scholarly journals Identification of New Antifungal Agents Targeting Chitin Synthesis by a Chemical-Genetic Method

Molecules ◽  
2019 ◽  
Vol 24 (17) ◽  
pp. 3155
Author(s):  
Li ◽  
Sun ◽  
Zhu ◽  
Bian ◽  
Wang ◽  
...  
Keyword(s):  
Cell Wall ◽  
Antifungal Agents ◽  
Antifungal Drugs ◽  
Chitin Synthase ◽  
Yeast Cells ◽  
Mass Spectrometry Analysis ◽  
Chitin Synthesis ◽  
Glucan Synthase ◽  
Genetic Method ◽  
Chemical Genetic

Fungal infection is a leading cause of mortality in immunocompromised population; thus, it is urgent to develop new and safe antifungal agents. Different from human cells, fungi have a cell wall, which is composed mainly of polysaccharide glucan and chitin. The unique cell wall structure is an ideal target for antifungal drugs. In this research, a chemical-genetic method was used to isolate antifungal agents that target chitin synthesis in yeast cells. From a compound library, we isolated two benzothiazole compounds that showed greater toxicity to yeast mutants lacking glucan synthase Fks1 compared to wild-type yeast cells and mutants lacking chitin synthase Chs3. Both of them inhibited the activity of chitin synthase in vitro and reduced chitin level in yeast cells. Besides, these compounds showed clear synergistic antifungal effect with a glucan synthase inhibitors caspofungin. Furthermore, these compounds inhibited the growth of Saccharomyces cerevisiae and opportunistic pathogen Candida albicans. Surprisingly, the genome-wide mass-spectrometry analysis showed decreased protein level of chitin synthases in cells treated with one of these drugs, and this decrease was not a result of downregulation of gene transcription. Therefore, we successfully identified two new antifungal agents that inhibit chitin synthesis using a chemical-genetic method.

scholarly journals A Chitin Synthase and Its Regulator Protein Are Critical for Chitosan Production and Growth of the Fungal Pathogen Cryptococcus neoformans

2005 ◽  
Vol 4 (11) ◽  
pp. 1902-1912 ◽  
Author(s):  
Isaac R. Banks ◽  
Charles A. Specht ◽  
Maureen J. Donlin ◽  
Kimberly J. Gerik ◽  
Stuart M. Levitz ◽  
...  
Keyword(s):  
Cell Wall ◽  
Cryptococcus Neoformans ◽  
Fungal Pathogen ◽  
Liquid Culture ◽  
Chitin Synthase ◽  
Yeast Cells ◽  
Chitin Synthesis ◽  
Chitin Synthases ◽  
Regulator Protein ◽  
Chitin And Chitosan

ABSTRACT Chitin is an essential component of the cell wall of many fungi. Chitin also can be enzymatically deacetylated to chitosan, a more flexible and soluble polymer. Cryptococcus neoformans is a fungal pathogen that causes cryptococcal meningoencephalitis, particularly in immunocompromised patients. In this work, we show that both chitin and chitosan are present in the cell wall of vegetatively growing C. neoformans yeast cells and that the levels of both rise dramatically as cells grow to higher density in liquid culture. C. neoformans has eight putative chitin synthases, and strains with any one chitin synthase deleted are viable at 30°C. In addition, C. neoformans genes encode three putative regulator proteins, which are homologs of Saccharomyces cerevisiae Skt5p. None of these three is essential for viability. However, one of the chitin synthases (Chs3) and one of the regulators (Csr2) are important for growth. Cells with deletions in either CHS3 or CSR2 have several shared phenotypes, including sensitivity to growth at 37°C. The similarity of their phenotypes also suggests that Csr2 specifically regulates chitin synthesis by Chs3. Lastly, both chs3Δ and the csr2Δ mutants are defective in chitosan production, predicting that Chs3-Csr2 complex with chitin deacetylases for conversion of chitin to chitosan. These data suggest that chitin synthesis could be an excellent antifungal target.

scholarly journals Crosstalk between calcineurin and the cell wall integrity pathways prevents chitin overexpression in Candida albicans

