scholarly journals The Paradoxical Effect of Echinocandins in Aspergillus fumigatus Relies on Recovery of the β-1,3-Glucan Synthase Fks1

2016 ◽  
Vol 61 (2) ◽  
Author(s):  
Veronika Loiko ◽  
Johannes Wagener
Keyword(s):  
Cell Wall ◽  
Aspergillus Fumigatus ◽  
Minor Importance ◽  
Initial Growth ◽  
Paradoxical Effect ◽  
Fungal Cell ◽  
Glucan Synthase ◽  
Content Type ◽  
Drug Concentrations ◽  
Very High

ABSTRACT Echinocandins target the fungal cell wall by inhibiting biosynthesis of the cell wall carbohydrate β-1,3-glucan. This antifungal drug class exhibits a paradoxical effect that is characterized by the resumption of growth of otherwise susceptible strains at higher drug concentrations (approximately 4 to 32 μg/ml). The nature of this phenomenon is still unknown. In this study, we analyzed the paradoxical effect of the echinocandin caspofungin on the pathogenic mold Aspergillus fumigatus. Using a conditional fks1 mutant, we show that very high caspofungin concentrations exert an additional antifungal activity besides inhibition of the β-1,3-glucan synthase. This activity could explain the suppression of paradoxical growth at very high caspofungin concentrations. Additionally, we found that exposure to inhibitory caspofungin concentrations always causes initial growth deprivation independently of the capability of the drug concentration to induce the paradoxical effect. Paradoxically growing hyphae emerge from microcolonies essentially devoid of β-1,3-glucan. However, these hyphae expose β-1,3-glucan again, suggesting that β-1,3-glucan synthesis is restored. In agreement with this hypothesis, we found that expression of the β-1,3-glucan synthase Fks1 is an essential requirement for the paradoxical effect. Surprisingly, overexpression of fks1 renders A. fumigatus more susceptible, whereas reduced expression leads to hyphae that are more resistant to the growth-inhibitory and limited fungicidal activity of caspofungin. Upregulation of chitin synthesis appears to be of minor importance for the paradoxical effect, since paradoxically growing hyphae exhibit significantly less chitin than the growth-deprived parental microcolonies. Our results argue for a model where the paradoxical effect primarily relies on recovery of β-1,3-glucan synthase activity.

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 The Glycosylphosphatidylinositol-Anchored DFG Family Is Essential for the Insertion of Galactomannan into the β-(1,3)-Glucan–Chitin Core of the Cell Wall of Aspergillus fumigatus

mSphere ◽  
2019 ◽  
Vol 4 (4) ◽  
Author(s):  
Laetitia Muszkieta ◽  
Thierry Fontaine ◽  
Rémi Beau ◽  
Isabelle Mouyna ◽  
Marian Samuel Vogt ◽  
...  
Keyword(s):  
Cell Wall ◽  
Aspergillus Fumigatus ◽  
Gene Family ◽  
Protein Complexes ◽  
Growth Conditions ◽  
Fungal Cell Wall ◽  
Cross Linking ◽  
Fungal Cell ◽  
Content Type

ABSTRACT The fungal cell wall is a complex and dynamic entity essential for the development of fungi. It is composed mainly of polysaccharides that are synthetized by protein complexes. At the cell wall level, enzyme activities are involved in postsynthesis polysaccharide modifications such as cleavage, elongation, branching, and cross-linking. Glycosylphosphatidylinositol (GPI)-anchored proteins have been shown to participate in cell wall biosynthesis and specifically in polysaccharide remodeling. Among these proteins, the DFG family plays an essential role in controlling polar growth in yeast. In the filamentous fungus and opportunistic human pathogen Aspergillus fumigatus, the DFG gene family contains seven orthologous DFG genes among which only six are expressed under in vitro growth conditions. Deletions of single DFG genes revealed that DFG3 plays the most important morphogenetic role in this gene family. A sextuple-deletion mutant resulting from the deletion of all in vitro expressed DFG genes did not contain galactomannan in the cell wall and has severe growth defects. This study has shown that DFG members are absolutely necessary for the insertion of galactomannan into the cell wall of A. fumigatus and that the proper cell wall localization of the galactomannan is essential for correct fungal morphogenesis in A. fumigatus. IMPORTANCE The fungal cell wall is a complex and dynamic entity essential for the development of fungi. It is composed mainly of polysaccharides that are synthetized by protein complexes. Enzymes involved in postsynthesis polysaccharide modifications, such as cleavage, elongation, branching, and cross-linking, are essential for fungal life. Here, we investigated in Aspergillus fumigatus the role of the members of the Dfg family, one of the 4 GPI-anchored protein families common to yeast and molds involved in cell wall remodeling. Molecular and biochemical approaches showed that DFG members are required for filamentous growth, conidiation, and cell wall organization and are essential for the life of this fungal pathogen.

