scholarly journals Caspofungin-Mediated Growth Inhibition and Paradoxical Growth in Aspergillus fumigatus Involve Fungicidal Hyphal Tip Lysis Coupled with Regenerative Intrahyphal Growth and Dynamic Changes in β-1,3-Glucan Synthase Localization

2017 ◽  
Vol 61 (10) ◽  
Author(s):  
Sergio D. Moreno-Velásquez ◽  
Constanze Seidel ◽  
Praveen R. Juvvadi ◽  
William J. Steinbach ◽  
Nick D. Read
Keyword(s):  
Growth Inhibition ◽  
Aspergillus Fumigatus ◽  
Continuous Exposure ◽  
High Concentration ◽  
International Consensus ◽  
Glucan Synthase ◽  
Content Type ◽  
Growth Inhibitory ◽  
Paradoxical Growth ◽  
Hyphal Tip

ABSTRACT Caspofungin targets cell wall β-1,3-glucan synthesis and is the international consensus guideline-recommended salvage therapy for invasive aspergillosis. Although caspofungin is inhibitory at low concentrations, it exhibits a paradoxical effect (reversal of growth inhibition) at high concentrations by an undetermined mechanism. Treatment with caspofungin at either the growth-inhibitory concentration (0.5 μg/ml) or paradoxical growth-inducing concentration (4 μg/ml) for 24 h caused similar abnormalities, including wider, hyperbranched hyphae, increased septation, and repeated hyphal tip lysis, followed by regenerative intrahyphal growth. By 48 h, only hyphae at the colony periphery treated with the high caspofungin concentration displayed paradoxical growth. A similar high concentration of caspofungin also induced the paradoxical growth of Aspergillus fumigatus during human A549 alveolar cell invasion. Localization of the β-1,3-glucan synthase complex (Fks1 and Rho1) revealed significant differences between cells exposed to the growth-inhibitory and paradoxical growth-inducing concentrations of caspofungin. At both concentrations, Fks1 initially mislocalized from the hyphal tips to vacuoles. However, only continuous exposure to 4 μg/ml of caspofungin for 48 h led to recovery of the normal hyphal morphology with renewed localization of Fks1 to hyphal tips. Rho1 remained at the hyphal tip after treatment with both caspofungin concentrations but was required for paradoxical growth. Farnesol blocked paradoxical growth and relocalized Fks1 and Rho1 to vacuoles. Our results highlight the importance of regenerative intrahyphal growth as a rapid adaptation to the fungicidal lytic effects of caspofungin on hyphal tips and the dynamic localization of Fks1 as part of the mechanism for the caspofungin-mediated paradoxical response in A. fumigatus.

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 Evaluation of MIC Strip Isavuconazole Test for Susceptibility Testing of Wild-Type and Non-Wild-Type Aspergillus fumigatus Isolates

2016 ◽  
Vol 61 (1) ◽  
Author(s):  
Maiken Cavling Arendrup ◽  
Paul Verweij ◽  
Henrik Vedel Nielsen
Keyword(s):  
Growth Inhibition ◽  
Aspergillus Fumigatus ◽  
Susceptibility Testing ◽  
Wild Type ◽  
Content Type

ABSTRACT We evaluated the MIC Strip Isavuconazole test against EUCAST E.Def 9.3 by using 40 wild-type and 39 CYP51A mutant Aspergillus fumigatus strains. The strip full inhibition endpoint (FIE) and 80% growth inhibition endpoint were determined by two independent readers, reader 1 (R1) and R2. The essential (within ±0, ±1, and ±2 twofold dilutions) and categorical agreements were best with the FIE (for R1/R2, 42%/41%, 75%/73%, and 90%/89% for essential agreement, and 91.1%/92.4% categorical agreement, with 6.3/8.9% very major errors and 0/1.3% major errors, respectively). The MIC Strip Isavuconazole test with the FIE appears to be useful.

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 Growth-Inhibitory Effect of D-Tryptophan onVibriospp. in Shucked and Live Oysters

2018 ◽  
Vol 84 (19) ◽  
Author(s):  
Jian Chen ◽  
Hiroko Kudo ◽  
Kaito Kan ◽  
Shuso Kawamura ◽  
Shige Koseki
Keyword(s):  
Growth Inhibition ◽  
Ambient Temperature ◽  
Vibrio Parahaemolyticus ◽  
Vibrio Vulnificus ◽  
Inhibitory Effect ◽  
Artificial Seawater ◽  
Growth Inhibitory Effect ◽  
Content Type ◽  
Growth Inhibitory ◽  
Salinity Levels