2021 ◽  
Author(s):  
Alessandra da Silva Dantas ◽  
Filomena Nogueira ◽  
Keunsook K. Lee ◽  
Louise A. Walker ◽  
Matt Edmondson ◽  
...  
Keyword(s):  
Cell Wall ◽  
Candida Albicans ◽  
Antifungal Drugs ◽  
Cell Wall Integrity ◽  
Yeast Cells ◽  
Outer Cell ◽  
Chitin Synthesis ◽  
Simultaneous Activation ◽  
Glucan Synthesis ◽  
Calcineurin Pathway

Echinocandins such as caspofungin are front line antifungal drugs that compromise β-1,3 glucan synthesis in the cell wall. Recent reports have shown that fungal cells can resist killing by caspofungin by up-regulation of chitin synthesis, thereby sustaining cell wall integrity. When echinocandins are removed, the chitin content of cells quickly returns to basal levels, suggesting that there is a fitness cost associated with having elevated levels of chitin in the cell wall. We show here that simultaneous activation of the calcineurin and CWI pathways generates a sub-population of Candida albicans yeast cells that have supra-normal chitin levels interspersed throughout the inner and outer cell wall, and that these cells are non-viable, perhaps due to loss of wall elasticity required for cell expansion and growth. Mutations in the Ca2+-calcineurin pathway prevented the formation of these non-viable super high chitin cells by negatively regulating chitin synthesis driven by the CWI pathway. The Ca2+-calcineurin pathway may therefore act as an attenuator that prevents the overproduction of chitin by coordinating both chitin upregulation and negative regulation of the CWI signaling pathway.

Potent Chitin Synthase Inhibitors from Plants

2020 ◽  
Vol 16 (1) ◽  
pp. 58-63
Author(s):  
Amrutha Vijayakumar ◽  
Ajith Madhavan ◽  
Chinchu Bose ◽  
Pandurangan Nanjan ◽  
Sindhu S. Kokkal ◽  
...  
Keyword(s):  
Cell Wall ◽  
Candida Albicans ◽  
Caenorhabditis Elegans ◽  
Aspergillus Niger ◽  
Small Molecules ◽  
Tip Growth ◽  
Chitin Synthase ◽  
Chitin Synthesis ◽  
Hyphal Tip Growth ◽  
Hyphal Tip

Background: Chitin is the main component of fungal, protozoan and helminth cell wall. They help to maintain the structural and functional characteristics of these organisms. The chitin wall is dynamic and is repaired, rearranged and synthesized as the cells develop. Active synthesis can be noticed during cytokinesis, laying of primary septum, maintenance of lateral cell wall integrity and hyphal tip growth. Chitin synthesis involves coordinated action of two enzymes namely, chitin synthase (that lays new cell wall) and chitinase (that removes the older ones). Since chitin synthase is conserved in different eukaryotic microorganisms that can be a ‘soft target’ for inhibition with small molecules. When chitin synthase is inhibited, it leads to the loss of viability of cells owing to the self- disruption of the cell wall by existing chitinase. Methods: In the described study, small molecules from plant sources were screened for their ability to interfere with hyphal tip growth, by employing Hyphal Tip Burst assay (HTB). Aspergillus niger was used as the model organism. The specific role of these small molecules in interfering with chitin synthesis was established with an in-vitro method. The enzyme required was isolated from Aspergillus niger and its activity was deduced through a novel method involving non-radioactively labelled substrate. The activity of the potential lead molecules were also checked against Candida albicans and Caenorhabditis elegans. The latter was adopted as a surrogate for the pathogenic helminths as it shares similarity with regard to cell wall structure and biochemistry. Moreover, it is widely studied and the methodologies are well established. Results: Out of the 11 compounds and extracts screened, 8 were found to be prospective. They were also found to be effective against Candida albicans and Caenorhabditis elegans. Conclusion: Purified Methyl Ethyl Ketone (MEK) Fraction1 (F1) of Coconut (Cocos nucifera) Shell Extract (COSE) was found to be more effective against Candida albicans with an IC50 value of 3.04 μg/mL and on L4 stage of Caenorhabditis elegans with an IC50 of 77.8 μg/mL.

