Host Lung Environment Limits Aspergillus fumigatus Germination through an SskA-Dependent Signaling Response

Aspergillus fumigatus is an important human fungal pathogen particularly in immunocompromised individuals. Initiation of growth by A. fumigatus in the lung is important for its pathogenicity in murine models. ABSTRACT Aspergillus fumigatus isolates display significant heterogeneity in growth, virulence, pathology, and inflammatory potential in multiple murine models of invasive aspergillosis. Previous studies have linked the initial germination of a fungal isolate in the airways to the inflammatory and pathological potential, but the mechanism(s) regulating A. fumigatus germination in the airways is unresolved. To explore the genetic basis for divergent germination phenotypes, we utilized a serial passaging strategy in which we cultured a slow germinating strain (AF293) in a murine-lung-based medium for multiple generations. Through this serial passaging approach, a strain emerged with an increased germination rate that induces more inflammation than the parental strain (herein named LH-EVOL for lung homogenate evolved). We identified a potential loss-of-function allele of Afu5g08390 (sskA) in the LH-EVOL strain. The LH-EVOL strain had a decreased ability to induce the SakA-dependent stress pathway, similar to AF293 ΔsskA and CEA10. In support of the whole-genome variant analyses, sskA, sakA, or mpkC loss-of-function strains in the AF293 parental strain increased germination both in vitro and in vivo. Since the airway surface liquid of the lungs contains low glucose levels, the relationship of low glucose concentration on germination of these mutant AF293 strains was examined; interestingly, in low glucose conditions, the sakA pathway mutants exhibited an enhanced germination rate. In conclusion, A. fumigatus germination in the airways is regulated by SskA through the SakA mitogen-activated protein kinase (MAPK) pathway and drives enhanced disease initiation and inflammation in the lungs. IMPORTANCE Aspergillus fumigatus is an important human fungal pathogen particularly in immunocompromised individuals. Initiation of growth by A. fumigatus in the lung is important for its pathogenicity in murine models. However, our understanding of what regulates fungal germination in the lung environment is lacking. Through a serial passage experiment using lung-based medium, we identified a new strain of A. fumigatus that has increased germination potential and inflammation in the lungs. Using this serially passaged strain, we found it had a decreased ability to mediate signaling through the osmotic stress response pathway. This finding was confirmed using genetic null mutants demonstrating that the osmotic stress response pathway is critical for regulating growth in the murine lungs. Our results contribute to the understanding of A. fumigatus adaptation and growth in the host lung environment.

[1]  G. Butler,et al.  Candida pathogens induce protective mitochondria-associated type I interferon signalling and a damage-driven response in vaginal epithelial cells , 2021, Nature Microbiology.

[2]  J. Stajich,et al.  Aspergillus fumigatus In-Host HOG Pathway Mutation for Cystic Fibrosis Lung Microenvironment Persistence , 2021, bioRxiv.

[3]  R. A. Cramer,et al.  Is It Time To Kill the Survival Curve? A Case for Disease Progression Factors in Microbial Pathogenesis and Host Defense Research , 2021, mBio.

[4]  A. Mitchell,et al.  Determining Aspergillus fumigatus transcription factor expression and function during invasion of the mammalian lung , 2020, bioRxiv.

[5]  C. Schwarz,et al.  Risk factors for respiratory Aspergillus fumigatus in German Cystic Fibrosis patients and impact on lung function , 2020, Scientific Reports.

[6]  R. A. Cramer,et al.  MDA5 Is an Essential Sensor of a Pathogen-Associated Molecular Pattern Associated with Vitality That Is Necessary for Host Resistance against Aspergillus fumigatus , 2020, The Journal of Immunology.

[7]  Ashutosh Kumar Singh,et al.  The Two-Component Response Regulator Ssk1 and the Mitogen-Activated Protein Kinase Hog1 Control Antifungal Drug Resistance and Cell Wall Architecture of Candida auris , 2020, mSphere.

[8]  G. Goldman,et al.  Putative Membrane Receptors Contribute to Activation and Efficient Signaling of Mitogen-Activated Protein Kinase Cascades during Adaptation of Aspergillus fumigatus to Different Stressors and Carbon Sources , 2020, mSphere.

[9]  J. Latgé,et al.  Aspergillus fumigatus and Aspergillosis in 2019 , 2019, Clinical Microbiology Reviews.

[10]  Rafael Silva-Rocha,et al.  Aspergillus fumigatus High Osmolarity Glycerol Mitogen Activated Protein Kinases SakA and MpkC Physically Interact During Osmotic and Cell Wall Stresses , 2019, Front. Microbiol..

[11]  G. Goldman,et al.  Mitogen-Activated Protein Kinase Cross-Talk Interaction Modulates the Production of Melanins in Aspergillus fumigatus , 2019, mBio.

[12]  H. Saito,et al.  Interaction between the transmembrane domains of Sho1 and Opy2 enhances the signaling efficiency of the Hog1 MAP kinase cascade in Saccharomyces cerevisiae , 2019, PloS one.