ABSTRACTVibrio vulnificusandVibrio parahaemolyticusare important human pathogens that are frequently transmitted via consumption of contaminated raw oysters. A small amount ofd-tryptophan (d-Trp) inhibits some foodborne pathogenic bacteria in high-salt environments. In this study, we aimed to evaluate the antibacterial effect ofd-Trp onV. vulnificusandV. parahaemolyticusin culture media, artificial seawater, and shucked and live oysters. The effectiveness ofd-Trp in growth inhibition ofVibriospp. was highly dependent on environmental NaCl concentrations. Higher levels of NaCl (>4.0%) withd-Trp (>20 mM) resulted in higher and more consistent growth inhibition of bothVibriospp. Treatment with 40 mMd-Trp significantly (P< 0.05) reduced viableV. parahaemolyticuscell counts in tryptic soy broth (TSB) with >4.0% NaCl at 25°C. In contrast,V. vulnificuswas more sensitive tod-Trp (20 mM) thanV. parahaemolyticus.d-Trp (40 mM) treatment with NaCl (>4.5%) significantly (P< 0.05) inhibited the growth ofV. parahaemolyticusandV. vulnificusin shucked oysters immersed in peptone water at 25°C throughout a 48-h incubation period. In artificial seawater,d-Trp exhibited a stronger growth-inhibitory effect onV. vulnificusandV. parahaemolyticusat 25°C than in TSB at the same level of salinity and inhibited the growth of bothV. parahaemolyticusandV. vulnificusin live oysters at 25°C for 48 h. Furthermore, we tested the synergistic effect ofd-Trp and salinity on the inhibition of total viable bacterial counts (TVC) at refrigeration temperature.d-Trp (40 mM) inhibited the growth of TVC in shucked oysters immersed in artificial seawater at 4°C. Therefore, these results revealed thatd-Trp will serve as a novel and alternative food preservative to controlVibriospp. in live oysters at ambient temperature and to extend the shelf-life of shucked oysters at refrigeration temperature.IMPORTANCEOysters are the primary transmission vehicles for humanVibrioinfections. Raw oyster consumption is frequently associated with gastroenteritis. The current postharvest methods, such as high-pressure processing, used to controlVibriospp. in fresh oysters are still insufficient because of limited facilities, high cost, and potential adverse effects on production. We demonstrate that adding a small amount ofd-tryptophan (d-Trp) inhibits the growths ofVibrio parahaemolyticusandVibrio vulnificusin a high-salt environment at even ambient temperature. We further investigated thed-Trp treatment conditions and clarified the relationship between salt andd-Trp concentrations for optimal growth-inhibitory effect ofVibriospp. The results will be useful for enhancing the effectiveness ofd-Trp by increasing salinity levels. Furthermore, in a nutrientfree environment (artificial seawater), a stronger inhibitory effect could be observed at relatively lower salinity levels, indicating thatd-Trp may be regarded as effective food preservation in terms of salinity reduction. Therefore, we suggest the use of exogenousd-Trp in a seawater environment as a novel and effective strategy not only for controllingVibrioin live oysters at even ambient temperature but also for effectively retarding spoilage bacterial growth and extending the shelf life of shucked oysters at refrigeration temperature.

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 Calcium-Mediated Induction of Paradoxical Growth following Caspofungin Treatment Is Associated with Calcineurin Activation and Phosphorylation in Aspergillus fumigatus

2015 ◽  
Vol 59 (8) ◽  
pp. 4946-4955 ◽  
Author(s):  
Praveen R. Juvvadi ◽  
Alberto Muñoz ◽  
Frédéric Lamoth ◽  
Erik J. Soderblom ◽  
M. Arthur Moseley ◽  
...  
Keyword(s):  
Aspergillus Fumigatus ◽  
Expression System ◽  
Fungal Growth ◽  
Hyphal Growth ◽  
Underlying Mechanism ◽  
Paradoxical Effect ◽  
Content Type ◽  
Paradoxical Growth ◽  
High Concentrations ◽  
Calcineurin Signaling

