scholarly journals Aspergillus fumigatus Cyp51A and Cyp51B Proteins Are Compensatory in Function and Localize Differentially in Response to Antifungals and Cell Wall Inhibitors

2020 ◽  
Vol 64 (10) ◽  
Author(s):  
Mark T. Roundtree ◽  
Praveen R. Juvvadi ◽  
E. Keats Shwab ◽  
D. Christopher Cole ◽  
William J. Steinbach
Keyword(s):  
Cell Wall ◽  
Aspergillus Fumigatus ◽  
Therapeutic Option ◽  
Point Mutations ◽  
Normal Growth ◽  
Wall Stress ◽  
Stress Conditions ◽  
Compensatory Response ◽  
Content Type ◽  
Red Led

ABSTRACT Triazole antifungals are the primary therapeutic option against invasive aspergillosis. However, resistance to azoles has increased dramatically over the last decade. Azole resistance is known to primarily occur due to point mutations in the azole target protein Cyp51A, one of two paralogous 14-α sterol demethylases found in Aspergillus fumigatus. Despite the importance of Cyp51A, little is known about the function of its paralog, Cyp51B, and the behavior of these proteins within the cell or their functional interrelationship. In this study, we addressed two important aspects of the Cyp51 proteins: (i) we characterized their localization patterns under normal growth versus stress conditions, and (ii) we determined how the proteins compensate for each other’s absence and respond to azole treatment. Both the Cyp51A and Cyp51B proteins were found to localize in distinct endoplasmic reticulum (ER) domains, including the perinuclear ER and the peripheral ER. Occasionally, the Cyp51 proteins concentrated in the peripheral ER network of tubules along the hyphal septa and at the hyphal tips. Exposure to voriconazole, caspofungin, and Congo red led to significant increases in fluorescence intensity in these alternative localization sites, indicative of Cyp51 protein translocation in response to cell wall stress. Furthermore, deletion of either Cyp51 paralog increased susceptibility to voriconazole, though a greater effect was observed following deletion of cyp51A, indicating a compensatory response to stress conditions.

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

mBio ◽  
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.

scholarly journals The Aspergillus fumigatus Phosphoproteome Reveals Roles of High-Osmolarity Glycerol Mitogen-Activated Protein Kinases in Promoting Cell Wall Damage and Caspofungin Tolerance

mBio ◽  
2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Eliciane Cevolani Mattos ◽  
Lilian Pereira Silva ◽  
Clara Valero ◽  
Patrícia Alves de Castro ◽  
Thaila Fernanda dos Reis ◽  
...  
Keyword(s):  
Cell Wall ◽  
Aspergillus Fumigatus ◽  
Protein Kinases ◽  
Wall Stress ◽  
Mapk Signaling ◽  
Fungal Cell ◽  
Content Type ◽  
Cell Wall Stress ◽  
Cell Wall Damage ◽  
Mitogen Activated Protein

ABSTRACT The filamentous fungus Aspergillus fumigatus can cause a distinct set of clinical disorders in humans. Invasive aspergillosis (IA) is the most common life-threatening fungal disease of immunocompromised humans. The mitogen-activated protein kinase (MAPK) signaling pathways are essential to the adaptation to the human host. Fungal cell survival is highly dependent on the organization, composition, and function of the cell wall. Here, an evaluation of the global A. fumigatus phosphoproteome under cell wall stress caused by the cell wall-damaging agent Congo red (CR) revealed 485 proteins potentially involved in the cell wall damage response. Comparative phosphoproteome analyses with the ΔsakA, ΔmpkC, and ΔsakA ΔmpkC mutant strains from the osmotic stress MAPK cascades identify their additional roles during the cell wall stress response. Our phosphoproteomics allowed the identification of novel kinases and transcription factors (TFs) involved in osmotic stress and in the cell wall integrity (CWI) pathway. Our global phosphoproteome network analysis showed an enrichment for protein kinases, RNA recognition motif domains, and the MAPK signaling pathway. In contrast to the wild-type strain, there is an overall decrease of differentially phosphorylated kinases and phosphatases in ΔsakA, ΔmpkC, and ΔsakA ΔmpkC mutants. We constructed phosphomutants for the phosphorylation sites of several proteins differentially phosphorylated in the wild-type and mutant strains. For all the phosphomutants, there is an increase in the sensitivity to cell wall-damaging agents and a reduction in the MpkA phosphorylation upon CR stress, suggesting these phosphosites could be important for the MpkA modulation and CWI pathway regulation. IMPORTANCE Aspergillus fumigatus is an opportunistic human pathogen causing allergic reactions or systemic infections, such as invasive pulmonary aspergillosis in immunocompromised patients. The mitogen-activated protein kinase (MAPK) signaling pathways are essential for fungal adaptation to the human host. Fungal cell survival, fungicide tolerance, and virulence are highly dependent on the organization, composition, and function of the cell wall. Upon cell wall stress, MAPKs phosphorylate multiple target proteins involved in the remodeling of the cell wall. Here, we investigate the global phosphoproteome of the ΔsakA and ΔmpkC A. fumigatus and high-osmolarity glycerol (HOG) pathway MAPK mutants upon cell wall damage. This showed the involvement of the HOG pathway and identified novel protein kinases and transcription factors, which were confirmed by fungal genetics to be involved in promoting tolerance of cell wall damage. Our results provide understanding of how fungal signal transduction networks modulate the cell wall. This may also lead to the discovery of new fungicide drug targets to impact fungal cell wall function, fungicide tolerance, and virulence.