[13]  L. Ries,et al.  Protein Kinase A and High-Osmolarity Glycerol Response Pathways Cooperatively Control Cell Wall Carbohydrate Mobilization in Aspergillus fumigatus , 2018, mBio.

[14]  J. Aguirre,et al.  SakA and MpkC Stress MAPKs Show Opposite and Common Functions During Stress Responses and Development in Aspergillus nidulans , 2018, Front. Microbiol..

[15]  S. Johnston,et al.  Role of airway glucose in bacterial infections in patients with chronic obstructive pulmonary disease , 2018, The Journal of allergy and clinical immunology.

[16]  J. Flowers,et al.  Origins and geographic diversification of African rice (Oryza glaberrima) , 2018, bioRxiv.

[17]  A. Huttenlocher,et al.  Macrophages inhibit Aspergillus fumigatus germination and neutrophil-mediated fungal killing , 2018, PLoS pathogens.

[18]  J. Latgé,et al.  Penetration of the Human Pulmonary Epithelium by Aspergillus fumigatus Hyphae , 2018, The Journal of infectious diseases.

[19]  Cristina Aurrecoechea,et al.  FungiDB: An Integrated Bioinformatic Resource for Fungi and Oomycetes , 2018, Journal of fungi.

[20]  R. A. Cramer,et al.  Host-Derived Leukotriene B4 Is Critical for Resistance against Invasive Pulmonary Aspergillosis , 2018, Front. Immunol..

[21]  Mauricio O. Carneiro,et al.  Scaling accurate genetic variant discovery to tens of thousands of samples , 2017, bioRxiv.

[22]  S. Kotenko,et al.  Type III interferon is a critical regulator of innate antifungal immunity , 2017, Science Immunology.

[23]  T. Hohl,et al.  Interleukin 1α Is Critical for Resistance against Highly Virulent Aspergillus fumigatus Isolates , 2017, Infection and Immunity.

[24]  M. Netea,et al.  Aspergillus fumigatus morphology and dynamic host interactions , 2017, Nature Reviews Microbiology.

[25]  D. Baines,et al.  Airway Glucose Homeostasis: A New Target in the Prevention and Treatment of Pulmonary Infection , 2017, Chest.

[26]  A. Knutsen.  Allergic bronchopulmonary aspergillosis in asthma , 2017, Expert review of clinical immunology.

[27]  K. Kalsi,et al.  Hyperglycaemia and Pseudomonas aeruginosa acidify cystic fibrosis airway surface liquid by elevating epithelial monocarboxylate transporter 2 dependent lactate-H+ secretion , 2016, Scientific Reports.

[28]  J. Panepinto,et al.  Opposing PKA and Hog1 signals control the post‐transcriptional response to glucose availability in Cryptococcus neoformans , 2016, Molecular microbiology.

[29]  D. Baines,et al.  Increased airway glucose increases airway bacterial load in hyperglycaemia , 2016, Scientific Reports.

[30]  G. Goldman,et al.  Mitogen activated protein kinases SakAHOG1 and MpkC collaborate for Aspergillus fumigatus virulence , 2016, Molecular microbiology.

[31]  Sarah R Beattie,et al.  RbdB, a Rhomboid Protease Critical for SREBP Activation and Virulence in Aspergillus fumigatus , 2016, mSphere.

[32]  L. Goulart,et al.  SGLT1 activity in lung alveolar cells of diabetic rats modulates airway surface liquid glucose concentration and bacterial proliferation , 2016, Scientific Reports.

[33]  Ping Xu,et al.  Robust network structure of the Sln1-Ypd1-Ssk1 three-component phospho-relay prevents unintended activation of the HOG MAPK pathway in Saccharomyces cerevisiae , 2015, BMC Systems Biology.

[34]  B. Barker,et al.  IL-1α Signaling Is Critical for Leukocyte Recruitment after Pulmonary Aspergillus fumigatus Challenge , 2015, PLoS pathogens.

[35]  Heng Li,et al.  Toward better understanding of artifacts in variant calling from high-coverage samples , 2014, Bioinform..

[36]  A. Yoshimi,et al.  NikA/TcsC Histidine Kinase Is Involved in Conidiation, Hyphal Morphology, and Responses to Osmotic Stress and Antifungal Chemicals in Aspergillus fumigatus , 2013, PloS one.

[37]  Mauricio O. Carneiro,et al.  From FastQ Data to High‐Confidence Variant Calls: The Genome Analysis Toolkit Best Practices Pipeline , 2013, Current protocols in bioinformatics.

[38]  D. Cavalieri,et al.  Strain Dependent Variation of Immune Responses to A. fumigatus: Definition of Pathogenic Species , 2013, PloS one.

[39]  P. Mayinger,et al.  Metabolic Activation of the HOG MAP Kinase Pathway by Snf1/AMPK Regulates Lipid Signaling at the Golgi , 2012, Traffic.

[40]  D. Baines,et al.  Sweet talk: insights into the nature and importance of glucose transport in lung epithelium , 2012, European Respiratory Journal.

[41]  K. Kalsi,et al.  Proinflammatory Mediators Disrupt Glucose Homeostasis in Airway Surface Liquid , 2012, The Journal of Immunology.