Localisation of the Schizosaccharomyces pombe rho1p GTPase and its involvement in the organisation of the actin cytoskeleton

1997 ◽  
Vol 110 (20) ◽  
pp. 2547-2555 ◽  
Author(s):  
M. Arellano ◽  
A. Duran ◽  
P. Perez
Keyword(s):  
Cell Wall ◽  
Actin Cytoskeleton ◽  
Schizosaccharomyces Pombe ◽  
Antifungal Drugs ◽  
Dominant Negative ◽  
Cortical Actin ◽  
Glucan Synthase ◽  
Cell Wall Growth ◽  
Wall Growth ◽  
Mutant Cells

The Schizosaccharomyces pombe rho1p GTPase directly activates the (1–3) beta-D-glucan synthase and participates in the regulation of cell wall growth and morphogenesis in this fission yeast. Indirect immunofluorescence experiments using rho1p tagged with hemagglutinin have revealed that rho1p was located at the growing tips during interphase and at the septum prior to cytokinesis, localising to the same areas as actin patches. In S. pombe cdc10-129 mutant cells, arrested in G1, HA-rho1p accumulates at one tip whereas in cdc25-22 mutants, arrested in G2, HA-rho1p accumulates at both tips. In tea1-1 and tea2-1 cdc11-119 mutant cells, HA-rho1p is localised to the new growing tips. Overexpression of different rho1 mutant alleles caused different effects on cortical actin patch distribution, (1–3) beta-D-glucan synthase activation, and sensitivity to cell wall specific antifungal drugs. These results indicate that multiple cellular components are activated by rho1p. Overexpression of the dominant negative rho1T20N allele was lethal as was the rho1+ deletion. Moreover, when rho1+ expression was repressed in actively growing S. pombe, cells died in about 10 to 12 hours. Under these conditions, normal cell morphology was maintained but the level of (1–3) beta-D-glucan synthase activity decreased and the actin patches disappeared. Most cells lysed after cytokinesis during the process of separation, and lysis was not prevented by an osmotic stabiliser. We conclude that rho1p localisation is restricted to growth areas and regulated during the cell cycle and that rho1p is involved in cell wall growth and actin cytoskeleton organisation in S. pombe.

scholarly journals The Antifungal Protein AFP from Aspergillus giganteus Inhibits Chitin Synthesis in Sensitive Fungi

2007 ◽  
Vol 73 (7) ◽  
pp. 2128-2134 ◽  
Author(s):  
Silke Hagen ◽  
Florentine Marx ◽  
Arthur F. Ram ◽  
Vera Meyer
Keyword(s):  
Cell Wall ◽  
Wall Stress ◽  
Chitin Synthase ◽  
Antifungal Protein ◽  
Cell Integrity ◽  
Class Iii ◽  
Chitin Synthesis ◽  
Aspergillus Giganteus ◽  
Cell Wall Stress ◽  
Sytox Green

ABSTRACT The antifungal protein AFP from Aspergillus giganteus is highly effective in restricting the growth of major human- and plant-pathogenic filamentous fungi. However, a fundamental prerequisite for the use of AFP as an antifungal drug is a complete understanding of its mode of action. In this study, we performed several analyses focusing on the assumption that the chitin biosynthesis of sensitive fungi is targeted by AFP. Here we show that the N-terminal domain of AFP (amino acids 1 to 33) is sufficient for efficient binding of AFP to chitin but is not adequate for inhibition of the growth of sensitive fungi. AFP susceptibility tests and SYTOX Green uptake experiments with class III and class V chitin synthase mutants of Fusarium oxysporum and Aspergillus oryzae showed that deletions made the fungi less sensitive to AFP and its membrane permeabilization effect. In situ chitin synthase activity assays revealed that chitin synthesis is specifically inhibited by AFP in sensitive fungi, indicating that AFP causes cell wall stress and disturbs cell integrity. Further evidence that there was AFP-induced cell wall stress was obtained by using an Aspergillus niger reporter strain in which the cell wall integrity pathway was strongly induced by AFP.

scholarly journals Fluconazole and Lipopeptide Surfactin Interplay During Candida albicans Plasma Membrane and Cell Wall Remodeling Increases Fungal Immune System Exposure