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

mBio ◽  
2019 ◽  
Vol 10 (2) ◽  
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.

scholarly journals Interaction of Platelets and Anidulafungin against Aspergillus fumigatus

2012 ◽  
Vol 57 (1) ◽  
pp. 626-628 ◽  
Author(s):  
Susanne Perkhofer ◽  
Barbara Striessnig ◽  
Bettina Sartori ◽  
Barbara Hausott ◽  
Helmut W. Ott ◽  
...  
Keyword(s):  
Cell Wall ◽  
Aspergillus Fumigatus ◽  
Untreated Control ◽  
Germination Rate ◽  
Human Platelets ◽  
Cell Wall Synthesis ◽  
Content Type

ABSTRACTThe combination of platelets and anidulafungin at 0.03 μg/ml significantly (P< 0.05) reduced the germination rate and hyphal elongation inAspergillus fumigatuscompared to those with either anidulafungin only or an untreated control. Platelets decreased the expression of thefksgene, which plays an important role in cell wall synthesis. Our results suggest that human platelets plus anidulafungin might contribute to defense againstA. fumigatus.

scholarly journals Effects of a Defective Endoplasmic Reticulum-Associated Degradation Pathway on the Stress Response, Virulence, and Antifungal Drug Susceptibility of the Mold Pathogen Aspergillus fumigatus

2013 ◽  
Vol 12 (4) ◽  
pp. 512-519 ◽  
Author(s):  
Karthik Krishnan ◽  
Xizhi Feng ◽  
Margaret V. Powers-Fletcher ◽  
Gregory Bick ◽  
Daryl L. Richie ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Aspergillus Fumigatus ◽  
Wall Stress ◽  
Drug Susceptibility ◽  
Degradation Pathway ◽  
Eukaryotic Cell ◽  
Unfolded Proteins ◽  
Content Type ◽  
Double Deletion ◽  
The Unfolded Protein Response

ABSTRACT Proteins that are destined for release outside the eukaryotic cell, insertion into the plasma membrane, or delivery to intracellular organelles are processed and folded in the endoplasmic reticulum (ER). An imbalance between the level of nascent proteins entering the ER and the organelle's ability to manage that load results in the accumulation of unfolded proteins. Terminally unfolded proteins are disposed of by ER-associated degradation (ERAD), a pathway that transports the aberrant proteins across the ER membrane into the cytosol for proteasomal degradation. The ERAD pathway was targeted in the mold pathogen Aspergillus fumigatus by deleting the hrdA gene, encoding the A. fumigatus ortholog of Hrd1, the E3 ubiquitin ligase previously shown to contribute to ERAD in other species. Loss of HrdA was associated with impaired degradation of a folding-defective ERAD substrate, CPY*, as well as activation of the unfolded-protein response (UPR). The Δ hrdA mutant showed resistance to voriconazole and reduced thermotolerance but was otherwise unaffected by a variety of environmental stressors. A double-deletion mutant deficient in both HrdA and another component of the same ERAD complex, DerA, was defective in secretion and showed hypersensitivity to ER, thermal, and cell wall stress. However, the Δ hrdA Δ derA mutant remained virulent in mouse and insect infection models. These data demonstrate that HrdA and DerA support complementary ERAD functions that promote survival under conditions of ER stress but are dispensable for virulence in the host environment.