scholarly journals Two KTR Mannosyltransferases Are Responsible for the Biosynthesis of Cell Wall Mannans and Control Polarized Growth inAspergillus fumigatus

mBio ◽  
2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Christine Henry ◽  
Jizhou Li ◽  
François Danion ◽  
Laura Alcazar-Fuoli ◽  
Emilia Mellado ◽  
...  
Keyword(s):  
Cell Wall ◽  
Invasive Aspergillosis ◽  
Aspergillus Fumigatus ◽  
Filamentous Fungi ◽  
Biosynthetic Pathway ◽  
Fungal Cell Wall ◽  
Polarized Growth ◽  
Biological Functions ◽  
Fungal Cell ◽  
Content Type

ABSTRACTFungal cell wall mannans are complex carbohydrate polysaccharides with different structures in yeasts and molds. In contrast to yeasts, their biosynthetic pathway has been poorly investigated in filamentous fungi. InAspergillus fumigatus, the major mannan structure is a galactomannan that is cross-linked to the β-1,3-glucan-chitin cell wall core. This polymer is composed of a linear mannan with a repeating unit composed of four α1,6-linked and α1,2-linked mannoses with side chains of galactofuran. Despite its use as a biomarker to diagnose invasive aspergillosis, its biosynthesis and biological function were unknown. Here, we have investigated the function of three members of the Ktr (also named Kre2/Mnt1) family (Ktr1, Ktr4, and Ktr7) inA. fumigatusand show that two of them are required for the biosynthesis of galactomannan. In particular, we describe a newly discovered form of α-1,2-mannosyltransferase activity encoded by theKTR4gene. Biochemical analyses showed that deletion of theKTR4gene or theKTR7gene leads to the absence of cell wall galactomannan. In comparison to parental strains, theΔktr4andΔktr7mutants showed a severe growth phenotype with defects in polarized growth and in conidiation, marked alteration of the conidial viability, and reduced virulence in a mouse model of invasive aspergillosis. In yeast, the KTR proteins are involved in protein 0- and N-glycosylation. This study provided another confirmation that orthologous genes can code for proteins that have very different biological functions in yeasts and filamentous fungi. Moreover, inA. fumigatus, cell wall mannans are as important structurally as β-glucans and chitin.IMPORTANCEThe fungal cell wall is a complex and dynamic entity essential for the development of fungi. It allows fungal pathogens to survive environmental challenge posed by nutrient stress and host defenses, and it also is central to polarized growth. The cell wall is mainly composed of polysaccharides organized in a three-dimensional network.Aspergillus fumigatusproduces a cell wall galactomannan whose biosynthetic pathway and biological functions remain poorly defined. Here, we described two new mannosyltransferases essential to the synthesis of the cell wall galactomannan. Their absence leads to a growth defect with misregulation of polarization and altered conidiation, with conidia which are bigger and more permeable than the conidia of the parental strain. This study showed that in spite of its low concentration in the cell wall, this polysaccharide is absolutely required for cell wall stability, for apical growth, and for the full virulence ofA. fumigatus.

scholarly journals Potential of Chemically Synthesized Oligosaccharides To Define the Carbohydrate Moieties of the Fungal Cell Wall Responsible for the Human Immune Response, Using Aspergillus fumigatus Galactomannan as a Model

mSphere ◽  
2020 ◽  
Vol 5 (1) ◽  
Author(s):  
Sarah Sze Wah Wong ◽  
Vadim B. Krylov ◽  
Dmitry A. Argunov ◽  
Alexander A. Karelin ◽  
Jean-Phillipe Bouchara ◽  
...  
Keyword(s):  
Immune Response ◽  
Cell Wall ◽  
Aspergillus Fumigatus ◽  
Fungal Cell Wall ◽  
Human Pathogens ◽  
Cell Wall Polysaccharides ◽  
Fungal Cell ◽  
Content Type ◽  
Human Immune Response ◽  
Synthetic Oligosaccharides