ABSTRACTThe echinocandin antifungal drug caspofungin at high concentrations reverses the growth inhibition ofAspergillus fumigatus, a phenomenon known as the “paradoxical effect,” which is not consistently observed with other echinocandins (micafungin and anidulafungin). Previous studies ofA. fumigatusrevealed the loss of the paradoxical effect following pharmacological or genetic inhibition of calcineurin, yet the underlying mechanism is poorly understood. Here, we utilized a codon-optimized bioluminescent Ca2+reporter aequorin expression system inA. fumigatusand showed that caspofungin elicits a transient increase in cytosolic free Ca2+([Ca2+]c) in the fungus that acts as the initial trigger of the paradoxical effect by activating calmodulin-calcineurin signaling. While the increase in [Ca2+]cwas also observed upon treatment with micafungin, another echinocandin without the paradoxical effect, a higher [Ca2+]cincrease was noted with the paradoxical-growth concentration of caspofungin. Treatments with a Ca2+-selective chelator, BAPTA [1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid], or the L-type Ca2+channel blocker verapamil abolished caspofungin-mediated paradoxical growth in both the wild-type and the echinocandin-resistant (EMFR-S678P) strains. Concomitant with increased [Ca2+]clevels at higher concentrations of caspofungin, calmodulin and calcineurin gene expression was enhanced. Phosphoproteomic analysis revealed that calcineurin is activated through phosphorylation at its serine-proline-rich region (SPRR), a domain previously shown to be essential for regulation of hyphal growth, only at a paradoxical-growth concentration of caspofungin. Our results indicate that as opposed to micafungin, the increased [Ca2+]cat high concentrations of caspofungin activates calmodulin-calcineurin signaling at both a transcriptional and a posttranslational level and ultimately leads to paradoxical fungal growth.

scholarly journals Semi-Synthesis of Small Molecules of Aminocarbazoles: Tumor Growth Inhibition and Potential Impact on p53

Molecules ◽  
2021 ◽  
Vol 26 (6) ◽  
pp. 1637
Author(s):  
Solida Long ◽  
Joana B. Loureiro ◽  
Carla Carvalho ◽  
Luís Gales ◽  
Lucília Saraiva ◽  
...  
Keyword(s):  
Growth Inhibition ◽  
Small Molecules ◽  
Tumor Cells ◽  
Mutant P53 ◽  
Wild Type ◽  
Naturally Occurring ◽  
Growth Inhibitory ◽  
Growth Inhibitory Activity ◽  
Amino Derivatives ◽  
Carbazole Alkaloids

The tumor suppressor p53 is inactivated by mutation in approximately 50% of human cancers. Small molecules that bind and stabilize those mutants may represent effective anticancer drugs. Herein, we report the tumor cell growth inhibitory activity of carbazole alkaloids and amino derivatives, as well as their potential activation of p53. Twelve aminocarbazole alkaloids were semi-synthesized from heptaphylline (1), 7-methoxy heptaphylline (2), and 7-methoxymukonal (3), isolated from Clausena harmandiana, using a reductive amination protocol. Naturally-occurring carbazoles 1–3 and their amino derivatives were evaluated for their potential effect on wild-type and mutant p53 activity using a yeast screening assay and on human tumor cell lines. Naturally-occurring carbazoles 1–3 showed the most potent growth inhibitory effects on wild-type p53-expressing cells, being heptaphylline (1) the most promising in all the investigated cell lines. However, compound 1 also showed growth inhibition against non-tumor cells. Conversely, semi-synthetic aminocarbazole 1d showed an interesting growth inhibitory activity in tumor cells expressing both wild-type and mutant p53, exhibiting low growth inhibition on non-tumor cells. The yeast assay showed a potential reactivation of mutant p53 by heptaphylline derivatives, including compound 1d. The results obtained indicate that carbazole alkaloids may represent a promising starting point to search for new mutp53-reactivating agents with promising applications in cancer therapy.

scholarly journals Dectin-2-Targeted Antifungal Liposomes Exhibit Enhanced Efficacy

mSphere ◽  
2019 ◽  
Vol 4 (5) ◽  
Author(s):  
Suresh Ambati ◽  
Emma C. Ellis ◽  
Jianfeng Lin ◽  
Xiaorong Lin ◽  
Zachary A. Lewis ◽  
...  
Keyword(s):  
Candida Albicans ◽  
Aspergillus Fumigatus ◽  
Effective Dose ◽  
Cryptococcus Neoformans ◽  
Amphotericin B ◽  
Antifungal Drugs ◽  
Extracellular Matrices ◽  
Content Type ◽  
Order Of Magnitude ◽  
Life Threatening