scholarly journals Surface Structure Characterization of Aspergillus fumigatus Conidia Mutated in the Melanin Synthesis Pathway and Their Human Cellular Immune Response

2014 ◽  
Vol 82 (8) ◽  
pp. 3141-3153 ◽  
Author(s):  
Jagadeesh Bayry ◽  
Audrey Beaussart ◽  
Yves F. Dufrêne ◽  
Meenu Sharma ◽  
Kushagra Bansal ◽  
...  
Keyword(s):  
Cell Wall ◽  
Cell Surface ◽  
Aspergillus Fumigatus ◽  
Surface Defects ◽  
Structure Characterization ◽  
Inert Material ◽  
Cell Wall Polysaccharides ◽  
Melanin Biosynthesis ◽  
Content Type ◽  
Conidial Surface

ABSTRACTInAspergillus fumigatus, the conidial surface contains dihydroxynaphthalene (DHN)-melanin. Six-clustered gene products have been identified that mediate sequential catalysis of DHN-melanin biosynthesis. Melanin thus produced is known to be a virulence factor, protecting the fungus from the host defense mechanisms. In the present study, individual deletion of the genes involved in the initial three steps of melanin biosynthesis resulted in an altered conidial surface with masked surface rodlet layer, leaky cell wall allowing the deposition of proteins on the cell surface and exposing the otherwise-masked cell wall polysaccharides at the surface. Melanin as such was immunologically inert; however, deletion mutant conidia with modified surfaces could activate human dendritic cells and the subsequent cytokine production in contrast to the wild-type conidia. Cell surface defects were rectified in the conidia mutated in downstream melanin biosynthetic pathway, and maximum immune inertness was observed upon synthesis of vermelone onward. These observations suggest that although melanin as such is an immunologically inert material, it confers virulence by facilitating proper formation of theA. fumigatusconidial surface.

scholarly journals Dual Roles of FmtA in Staphylococcus aureus Cell Wall Biosynthesis and Autolysis

2012 ◽  
Vol 56 (7) ◽  
pp. 3797-3805 ◽  
Author(s):  
Aneela Qamar ◽  
Dasantila Golemi-Kotra
Keyword(s):  
Staphylococcus Aureus ◽  
Cell Wall ◽  
Wall Stress ◽  
Microscopy Study ◽  
Dual Roles ◽  
Content Type ◽  
Teichoic Acids ◽  
Low Concentrations ◽  
High Concentrations ◽  
Wall Teichoic Acids

ABSTRACTThefmtAgene is a member of theStaphylococcus aureuscore cell wall stimulon. The FmtA protein interacts with β-lactams through formation of covalent species. Here, we show that FmtA has weakd-Ala-d-Ala-carboxypeptidase activity and is capable of covalently incorporating C14-Gly into cell walls. The fluorescence microscopy study showed that the protein is localized to the cell division septum. Furthermore, we show that wall teichoic acids interact specifically with FmtA and mediate recruitment of FmtA to theS. aureuscell wall. Subjection ofS. aureusto FmtA concentrations of 0.1 μM or less induces autolysis and biofilm production. This effect requires the presence of wall teichoic acids. At FmtA concentrations greater than 0.2 μM, autolysis and biofilm formation inS. aureusare repressed and growth is enhanced. Our findings indicate dual roles of FmtA inS. aureusgrowth, whereby at low concentrations, FmtA may modulate the activity of the major autolysin (AtlA) ofS. aureusand, at high concentrations, may participate in synthesis of cell wall peptidoglycan. These two roles of FmtA may reflect dual functions of FmtA in the absence and presence of cell wall stress, respectively.

scholarly journals The Posttranslocational Chaperone Lipoprotein PrsA Is Involved in both Glycopeptide and Oxacillin Resistance in Staphylococcus aureus

2012 ◽  
Vol 56 (7) ◽  
pp. 3629-3640 ◽  
Author(s):  
Ambre Jousselin ◽  
Adriana Renzoni ◽  
Diego O. Andrey ◽  
Antoinette Monod ◽  
Daniel P. Lew ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Cell Wall ◽  
Wall Stress ◽  
Growth Conditions ◽  
Transcriptional Analysis ◽  
Pharmacological Intervention ◽  
Content Type ◽  
Cell Wall Stress ◽  
Oxacillin Resistance ◽  
Mrsa Strains