ABSTRACT Methodologies to identify epitopes or ligands of the fungal cell wall polysaccharides influencing the immune response of human pathogens have to date been imperfect. Using the galactomannan (GM) of Aspergillus fumigatus as a model, we have shown that synthetic oligosaccharides of distinct structures representing key fragments of cell wall polysaccharides are the most precise tools to study the serological and immunomodulatory properties of a fungal polysaccharide.

scholarly journals α1,3 Glucans Are Dispensable in Aspergillus fumigatus

2011 ◽  
Vol 11 (1) ◽  
pp. 26-29 ◽  
Author(s):  
Christine Henry ◽  
Jean-Paul Latgé ◽  
Anne Beauvais
Keyword(s):  
Cell Wall ◽  
Aspergillus Fumigatus ◽  
Vegetative Growth ◽  
Conidial Germination ◽  
Glucan Synthase ◽  
Content Type ◽  
Chitin Content ◽  
A Cell

ABSTRACTA triple α1,3 glucan synthase mutant ofAspergillus fumigatusobtained by successive deletions of the three α1,3 glucan synthase genes (AGS1,AGS2, andAGS3) has a cell wall devoid of α1,3 glucans. The lack of α1,3 glucans affects neither conidial germination nor mycelial vegetative growth and is compensated by an increase in β1,3 glucan and/or chitin content.

scholarly journals An Ssd1 Homolog Impacts Trehalose and Chitin Biosynthesis and Contributes to Virulence inAspergillus fumigatus

mSphere ◽  
2019 ◽  
Vol 4 (3) ◽  
Author(s):  
Arsa Thammahong ◽  
Sourabh Dhingra ◽  
Katherine M. Bultman ◽  
Joshua D. Kerkaert ◽  
Robert A. Cramer
Keyword(s):  
Cell Wall ◽  
Aspergillus Fumigatus ◽  
Mutant Strain ◽  
Filamentous Fungus ◽  
Fungal Cell Wall ◽  
Cell Wall Biosynthesis ◽  
Null Mutant ◽  
Fungal Cell ◽  
Fungal Virulence ◽  
Content Type

ABSTRACTRegulation of fungal cell wall biosynthesis is critical to maintain cell wall integrity in dynamic fungal infection microenvironments. Genes involved in this response that impact fungal fitness and host immune responses remain to be fully defined. In this study, we observed that a yeastssd1homolog,ssdA, in the filamentous fungusAspergillus fumigatusis involved in trehalose and cell wall homeostasis. AnssdAnull mutant strain exhibited an increase in trehalose levels and a reduction in fungal colony growth rate. In contrast, overexpression ofssdAperturbed trehalose biosynthesis and reduced germination of conidia. ThessdAnull mutant strain was more resistant to cell wall-perturbing agents, while overexpression ofssdAincreased sensitivity. Overexpression ofssdAsignificantly increased chitin levels, and both loss and overexpression ofssdAaltered subcellular localization of the class V chitin synthase CsmA. Strikingly, overexpression ofssdAabolished adherence to abiotic surfaces and severely attenuated the virulence ofA. fumigatusin a murine model of invasive pulmonary aspergillosis. Despite the severein vitrofitness defects observed upon loss ofssdA, neither surface adherence nor murine survival was impacted. In conclusion,A. fumigatusSsdA plays a critical role in cell wall homeostasis impactingA. fumigatus-host interactions.IMPORTANCEThe incidence of life-threatening infections caused by the filamentous fungusAspergillus fumigatusis increasing along with an increase in the number of fungal strains resistant to contemporary antifungal therapies. The fungal cell wall and the associated carbohydrates required for its synthesis and maintenance are attractive drug targets given that many genes encoding proteins involved in cell wall biosynthesis and integrity are absent in humans. Importantly, genes and associated cell wall biosynthesis and homeostasis regulatory pathways remain to be fully defined inA. fumigatus. In this report, we identify SsdA as an important component of trehalose and fungal cell wall biosynthesis inA. fumigatusthat consequently impacts the host immune response and fungal virulence in animal models of infection.

scholarly journals Biosynthesis of β-(1→5)-Galactofuranosyl Chains of Fungal-Type and O-Mannose-Type Galactomannans within the Invasive Pathogen Aspergillus fumigatus

mSphere ◽  
2020 ◽  
Vol 5 (1) ◽  
Author(s):  
Yuria Chihara ◽  
Yutaka Tanaka ◽  
Minoru Izumi ◽  
Daisuke Hagiwara ◽  
Akira Watanabe ◽  
...  
Keyword(s):  
Cell Wall ◽  
Cell Surface ◽  
Aspergillus Fumigatus ◽  
Pathogenic Fungus ◽  
Cell Surface Hydrophobicity ◽  
Surface Hydrophobicity ◽  
Wall Structure ◽  
Fungal Cell ◽  
Content Type ◽  
Animal Pathogens

