Aspergillus fumigatus calcium-responsive transcription factors regulate cell wall architecture promoting stress tolerance, virulence and caspofungin resistance

ABSTRACT Aspergillus fumigatus is the leading cause of pulmonary fungal diseases. Azoles have been used for many years as the main antifungal agents to treat and prevent invasive aspergillosis. However, in the last 10 years there have been several reports of azole resistance in A. fumigatus and new strategies are needed to combat invasive aspergillosis. Caspofungin is effective against other human-pathogenic fungal species, but it is fungistatic only against A. fumigatus. Resistance to caspofungin in A. fumigatus has been linked to mutations in the fksA gene that encodes the target enzyme of the drug β-1,3-glucan synthase. However, tolerance of high caspofungin concentrations, a phenomenon known as the caspofungin paradoxical effect (CPE), is also important for subsequent adaptation and drug resistance evolution. Here, we identified and characterized the transcription factors involved in the response to CPE by screening an A. fumigatus library of 484 null transcription factors (TFs) in CPE drug concentrations. We identified 11 TFs that had reduced CPE and that encoded proteins involved in the basal modulation of the RNA polymerase II initiation sites, calcium metabolism, and cell wall remodeling. One of these TFs, FhdA, was important for mitochondrial respiratory function and iron metabolism. The ΔfhdA mutant showed decreased growth when exposed to Congo red or to high temperature. Transcriptome sequencing (RNA-seq) analysis and further experimental validation indicated that the ΔfhdA mutant showed diminished respiratory capacity, probably affecting several pathways related to the caspofungin tolerance and resistance. Our results provide the foundation to understand signaling pathways that are important for caspofungin tolerance and resistance. IMPORTANCE Aspergillus fumigatus, one of the most important human-pathogenic fungal species, is able to cause aspergillosis, a heterogeneous group of diseases that presents a wide range of clinical manifestations. Invasive pulmonary aspergillosis is the most serious pathology in terms of patient outcome and treatment, with a high mortality rate ranging from 50% to 95% primarily affecting immunocompromised patients. Azoles have been used for many years as the main antifungal agents to treat and prevent invasive aspergillosis. However, there were several reports of evolution of clinical azole resistance in the last decade. Caspofungin, a noncompetitive β-1,3-glucan synthase inhibitor, has been used against A. fumigatus, but it is fungistatic and is recommended as second-line therapy for invasive aspergillosis. More information about caspofungin tolerance and resistance is necessary in order to refine antifungal strategies that target the fungal cell wall. Here, we screened a transcription factor (TF) deletion library for TFs that can mediate caspofungin tolerance and resistance. We have identified 11 TFs that are important for caspofungin sensitivity and/or for the caspofungin paradoxical effect (CPE). These TFs encode proteins involved in the basal modulation of the RNA polymerase II initiation sites, calcium metabolism or cell wall remodeling, and mitochondrial respiratory function. The study of those genes regulated by TFs identified in this work will provide a better understanding of the signaling pathways that are important for caspofungin tolerance and resistance.

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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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Reprogramming of ethanol stress response in S. cerevisiae by the transcription factor Znf1 and the effect on the biosynthesis of glycerol and ethanol

Applied and Environmental Microbiology ◽

10.1128/aem.00588-21 ◽

2021 ◽

Author(s):

Wiwan Samakkarn ◽

Khanok Ratanakhanokchai ◽

Nitnipa Soontorngun

Keyword(s):