Pharmaceutics ◽  
2020 ◽  
Vol 12 (4) ◽  
pp. 314 ◽  
Author(s):  
Jakub Suchodolski ◽  
Daria Derkacz ◽  
Jakub Muraszko ◽  
Jarosław J. Panek ◽  
Aneta Jezierska ◽  
...  
Keyword(s):  
Cell Wall ◽  
Candida Albicans ◽  
Plasma Membrane ◽  
Immune System ◽  
Antifungal Drugs ◽  
Stable Complex ◽  
Host Immune System ◽  
Chitin Synthesis ◽  
Fungal Cell ◽  
In Silico Studies

Recognizing the β-glucan component of the Candida albicans cell wall is a necessary step involved in host immune system recognition. Compounds that result in exposed β-glucan recognizable to the immune system could be valuable antifungal drugs. Antifungal development is especially important because fungi are becoming increasingly drug resistant. This study demonstrates that lipopeptide, surfactin, unmasks β-glucan when the C. albicans cells lack ergosterol. This observation also holds when ergosterol is depleted by fluconazole. Surfactin does not enhance the effects of local chitin accumulation in the presence of fluconazole. Expression of the CHS3 gene, encoding a gene product resulting in 80% of cellular chitin, is downregulated. C. albicans exposure to fluconazole changes the composition and structure of the fungal plasma membrane. At the same time, the fungal cell wall is altered and remodeled in a way that makes the fungi susceptible to surfactin. In silico studies show that surfactin can form a complex with β-glucan. Surfactin forms a less stable complex with chitin, which in combination with lowering chitin synthesis, could be a second anti-fungal mechanism of action of this lipopeptide.

scholarly journals Drug Interaction Studies of a Glucan Synthase Inhibitor (LY 303366) and a Chitin Synthase Inhibitor (Nikkomycin Z) for Inhibition and Killing of Fungal Pathogens

2000 ◽  
Vol 44 (9) ◽  
pp. 2547-2548 ◽  
Author(s):  
David A. Stevens
Keyword(s):  
Cell Wall ◽  
Fungal Pathogens ◽  
Enzyme Inhibitors ◽  
Chitin Synthase ◽  
Synergistic Activity ◽  
Fungal Cell ◽  
Glucan Synthase ◽  
Nikkomycin Z ◽  
Synthase Inhibitor

ABSTRACT The interaction between inhibitors of components of the fungal cell wall, glucan and chitin, was studied in vitro with the respective synthase enzyme inhibitors LY 303366 and nikkomycin Z. WithAspergillus fumigatus synergy was noted for inhibition and killing, and synergistic activity was also noted for some isolates of other species presently regarded as difficult to treat.

scholarly journals Caspofungin Treatment of Aspergillus fumigatus Results in ChsG-Dependent Upregulation of Chitin Synthesis and the Formation of Chitin-Rich Microcolonies

2015 ◽  
Vol 59 (10) ◽  
pp. 5932-5941 ◽  
Author(s):  
Louise A. Walker ◽  
Keunsook K. Lee ◽  
Carol A. Munro ◽  
Neil A. R. Gow
Keyword(s):  
Cell Wall ◽  
Aspergillus Fumigatus ◽  
Dynamic Imaging ◽  
Chitin Synthase ◽  
Response To Treatment ◽  
Chitin Synthesis ◽  
Calcofluor White ◽  
Content Type ◽  
Chitin Content

ABSTRACTTreatment ofAspergillus fumigatuswith echinocandins such as caspofungin inhibits the synthesis of cell wall β-1,3-glucan, which triggers a compensatory stimulation of chitin synthesis. Activation of chitin synthesis can occur in response to sub-MICs of caspofungin and to CaCl2and calcofluor white (CFW), agonists of the protein kinase C (PKC), and Ca2+-calcineurin signaling pathways.A. fumigatusmutants with thechsgene (encoding chitin synthase) deleted (ΔAfchs) were tested for their response to these agonists to determine the chitin synthase enzymes that were required for the compensatory upregulation of chitin synthesis. Only the ΔAfchsGmutant was hypersensitive to caspofungin, and all other ΔAfchsmutants tested remained capable of increasing their chitin content in response to treatment with CaCl2and CFW and caspofungin. The resulting increase in cell wall chitin content correlated with reduced susceptibility to caspofungin in the wild type and all ΔAfchsmutants tested, with the exception of the ΔAfchsGmutant, which remained sensitive to caspofungin.In vitroexposure to the chitin synthase inhibitor, nikkomycin Z, along with caspofungin demonstrated synergistic efficacy that was againAfChsG dependent. Dynamic imaging using microfluidic perfusion chambers demonstrated that treatment with sub-MIC caspofungin resulted initially in hyphal tip lysis. However, thickened hyphae emerged that formed aberrant microcolonies in the continued presence of caspofungin. In addition, intrahyphal hyphae were formed in response to echinocandin treatment. Thesein vitrodata demonstrate thatA. fumigatushas the potential to survive echinocandin treatmentin vivobyAfChsG-dependent upregulation of chitin synthesis. Chitin-rich cells may, therefore, persist in human tissues and act as the focus for breakthrough infections.