[42]  N. Høiby,et al.  Phenotypes selected during chronic lung infection in cystic fibrosis patients: implications for the treatment of Pseudomonas aeruginosa biofilm infections. , 2012, FEMS immunology and medical microbiology.

[43]  J. Latgé,et al.  TLR3 essentially promotes protective class I-restricted memory CD8⁺ T-cell responses to Aspergillus fumigatus in hematopoietic transplanted patients. , 2012, Blood.

[44]  S. Randell,et al.  Localization and activity of the calcineurin catalytic and regulatory subunit complex at the septum is essential for hyphal elongation and proper septation in Aspergillus fumigatus , 2011, Molecular microbiology.

[45]  Brian P. Brunk,et al.  FungiDB: an integrated functional genomics database for fungi , 2011, Nucleic Acids Res..

[46]  R. Bals,et al.  Aspergillus fumigatus conidia induce interferon-&bgr; signalling in respiratory epithelial cells , 2011, European Respiratory Journal.

[47]  Jeffrey M. Macdonald,et al.  In vivo Hypoxia and a Fungal Alcohol Dehydrogenase Influence the Pathogenesis of Invasive Pulmonary Aspergillosis , 2011, PLoS pathogens.

[48]  J. Baddley.  Clinical risk factors for invasive aspergillosis. , 2011, Medical mycology.

[49]  K. Wikenheiser-Brokamp,et al.  Divergent Protein Kinase A isoforms co‐ordinately regulate conidial germination, carbohydrate metabolism and virulence in Aspergillus fumigatus , 2011, Molecular microbiology.

[50]  K. Rahmouni,et al.  Glucose Depletion in the Airway Surface Liquid Is Essential for Sterility of the Airways , 2011, PloS one.

[51]  Jae-Hyuk Yu,et al.  Gβγ-mediated growth and developmental control in Aspergillus fumigatus , 2009, Current Genetics.

[52]  Gonçalo R. Abecasis,et al.  The Sequence Alignment/Map format and SAMtools , 2009, Bioinform..

[53]  J. Heitman,et al.  Calcineurin Target CrzA Regulates Conidial Germination, Hyphal Growth, and Pathogenesis of Aspergillus fumigatus , 2008, Eukaryotic Cell.

[54]  J. Quinn,et al.  A single MAPKKK regulates the Hog1 MAPK pathway in the pathogenic fungus Candida albicans. , 2007, Molecular biology of the cell.

[55]  Yi Xiong,et al.  Fusion PCR and gene targeting in Aspergillus nidulans , 2006, Nature Protocols.

[56]  G. May,et al.  Novel Mitogen-Activated Protein Kinase MpkC of Aspergillus fumigatus Is Required for Utilization of Polyalcohol Sugars , 2006, Eukaryotic Cell.

[57]  M. Millar,et al.  Comparison of DNA extraction methods for Aspergillus fumigatus using real-time PCR. , 2006, Journal of medical microbiology.

[58]  Wei Zhao,et al.  Deletion of the Regulatory Subunit of Protein Kinase A in Aspergillus fumigatus Alters Morphology, Sensitivity to Oxidative Damage, andVirulence , 2006, Infection and Immunity.

[59]  C. d’Enfert,et al.  The Heterotrimeric G-Protein GanB(α)-SfaD(β)-GpgA(γ) Is a Carbon Source Sensor Involved in Early cAMP-Dependent Germination in Aspergillus nidulans , 2005, Genetics.

[60]  G. May,et al.  A Mitogen-Activated Protein Kinase That Senses Nitrogen Regulates Conidial Germination and Growth in Aspergillus fumigatus , 2004, Eukaryotic Cell.

[61]  Thomas D. Schmittgen,et al.  Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. , 2001, Methods.

[62]  N. Osherov,et al.  Conidial germination in Aspergillus nidulans requires RAS signaling and protein synthesis. , 2000, Genetics.

[63]  J. Latgé,et al.  Aspergillus fumigatus and Aspergillosis , 1999, Clinical Microbiology Reviews.

[64]  Masatoshi Suzuki,et al.  Production of Mice Deficient in Genes for Interleukin (IL)-1α, IL-1β, IL-1α/β, and IL-1 Receptor Antagonist Shows that IL-1β Is Crucial in Turpentine-induced Fever Development and Glucocorticoid Secretion , 1998, The Journal of experimental medicine.

[65]  Francesc Posas,et al.  Activation of the yeast SSK2 MAP kinase kinase kinase by the SSK1 two‐component response regulator , 1998, The EMBO journal.

[66]  Francesc Posas,et al.  Yeast HOG1 MAP Kinase Cascade Is Regulated by a Multistep Phosphorelay Mechanism in the SLN1–YPD1–SSK1 “Two-Component” Osmosensor , 1996, Cell.

[67]  J. Tayek,et al.  Weight loss and diabetes are new risk factors for the development of invasive aspergillosis infection in non-immunocompromized humans. , 2017, Clinical practice.

[68]  Thomas D. Schmittgen,et al.  Analysis of Relative Gene Expression Data Using Real-Time Quantitative PCR and the 2 2 DD C T Method , 2022 .