Cell Wall ◽

Transcription Factor ◽

Transcription Factors ◽

Stress Response ◽

Ethanol Production ◽

Stress Tolerance ◽

Target Genes ◽

Stress Adaptation ◽

Ethanol Stress ◽

High Ethanol

High ethanol can severely inhibit the growth of yeast cells and fermentation productivity. The ethanologenic yeast Saccharomyces cerevisiae activates several well-defined cellular mechanisms of ethanol stress response (ESR); however, the involved regulatory control remains to be characterized. Here, we report a new transcription factor of ethanol stress adaptation called Znf1. It plays a central role in ESR by activating genes for glycerol and fatty acid production ( GUP1 , GPP1/2 , GPD1 , GAT1 , and OLE1 ) to preserve plasma membrane integrity. Importantly, Znf1 also activates genes implicated in cell wall biosynthesis ( FKS1 , SED1 , and SMI1 ) and the unfolded protein response ( HSP30/104 , KAR1 , and LHS1 ) to protect cells from proteotoxic stress. The znf1 Δ strain displays increased sensitivity to ethanol, ER-stressor β-mercaptoethanol, and cell wall-perturbing agent calcofluor white. To compensate for defective cell wall, the strain lacking ZNF1 or its target SMI1 displays increased glycerol levels of 16.4% and 29.2%, respectively. Znf1 collectively regulates an intricate network of target genes essential for growth, protein refolding, and production of key metabolites. Overexpression of ZNF1 not only confers tolerance to high ethanol but also increases ethanol production by 4.6% (8.43 g/L) or 2.8% (75.78 g/L) when using 2% or 20% (w/v) glucose as a substrate, respectively, compared to the wild-type strain. Mutually, stress-responsive transcription factors Msn2/4, Hsf1, and Yap1 are associated with some promoters of Znf1’s target genes to promote ethanol stress tolerance. In conclusion, this work has implicated the novel regulator Znf1 in coordinating expression of ESR genes and illuminates the unifying transcriptional reprogramming during alcoholic fermentation. IMPORTANCE The yeast S. cerevisiae is a major microbe widely used in food and non-food industries. However, accumulation of ethanol has a negative effect on its growth and limits ethanol production. Znf1 transcription factor has been implicated in utilization of different carbon sources, including glucose, the most abundant sugar on earth, and non-fermentable substrates, as a key regulator of glycolysis and gluconeogenesis. Here, role of Znf1 in ethanol stress response is defined. Znf1 actively reprograms expression of genes linked to UPR, heat shock response, glycerol and carbohydrate metabolism, and biosyntheses of cell membrane and cell wall components. A complex interplay among transcription factors of ESR indicates transcriptional fine-tuning as the main mechanism of stress adaptation, and Znf1 plays a major regulatory role in the coordination. Understanding the adaptive ethanol stress mechanism is crucial to engineering robust yeast strains for enhanced stress tolerance or increased ethanol production.

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Genetic and structural validation of phosphomannomutase as a cell wall target in Aspergillus fumigatus

Molecular Microbiology ◽

10.1111/mmi.14706 ◽

2021 ◽

Cited By ~ 1

Author(s):

Yuanwei Zhang ◽

Wenxia Fang ◽

Olawale G. Raimi ◽

Deborah E. A. Lockhart ◽

Andrew T. Ferenbach ◽

...

Keyword(s):

Cell Wall ◽

Aspergillus Fumigatus ◽

A Cell ◽

Structural Validation

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MYB Transcription Factors and Its Regulation in Secondary Cell Wall Formation and Lignin Biosynthesis during Xylem Development

International Journal of Molecular Sciences ◽

10.3390/ijms22073560 ◽

2021 ◽

Vol 22 (7) ◽

pp. 3560

Author(s):

Ruixue Xiao ◽

Chong Zhang ◽

Xiaorui Guo ◽

Hui Li ◽

Hai Lu

Keyword(s):

Cell Wall ◽

Transcription Factors ◽

Secondary Wall ◽

Secondary Cell Wall ◽

Lignin Biosynthesis ◽

Cell Wall Formation ◽

Long Distance ◽

Secondary Cell Wall Formation ◽

Myb Transcription Factors ◽

Wall Formation

The secondary wall is the main part of wood and is composed of cellulose, xylan, lignin, and small amounts of structural proteins and enzymes. Lignin molecules can interact directly or indirectly with cellulose, xylan and other polysaccharide molecules in the cell wall, increasing the mechanical strength and hydrophobicity of plant cells and tissues and facilitating the long-distance transportation of water in plants. MYBs (v-myb avian myeloblastosis viral oncogene homolog) belong to one of the largest superfamilies of transcription factors, the members of which regulate secondary cell-wall formation by promoting/inhibiting the biosynthesis of lignin, cellulose, and xylan. Among them, MYB46 and MYB83, which comprise the second layer of the main switch of secondary cell-wall biosynthesis, coordinate upstream and downstream secondary wall synthesis-related transcription factors. In addition, MYB transcription factors other than MYB46/83, as well as noncoding RNAs, hormones, and other factors, interact with one another to regulate the biosynthesis of the secondary wall. Here, we discuss the biosynthesis of secondary wall, classification and functions of MYB transcription factors and their regulation of lignin polymerization and secondary cell-wall formation during wood formation.

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Expression Analyses of Soybean VOZ Transcription Factors and the Role of GmVOZ1G in Drought and Salt Stress Tolerance

International Journal of Molecular Sciences ◽

10.3390/ijms21062177 ◽

2020 ◽

Vol 21 (6) ◽

pp. 2177 ◽

Cited By ~ 1

Author(s):

Bo Li ◽

Jia-Cheng Zheng ◽

Ting-Ting Wang ◽

Dong-Hong Min ◽

Wen-Liang Wei ◽

...