ABSTRACT Candida albicans, Cryptococcus neoformans, and Aspergillus fumigatus cause life-threatening candidiasis, cryptococcosis, and aspergillosis, resulting in several hundred thousand deaths annually. The patients at the greatest risk of developing these life-threatening invasive fungal infections have weakened immune systems. The vulnerable population is increasing due to rising numbers of immunocompromised individuals as a result of HIV infection or immunosuppressed individuals receiving anticancer therapies and/or stem cell or organ transplants. While patients are treated with antifungals such as amphotericin B, all antifungals have serious limitations due to lack of sufficient fungicidal effect and/or host toxicity. Even with treatment, 1-year survival rates are low. We explored methods of increasing drug effectiveness by designing fungicide-loaded liposomes specifically targeted to fungal cells. Most pathogenic fungi are encased in cell walls and exopolysaccharide matrices rich in mannans. Dectin-2 is a mammalian innate immune membrane receptor that binds as a dimer to mannans and signals fungal infection. We coated amphotericin-loaded liposomes with monomers of Dectin-2’s mannan-binding domain, sDectin-2. sDectin monomers were free to float in the lipid membrane and form dimers that bind mannan substrates. sDectin-2-coated liposomes bound orders of magnitude more efficiently to the extracellular matrices of several developmental stages of C. albicans, C. neoformans, and A. fumigatus than untargeted control liposomes. Dectin-2-coated amphotericin B-loaded liposomes reduced the growth and viability of all three species more than an order of magnitude more efficiently than untargeted control liposomes and dramatically decreased the effective dose. Future efforts focus on examining pan-antifungal targeted liposomal drugs in animal models of fungal diseases. IMPORTANCE Invasive fungal diseases caused by Candida albicans, Cryptococcus neoformans, and Aspergillus fumigatus have mortality rates ranging from 10 to 95%. Individual patient costs may exceed $100,000 in the United States. All antifungals in current use have serious limitations due to host toxicity and/or insufficient fungal cell killing that results in recurrent infections. Few new antifungal drugs have been introduced in the last 2 decades. Hence, there is a critical need for improved antifungal therapeutics. By targeting antifungal-loaded liposomes to α-mannans in the extracellular matrices secreted by these fungi, we dramatically reduced the effective dose of drug. Dectin-2-coated liposomes loaded with amphotericin B bound 50- to 150-fold more strongly to C. albicans, C. neoformans, and A. fumigatus than untargeted liposomes and killed these fungi more than an order of magnitude more efficiently. Targeting drug-loaded liposomes specifically to fungal cells has the potential to greatly enhance the efficacy of most antifungal drugs.

scholarly journals In Vitro Activity of Ibrexafungerp, a Novel Glucan Synthase Inhibitor against Candida glabrata Isolates with FKS Mutations

2019 ◽  
Vol 63 (11) ◽  
Author(s):  
Natalie S. Nunnally ◽  
Kizee A. Etienne ◽  
David Angulo ◽  
Shawn R. Lockhart ◽  
Elizabeth L. Berkow
Keyword(s):  
Candida Glabrata ◽  
Vitro Activity ◽  
Good Activity ◽  
In Vitro Activity ◽  
Broth Microdilution ◽  
Glucan Synthase ◽  
Content Type ◽  
Synthase Inhibitor

ABSTRACT Ibrexafungerp is a first-in-class glucan synthase inhibitor. In vitro activity was determined for 89 Candida glabrata isolates with molecularly identified FKS1 or FKS2 mutations conferring resistance to the echinocandins. All isolates were resistant to at least one echinocandin (i.e., anidulafungin, caspofungin, or micafungin) by broth microdilution. Results for ibrexafungerp were compared with those for each echinocandin. Ibrexafungerp had good activity against all echinocandin-resistant C. glabrata isolates.

scholarly journals Discovery of a Novel Class of Orally Active Antifungal β-1,3-d-Glucan Synthase Inhibitors

2011 ◽  
Vol 55 (11) ◽  
pp. 5099-5106 ◽  
Author(s):  
Scott S. Walker ◽  
Yiming Xu ◽  
Ilias Triantafyllou ◽  
Michelle F. Waldman ◽  
Cara Mendrick ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Candida Glabrata ◽  
Pharmacokinetic Data ◽  
Morphological Response ◽  
Glucan Synthase ◽  
Content Type ◽  
Safety Issues ◽  
Spontaneous Mutants ◽  
Orally Active

ABSTRACTThe echinocandins are a class of semisynthetic natural products that target β-1,3-glucan synthase (GS). Their proven clinical efficacy combined with minimal safety issues has made the echinocandins an important asset in the management of fungal infection in a variety of patient populations. However, the echinocandins are delivered only parenterally. A screen for antifungal bioactivities combined with mechanism-of-action studies identified a class of piperazinyl-pyridazinones that target GS. The compounds exhibitedin vitroactivity comparable, and in some cases superior, to that of the echinocandins. The compounds inhibit GSin vitro, and there was a strong correlation between enzyme inhibition andin vitroantifungal activity. In addition, like the echinocandins, the compounds caused a leakage of cytoplasmic contents from yeast and produced a morphological response in molds characteristic of GS inhibitors. Spontaneous mutants ofSaccharomyces cerevisiaewith reduced susceptibility to the piperazinyl-pyridazinones had substitutions inFKS1. The sites of these substitutions were distinct from those conferring resistance to echinocandins; likewise, echinocandin-resistant isolates remained susceptible to the test compounds. Finally, we present efficacy and pharmacokinetic data on an example of the piperazinyl-pyridazinone compounds that demonstrated efficacy in a murine model ofCandida glabratainfection.

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