phagocytosing and eliminating these conidia, effectively curbing their germination and consequent invasion of pulmonary tissue. However, under some circumstances, the fungus evades phagocyte-mediated immunity and persists in the respiratory tree. Here, we report that A. fumigatus escapes macrophage recognition by strategically targeting phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P 3 ] metabolism through gliotoxin, a potent immunosuppressive mycotoxin. Time-lapse microscopy revealed that, in response to the toxin, macrophages cease to ruffle, undergo abrupt membrane retraction, and fail to phagocytose large targets effectively. Gliotoxin was found to prevent integrin activation and interfere with actin dynamics, both of which are instrumental for phagocytosis; similar effects were noted in immortalized and primary phagocytes. Detailed studies of the underlying molecular mechanisms of toxicity revealed that inhibition of phagocytosis is attributable to impaired accumulation of PtdIns(3,4,5)P 3 and the associated dysregulation of downstream effectors, including Rac and/or Cdc42. Strikingly, in response to the diacylglycerol mimetic phorbol 12-myristate 13-acetate, gliotoxin-treated macrophages reactivate beta integrins, reestablish actin dynamics, and regain phagocytic capacity, despite the overt absence of plasmalemmal PtdIns(3,4,5)P 3 . Together, our findings identify phosphoinositide metabolism as a critical upstream target of gliotoxin and also indicate that increased diacylglycerol levels can bypass the requirement for PtdIns(3,4,5)P 3 signaling during membrane ruffling and phagocytosis. IMPORTANCE Aspergillus fumigatus is the most frequent cause of human infections in the Aspergillus genus. In immunocompromised populations, invasive aspergillosis (IA) is associated with a mortality rate of up to 90%, and current antifungal therapies have failed to prevent or reverse the infection. Therefore, a deeper understanding of the interactions between A. fumigatus and its host is required. In healthy humans, alveolar macrophages can ingest and eliminate fungal spores, thus limiting their germination into mycotoxin-producing hyphae. Our studies reveal that gliotoxin—the most abundant Aspergillus mycotoxin—undermines the ability of phagocytes to carry out their protective functions. By targeting PtdIns(3,4,5)P 3 signaling and downregulating phagocytic immune defenses, the toxin could also exacerbate polymicrobial infections. Notably, we were able to reverse gliotoxin toxicity by addition of diacylglycerol analogues, which may provide the basis for therapeutic interventions.

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Growth of Aspergillus fumigatus in Biofilms in Comparison to Candida albicans

Journal of Fungi ◽

10.3390/jof8010048 ◽

2022 ◽

Vol 8 (1) ◽

pp. 48

Author(s):

Eefje Subroto ◽

Jacq van Neer ◽

Ivan Valdes ◽

Hans de Cock

Keyword(s):

Aspergillus Fumigatus ◽

Defense Mechanisms ◽

Molecular Mechanisms ◽

Fungal Infections ◽

Opportunistic Pathogen ◽

Immune Recognition ◽

Genetic Changes ◽

Host Environment ◽

Cell Dispersion ◽

Protective Environment

Biofilm formation during infections with the opportunistic pathogen Aspergillus fumigatus can be very problematic in clinical settings, since it provides the fungal cells with a protective environment. Resistance against drug treatments, immune recognition as well as adaptation to the host environment allows fungal survival in the host. The exact molecular mechanisms behind most processes in the formation of biofilms are unclear. In general, the formation of biofilms can be categorized roughly in a few stages; adhesion, conidial germination and development of hyphae, biofilm maturation and cell dispersion. Fungi in biofilms can adapt to the in-host environment. These adaptations can occur on a level of phenotypic plasticity via gene regulation. However, also more substantial genetic changes of the genome can result in increased resistance and adaptation in the host, enhancing the survival chances of fungi in biofilms. Most research has focused on the development of biofilms. However, to tackle developing microbial resistance and adaptation in biofilms, more insight in mechanisms behind genetic adaptations is required to predict which defense mechanisms can be expected. This can be helpful in the development of novel and more targeted antifungal treatments to combat fungal infections.

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Heterogeneity in the transcriptional response of the human pathogen Aspergillus fumigatus to the antifungal agent caspofungin

Genetics ◽

10.1093/genetics/iyab183 ◽

2021 ◽

Author(s):

Ana Cristina Colabardini ◽

Fang Wang ◽

Zhiqiang Dong ◽

Lakhansing Pardeshi ◽

Marina Campos Rocha ◽

...

Keyword(s):