ABSTRACTUnderstanding in detail the factors which permitStaphylococcus aureusto counteract cell wall-active antibiotics is a prerequisite to elaborating effective strategies to prolong the usefulness of these drugs and define new targets for pharmacological intervention. Methicillin-resistantS. aureus(MRSA) strains are major pathogens of hospital-acquired and community-acquired infections and are most often treated with glycopeptides (vancomycin and teicoplanin) because of their resistance to most penicillins and a limited arsenal of clinically proven alternatives. In this study, we examined PrsA, a lipid-anchored protein of the parvulin PPIase family (peptidyl-prolylcis/transisomerase) found ubiquitously in all Gram-positive species, in which it assists posttranslocational folding at the outer surface of the cytoplasmic membrane. We show by both genetic and biochemical assays thatprsAis directly regulated by the VraRS two-component sentinel system of cell wall stress. Disruption ofprsAis tolerated byS. aureus, and its loss results in no detectable overt macroscopic changes in cell wall architecture or growth rate under nonstressed growth conditions. Disruption ofprsAleads, however, to notable alterations in the sensitivity to glycopeptides and dramatically decreases the resistance of COL (MRSA) to oxacillin. Quantitative transcriptional analysis reveals thatprsAandvraRare coordinately upregulated in a panel of stable laboratory and clinical glycopeptide-intermediateS. aureus(GISA) strains compared to their susceptible parents. Collectively, our results point to a role forprsAas a facultative facilitator of protein secretion or extracellular folding and provide a framework for understanding whyprsAis a key element of the VraRS-mediated cell wall stress response.

scholarly journals Single-Center Evaluation of an Agar-Based Screening for Azole Resistance in Aspergillus fumigatus by Using VIPcheck

2017 ◽  
Vol 61 (12) ◽  
Author(s):  
J. B. Buil ◽  
H. A. L. van der Lee ◽  
A. J. M. M. Rijs ◽  
J. Zoll ◽  
J. A. M. F. Hovestadt ◽  
...  
Keyword(s):  
Aspergillus Fumigatus ◽  
Sensitivity And Specificity ◽  
Antifungal Susceptibility ◽  
Screening Method ◽  
Point Mutations ◽  
Azole Resistance ◽  
Minimal Growth ◽  
Content Type ◽  
Mean Sensitivity ◽  
Selection Of

ABSTRACT Antifungal susceptibility testing is an essential tool for guiding therapy, although EUCAST and CLSI reference methods are often available only in specialized centers. We studied the performance of an agar-based screening method for the detection of azole resistance in Aspergillus fumigatus cultures. The VIPcheck consists of four wells containing voriconazole, itraconazole, posaconazole, or a growth control. Ninety-six A. fumigatus isolates were used. Thirty-three isolates harbored a known resistance mechanism: TR34/L98H (11 isolates), TR46/Y121F/T289A (6 isolates), TR53 (2 isolates), and 14 isolates with other cyp51A gene point mutations. Eighteen resistant isolates had no cyp51A-mediated azole resistance. Forty-five isolates had a wild-type (WT) azole phenotype. Four technicians and two inexperienced interns, blinded to the genotype/phenotype, read the plates visually after 24 h and 48 h and documented minimal growth, uninhibited growth, and no growth. The performance was compared to the EUCAST method. After 24 h of incubation, the mean sensitivity and specificity were 0.54 and 1.00, respectively, with uninhibited growth as the threshold. After 48 h of incubation, the performance mean sensitivity and specificity were 0.98 and 0.93, respectively, with minimal growth. The performance was not affected by observer experience in mycology. The interclass correlation coefficient was 0.87 after 24 h and 0.85 after 48 h. VIPcheck enabled the selection of azole-resistant A. fumigatus colonies, with a mean sensitivity and specificity of 0.98 and 0.93, respectively. Uninhibited growth on any azole-containing well after 24 h and minimal growth after 48 h were indicative of resistance. These results indicate that the VIPcheck is an easy-to-use tool for azole resistance screening and the selection of colonies that require MIC testing.

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