Keyword(s):

Transcription Factor ◽

Transcription Factors ◽

Salt Stress ◽

Abiotic Stress ◽

Stress Tolerance ◽

Zinc Finger ◽

Hairy Roots ◽

Yeast Cells ◽

Salt Stress Tolerance ◽

Drought And Salt Stress

Vascular plant one-zinc-finger (VOZ) transcription factor, a plant specific one-zinc-finger-type transcriptional activator, is involved in regulating numerous biological processes such as floral induction and development, defense against pathogens, and response to multiple types of abiotic stress. Six VOZ transcription factor-encoding genes (GmVOZs) have been reported to exist in the soybean (Glycine max) genome. In spite of this, little information is currently available regarding GmVOZs. In this study, GmVOZs were cloned and characterized. GmVOZ genes encode proteins possessing transcriptional activation activity in yeast cells. GmVOZ1E, GmVOZ2B, and GmVOZ2D gene products were widely dispersed in the cytosol, while GmVOZ1G was primarily located in the nucleus. GmVOZs displayed a differential expression profile under dehydration, salt, and salicylic acid (SA) stress conditions. Among them, GmVOZ1G showed a significantly induced expression in response to all stress treatments. Overexpression of GmVOZ1G in soybean hairy roots resulted in a greater tolerance to drought and salt stress. In contrast, RNA interference (RNAi) soybean hairy roots suppressing GmVOZ1G were more sensitive to both of these stresses. Under drought treatment, soybean composite plants with an overexpression of hairy roots had higher relative water content (RWC). In response to drought and salt stress, lower malondialdehyde (MDA) accumulation and higher peroxidase (POD) and superoxide dismutase (SOD) activities were observed in soybean composite seedlings with an overexpression of hairy roots. The opposite results for each physiological parameter were obtained in RNAi lines. In conclusion, GmVOZ1G positively regulates drought and salt stress tolerance in soybean hairy roots. Our results will be valuable for the functional characterization of soybean VOZ transcription factors under abiotic stress.

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Screening and Genetic Network Analysis of Genes Involved in Freezing and Thawing Resistance in DaMDHAR—Expressing Saccharomyces cerevisiae Using Gene Expression Profiling

Genes ◽

10.3390/genes12020219 ◽

2021 ◽

Vol 12 (2) ◽

pp. 219

Author(s):

Il-Sup Kim ◽

Woong Choi ◽

Jonghyeon Son ◽

Jun Hyuck Lee ◽

Hyoungseok Lee ◽

...

Keyword(s):

Gene Expression ◽

Saccharomyces Cerevisiae ◽

Transcription Factors ◽

Stress Tolerance ◽

Genetic Network ◽

Yeast Cells ◽

Enzymatic Antioxidants ◽

Freezing And Thawing ◽

Metal Ion Homeostasis ◽

Transgenic Yeast

The cryoprotection of cell activity is a key determinant in frozen-dough technology. Although several factors that contribute to freezing tolerance have been reported, the mechanism underlying the manner in which yeast cells respond to freezing and thawing (FT) stress is not well established. Therefore, the present study demonstrated the relationship between DaMDHAR encoding monodehydroascorbate reductase from Antarctic hairgrass Deschampsia antarctica and stress tolerance to repeated FT cycles (FT2) in transgenic yeast Saccharomyces cerevisiae. DaMDHAR-expressing yeast (DM) cells identified by immunoblotting analysis showed high tolerance to FT stress conditions, thereby causing lower damage for yeast cells than wild-type (WT) cells with empty vector alone. To detect FT2 tolerance-associated genes, 3′-quant RNA sequencing was employed using mRNA isolated from DM and WT cells exposed to FT (FT2) conditions. Approximately 332 genes showed ≥2-fold changes in DM cells and were classified into various groups according to their gene expression. The expressions of the changed genes were further confirmed using western blot analysis and biochemical assay. The upregulated expression of 197 genes was associated with pentose phosphate pathway, NADP metabolic process, metal ion homeostasis, sulfate assimilation, β-alanine metabolism, glycerol synthesis, and integral component of mitochondrial and plasma membrane (PM) in DM cells under FT2 stress, whereas the expression of the remaining 135 genes was partially related to protein processing, selenocompound metabolism, cell cycle arrest, oxidative phosphorylation, and α-glucoside transport under the same condition. With regard to transcription factors in DM cells, MSN4 and CIN5 were activated, but MSN2 and MGA1 were not. Regarding antioxidant systems and protein kinases in DM cells under FT stress, CTT1, GTO, GEX1, and YOL024W were upregulated, whereas AIF1, COX2, and TRX3 were not. Gene activation represented by transcription factors and enzymatic antioxidants appears to be associated with FT2-stress tolerance in transgenic yeast cells. RCK1, MET14, and SIP18, but not YPK2, have been known to be involved in the protein kinase-mediated signalling pathway and glycogen synthesis. Moreover, SPI18 and HSP12 encoding hydrophilin in the PM were detected. Therefore, it was concluded that the genetic network via the change of gene expression levels of multiple genes contributing to the stabilization and functionality of the mitochondria and PM, not of a single gene, might be the crucial determinant for FT tolerance in DaMDAHR-expressing transgenic yeast. These findings provide a foundation for elucidating the DaMDHAR-dependent molecular mechanism of the complex functional resistance in the cellular response to FT stress.

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