Cell Wall ◽

Transcription Factor ◽

Aspergillus Fumigatus ◽

Antifungal Agent ◽

Iron Homeostasis ◽

Molecular Mechanisms ◽

Target Genes ◽

Invasive Pulmonary Aspergillosis ◽

Transcriptional Response ◽

Cell Wall Remodeling

Abstract Aspergillus fumigatus is the main causative agent of invasive pulmonary aspergillosis (IPA), a severe disease that affects immunosuppressed patients worldwide. The fungistatic drug caspofungin is the second line of therapy against IPA but has increasingly been used against clinical strains that are resistant to azoles, the first line antifungal therapy. In high concentrations, caspofungin induces a tolerance phenotype with partial reestablishment of fungal growth called caspofungin paradoxical effect (CPE), resulting from a change in the composition of the cell wall. An increasing number of studies has shown that different isolates of A. fumigatus exhibit phenotypic heterogeneity, including heterogeneity in their CPE response. To gain insights into the underlying molecular mechanisms of CPE response heterogeneity, we analyzed the transcriptomes of two A. fumigatus reference strains, Af293 and CEA17, exposed to low and high caspofungin concentrations. We found that there is a core transcriptional response that involves genes related to cell wall remodeling processes, mitochondrial function, transmembrane transport, and amino acid and ergosterol metabolism, and a variable response related to secondary metabolite (SM) biosynthesis and iron homeostasis. Specifically, we show here that the overexpression of a SM pathway that works as an iron chelator extinguishes the CPE in both backgrounds, whereas iron depletion is detrimental for the CPE in Af293 but not in CEA17. We next investigated the function of the transcription factor CrzA, whose deletion was previously shown to result in heterogeneity in the CPE response of the Af293 and CEA17 strains. We found that CrzA constitutively binds to and modulates the expression of several genes related to processes involved in caspofungin tolerance, and that crzA deletion differentially impacts the SM production and growth of Af293 and CEA17. As opposed to the ΔcrzACEA17 mutant, the ΔcrzA Af293 mutant fails to activate cell wall remodeling genes upon caspofungin exposure, which most likely severely affects its macrostructure and extinguishes its CPE. This work describes how heterogeneity in the response to an antifungal agent between A. fumigatus strains stems from heterogeneity in the function of a transcription factor and its downstream target genes.

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A heterogeneously expressed gene family modulates biofilm architecture and hypoxic growth of Aspergillus fumigatus

10.1101/2020.12.23.424276 ◽

2020 ◽

Author(s):

Caitlin H. Kowalski ◽

Kaesi A. Morelli ◽

Jason E. Stajich ◽

Carey D. Nadell ◽

Robert A. Cramer

Keyword(s):

Aspergillus Fumigatus ◽

Gene Family ◽

Molecular Mechanisms ◽

Genetic Manipulation ◽

Large Population ◽

Colony Morphology ◽

Low Oxygen ◽

Biofilm Architecture ◽

Biofilm Development ◽

And Function

AbstractThe genus Aspergillus encompasses human pathogens such as Aspergillus fumigatus and industrial powerhouses such as Aspergillus niger. In both cases, Aspergillus biofilms have consequences for infection outcomes and yields of economically important products. Yet, the molecular components influencing filamentous fungal biofilm development, structure, and function remain ill-defined. Macroscopic colony morphology is an indicator of underlying biofilm architecture and fungal physiology. A hypoxia-locked colony morphotype of A. fumigatus has abundant colony furrows that coincide with a reduction in vertically-oriented hyphae within biofilms and increased low oxygen growth and virulence. Investigation of this morphotype has led to the identification of the causative gene, biofilm architecture factor A (bafA), a small cryptic open reading frame within a subtelomeric gene cluster. BafA is sufficient to induce the hypoxia-locked colony morphology and biofilm architecture in A. fumigatus. Analysis across a large population of A. fumigatus isolates identified a larger family of baf genes, all of which have the capacity to modulate hyphal architecture, biofilm development, and hypoxic growth. Furthermore, introduction of A. fumigatus bafA into A. niger is sufficient to generate the hypoxia-locked colony morphology, biofilm architecture, and increased hypoxic growth. Together these data indicate the potential broad impacts of this previously uncharacterized family of small genes to modulate biofilm architecture and function in clinical and industrial settings.ImportanceThe manipulation of microbial biofilms in industrial and clinical applications remains a difficult task. The problem is particularly acute with regard to filamentous fungal biofilms for which molecular mechanisms of biofilm formation, maintenance, and function are only just being elucidated. Here we describe a family of small genes heterogeneously expressed across Aspergillus fumigatus strains that are capable of modifying colony biofilm morphology and microscopic hyphal architecture. Specifically, these genes are implicated in the formation of a hypoxia-locked colony morphotype that is associated with increased virulence of A. fumigatus. Synthetic introduction of these gene family members, here referred to as biofilm architecture factors, in both A. fumigatus and A. niger additionally modulates low oxygen growth and surface adherence. Thus, these genes are candidates for genetic manipulation of biofilm development in Aspergilli.

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Antifungal Resistance: Specific Focus on Multidrug Resistance in Candida auris and Secondary Azole Resistance in Aspergillus fumigatus

Journal of Fungi ◽

10.3390/jof4040129 ◽

2018 ◽

Vol 4 (4) ◽

pp. 129 ◽

Cited By ~ 16

Author(s):

Sevtap Arikan-Akdagli ◽

Mahmoud Ghannoum ◽

Jacques Meis

Keyword(s):

Aspergillus Fumigatus ◽

Molecular Mechanisms ◽

Multidrug Resistant ◽

Fungal Species ◽

Antifungal Resistance ◽

Current Status ◽

Clinical Implications ◽

Azole Resistance ◽

Candida Auris

Antifungal resistance is a topic of concern, particularly for specific fungal species and drugs. Among these are the multidrug-resistant Candida auris and azole-resistant Aspergillus fumigatus. While the knowledge on molecular mechanisms of resistance is now accumulating, further data are also available for the clinical implications and the extent of correlation of in vitro resistance to clinical outcomes. This review article summarizes the epidemiology of C. auris infections, animal models focusing on the activity of novel antifungal compounds in C. auris infections, virulence factors, and the mechanisms of antifungal resistance for this multi-resistant Candida species. Regarding A. fumigatus, the significance of azoles in the treatment of A. fumigatus infections, reference methods available for the detection of resistance in vitro, molecular mechanisms of secondary azole resistance, routes of acquisition, and clinical implications of in vitro resistance are covered to provide guidance for the current status of azole resistance in A. fumigatus.

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