ABSTRACT The pathogenic fungus Aspergillus fumigatus contains galactomannans localized on the surface layer of its cell walls, which are involved in various biological processes. Galactomannans comprise α-(1→2)-/α-(1→6)-mannan and β-(1→5)-/β-(1→6)-galactofuranosyl chains. We previously revealed that GfsA is a β-galactofuranoside β-(1→5)-galactofuranosyltransferase involved in the biosynthesis of β-(1→5)-galactofuranosyl chains. In this study, we clarified the biosynthesis of β-(1→5)-galactofuranosyl chains in A. fumigatus. Two paralogs exist within A. fumigatus: GfsB and GfsC. We show that GfsB and GfsC, in addition to GfsA, are β-galactofuranoside β-(1→5)-galactofuranosyltransferases by biochemical and genetic analyses. GfsA, GfsB, and GfsC can synthesize β-(1→5)-galactofuranosyl oligomers at up to lengths of 7, 3, and 5 galactofuranoses within an established in vitro highly efficient assay of galactofuranosyltransferase activity. Structural analyses of galactomannans extracted from ΔgfsB, ΔgfsC, ΔgfsAC, and ΔgfsABC strains revealed that GfsA and GfsC synthesized all β-(1→5)-galactofuranosyl residues of fungal-type and O-mannose-type galactomannans and that GfsB exhibited limited function in A. fumigatus. The loss of β-(1→5)-galactofuranosyl residues decreased the hyphal growth rate and conidium formation ability and increased the abnormal hyphal branching structure and cell surface hydrophobicity, but this loss is dispensable for sensitivity to antifungal agents and virulence toward immunocompromised mice. IMPORTANCE β-(1→5)-Galactofuranosyl residues are widely distributed in the subphylum Pezizomycotina of the phylum Ascomycota. Pezizomycotina includes many plant and animal pathogens. Although the structure of β-(1→5)-galactofuranosyl residues of galactomannans in filamentous fungi was discovered long ago, it remains unclear which enzyme is responsible for biosynthesis of this glycan. Fungal cell wall formation processes are complicated, and information concerning glycosyltransferases is essential for understanding them. In this study, we showed that GfsA and GfsC are responsible for the biosynthesis of all β-(1→5)-galactofuranosyl residues of fungal-type and O-mannose-type galactomannans. The data presented here indicate that β-(1→5)-galactofuranosyl residues are involved in cell growth, conidiation, polarity, and cell surface hydrophobicity. Our new understanding of β-(1→5)-galactofuranosyl residue biosynthesis provides important novel insights into the formation of the complex cell wall structure and the virulence of the members of the subphylum Pezizomycotina.

scholarly journals The Aspergillus fumigatus CrzA Transcription Factor Activates Chitin Synthase Gene Expression during the Caspofungin Paradoxical Effect

mBio ◽  
2017 ◽  
Vol 8 (3) ◽  
Author(s):  
Laure Nicolas Annick Ries ◽  
Marina Campos Rocha ◽  
Patrícia Alves de Castro ◽  
Rafael Silva-Rocha ◽  
Roberto Nascimento Silva ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Wall ◽  
Transcription Factor ◽  
Aspergillus Fumigatus ◽  
Chitin Synthase ◽  
Cell Wall Biosynthesis ◽  
Paradoxical Effect ◽  
Biosynthetic Genes ◽  
Fungal Cell ◽  
High Concentrations