scholarly journals Differential Aspergillus lentulus Echinocandin Susceptibilities Are Fksp Independent

2010 ◽  
Vol 54 (12) ◽  
pp. 4992-4998 ◽  
Author(s):  
Janet F. Staab ◽  
Jennifer Nielsen Kahn ◽  
Kieren A. Marr
Keyword(s):  
Cell Wall ◽  
Aspergillus Fumigatus ◽  
Hot Spot ◽  
Cellular Level ◽  
Antifungal Drugs ◽  
Gene Encoding ◽  
Glucan Synthase ◽  
Kinetic Inhibition ◽  
Aspergillus Lentulus

ABSTRACT The recently described species Aspergillus lentulus exhibits differential and reduced susceptibilities to echinocandins and other antifungal drugs in vitro. A. lentulus isolates overall are less susceptible to caspofungin, although they maintain susceptibility to anidulafungin and micafungin. Mutations or polymorphisms in fks, the gene encoding the catalytic subunit of β-1,3-glucan synthase, are known to confer decreased susceptibility to echinocandins in Candida spp. and Aspergillus fumigatus. The analysis of the A. lentulus fks sequence did not reveal a polymorphism at any of the known hot-spot regions of the gene. Caspofungin and micafungin kinetic inhibition profiles of the A. lentulus glucan synthase were comparable to those from susceptible A. fumigatus enzymes. Although the basal cell wall chitin levels in A. lentulus averaged 60% of those in A. fumigatus, echinocandin treatment promoted the increase of cell wall chitin in both organisms, indicating that A. lentulus displays a compensatory chitin response similar to that of A. fumigatus. The data suggest that differential echinocandin susceptibilities in A. lentulus are independent of the echinocandin target, Fksp, and they emphasize the potential that the drugs' capacity to inhibit the target enzyme is unequal at the cellular level.

scholarly journals S. cerevisiae Outer and Inner Membranes are Compromised upon Benzyl Alcohol Treatment

2021 ◽  
Vol 8 (2) ◽  
pp. 35-39
Author(s):  
Ergüden Bengü
Keyword(s):  
Cell Wall ◽  
Cell Membrane ◽  
Yeast Cell ◽  
Benzyl Alcohol ◽  
Cell Membranes ◽  
Antifungal Agents ◽  
Fungal Infections ◽  
New Drugs ◽  
Yeast Cells ◽  
Fungal Cell

Although there are innovations in the treatment of diseases caused by fungi and medicines with multiple targets have been developed, the search for a drug with a broad spectrum and without any side effects continues to date. It is generally accepted that determining the cellular target responsible for the toxic effect opens up new possibilities for the development of new drugs. Especially the effects of antifungal agents on the surface components of the fungal cell, on cell wall synthesis and the identification of the target site are crucial in antifungal drug development. Thus studies on the fungal cell membranes in connection with the antifungal agents, aim to develop new strategies for the therapy of fungal infections. Antifungal agents targeting fungal cell wall and cell membrane components have increased in importance in clinical studies. In this study, understanding the mechanism of action of benzyl alcohol, a known membrane fluidizer, and the determination of its cellular targets are aimed. We have shown that in the presence of sorbitol, the osmotic stabilizer, benzyl alcohol becomes less effective against yeast cell. Moreover, benzyl alcohol disrupts cell membrane, causing leakage of ions to the extracellular medium. Nuclear membrane is distorted upon treatment of yeast cells with benzyl alcohol. Thus, we conclude that both outer and inner yeast cell membranes are compromised by the action of benzyl alcohol.

Sign in / Sign up

Export Citation Format

Share Document