ABSTRACT Aspergillus fumigatus is an opportunistic fungal pathogen that causes invasive aspergillosis (IA), a life-threatening disease in immunocompromised humans. The echinocandin caspofungin, adopted as a second-line therapy in combating IA, is a β-1,3-glucan synthase inhibitor, which, when used in high concentrations, reverts the anticipated A. fumigatus growth inhibition, a phenomenon called the “caspofungin paradoxical effect” (CPE). The CPE has been widely associated with increased chitin content in the cell wall due to a compensatory upregulation of chitin synthase-encoding genes. Here, we demonstrate that the CPE is dependent on the cell wall integrity (CWI) mitogen-activated protein kinase MpkAMPK1 and its associated transcription factor (TF) RlmARLM1, which regulate chitin synthase gene expression in response to different concentrations of caspofungin. Furthermore, the calcium- and calcineurin-dependent TF CrzA binds to and regulates the expression of specific chitin synthase genes during the CPE. These results suggest that the regulation of cell wall biosynthetic genes occurs by several cellular signaling pathways. In addition, CrzA is also involved in cell wall organization in the absence of caspofungin. Differences in the CPE were also observed between two A. fumigatus clinical isolates, which led to the identification of a novel basic leucine zipper TF, termed ZipD. This TF functions in the calcium-calcineurin pathway and is involved in the regulation of cell wall biosynthesis genes. This study therefore unraveled additional mechanisms and novel factors governing the CPE response, which ultimately could aid in developing more effective antifungal therapies. IMPORTANCE Systemic Aspergillus fumigatus infections are often accompanied by high mortality rates. The fungal cell wall is important for infection as it has immunomodulatory and immunoevasive properties. Paradoxical growth of A. fumigatus in the presence of high concentrations of the cell wall-disturbing agent caspofungin has been observed for more than a decade, although the mechanistic nature of this phenomenon remains largely uncharacterized. Here, we show that the CWI pathway components MpkA and RlmA as well as the calcium/calcineurin-responsive transcription factor CrzA regulate the expression of cell wall biosynthetic genes during the caspofungin paradoxical effect (CPE). Furthermore, an additional, novel calcium/calcineurin-responsive transcription factor was identified to play a role in cell wall biosynthesis gene expression during the CPE. This work paints a crucial role for calcium metabolism in the CPE and provides further insight into the complex regulation of cell wall biosynthesis, which could ultimately lead to the development of more efficient antifungal therapies. IMPORTANCE Systemic Aspergillus fumigatus infections are often accompanied by high mortality rates. The fungal cell wall is important for infection as it has immunomodulatory and immunoevasive properties. Paradoxical growth of A. fumigatus in the presence of high concentrations of the cell wall-disturbing agent caspofungin has been observed for more than a decade, although the mechanistic nature of this phenomenon remains largely uncharacterized. Here, we show that the CWI pathway components MpkA and RlmA as well as the calcium/calcineurin-responsive transcription factor CrzA regulate the expression of cell wall biosynthetic genes during the caspofungin paradoxical effect (CPE). Furthermore, an additional, novel calcium/calcineurin-responsive transcription factor was identified to play a role in cell wall biosynthesis gene expression during the CPE. This work paints a crucial role for calcium metabolism in the CPE and provides further insight into the complex regulation of cell wall biosynthesis, which could ultimately lead to the development of more efficient antifungal therapies.

scholarly journals Paradoxical Effect of Caspofungin: Reduced Activity against Candida albicans at High Drug Concentrations

2004 ◽  
Vol 48 (9) ◽  
pp. 3407-3411 ◽  
Author(s):  
David A. Stevens ◽  
Marife Espiritu ◽  
Rachana Parmar
Keyword(s):  
Candida Albicans ◽  
Resistance Mechanisms ◽  
Paradoxical Effect ◽  
Fungal Cell ◽  
High Drug ◽  
Minimum Fungicidal Concentration ◽  
Drug Concentrations ◽  
High Concentrations ◽  
Glucan Synthesis

ABSTRACT Resistance problems with caspofungin, an echinocandin inhibitor of fungal cell wall glucan synthesis, have been rare. We noted paradoxical turbid growth of Candida albicans isolates in broth in some high (supra-MIC) concentrations. Among isolates submitted for susceptibility testing and screened at drug concentrations up to 12.5 μg/ml, the frequency was 16%. Analysis of the turbid growth indicated slowing of growth in the presence of drug but with numbers of CFU up to 72% those of drug-free controls. Clearing of growth again by the highest drug concentrations produced a quadriphasic pattern in a tube dilution series. Cells growing at high drug concentrations were not resistant on retesting but showed the paradoxical effect of the parent. Among a selected series of isolates tested at concentrations up to 50 μg/ml, an additional 53% showed a “mini-paradoxical effect”: no turbid growth but incomplete killing at high concentrations (supra-minimum fungicidal concentration). These effects were reproducible; medium dependent in extent; noted in macro- and microdilution, in the presence or absence of serum, and on agar containing drug (but not when drug concentrations were not constant, as in agar diffusion); not seen with other echinocandins and less commonly in other Candida species; and not due to destruction of drug in tubes showing the effect. Cooperative enhancement of inhibition by a second drug could eradicate the effect. We postulate that high drug concentrations derepress or activate resistance mechanisms. The abilities of subpopulations to survive at high drug concentrations could have in vivo consequences.

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