scholarly journals Stereoselective interaction of the azole antifungal agent SCH39304 with the cytochrome P-450 monooxygenase system isolated from Cryptococcus neoformans.

1997 ◽  
Vol 41 (7) ◽  
pp. 1465-1467 ◽  
Author(s):  
D C Lamb ◽  
B C Baldwin ◽  
K J Kwon-Chung ◽  
S L Kelly
Keyword(s):  
Cryptococcus Neoformans ◽  
Pathogenic Fungus ◽  
Ergosterol Biosynthesis ◽  
Classical Type ◽  
Monooxygenase System ◽  
Inhibition Of Growth ◽  
Fluconazole Therapy ◽  
Specific Localization ◽  
Cytochrome P 450

We investigated the stereoselective inhibition of growth and ergosterol biosynthesis by SCH39304 in the pathogenic fungus Cryptococcus neoformans obtained from four AIDS patients who failed fluconazole therapy and compared the results to those obtained with a wild-type strain. For all strains, the MICs of the RR isomer were approximately half those of the racemate, with the SS enantiomer showing no inhibitory activity. The 50% inhibitory concentrations for in vitro ergosterol biosynthesis correlated with the MIC data, indicating stereoselective inhibition of their target P-450 enzyme, sterol 14alpha-demethylase, as the cause of this difference. The RR enantiomer produced classical type II spectra on addition to microsomal extracts of the strains, whereas the SS enantiomer showed an absence of binding. Stereo- and regio-specific localization of N-1 substituent groups of SCH39304 within the active site of the enzyme determined the unique discrimination between its two enantiomers, and the inability to bind to sterol 14alpha-demethylase is also true of other P-450 enzymes contained in the microsomal fraction. As previously observed for other antifungal azoles, isolates obtained following failure of fluconazole therapy showed resistance to SCH39304 and its RR enantiomer. This resistance could be associated with an alteration in the sensitivity of ergosterol biosynthesis in vitro. These alterations did not cause any changes allowing the SS enantiomer to bind to the P-450 mediating sterol 14alpha-demethylation.

scholarly journals Structure and inhibition of Cryptococcus neoformans sterylglucosidase to develop antifungal agents

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Nivea Pereira de Sa ◽  
Adam Taouil ◽  
Jinwoo Kim ◽  
Timothy Clement ◽  
Reece M. Hoffmann ◽  
...  
Keyword(s):  
Cryptococcus Neoformans ◽  
Rational Design ◽  
Antifungal Agents ◽  
Pathogenic Fungus ◽  
Pathogenic Fungi ◽  
Growth Defect ◽  
Wild Type ◽  
Secondary Infections ◽  
Heavy Burden

AbstractPathogenic fungi exhibit a heavy burden on medical care and new therapies are needed. Here, we develop the fungal specific enzyme sterylglucosidase 1 (Sgl1) as a therapeutic target. Sgl1 converts the immunomodulatory glycolipid ergosterol 3β-D-glucoside to ergosterol and glucose. Previously, we found that genetic deletion of Sgl1 in the pathogenic fungus Cryptococcus neoformans (Cn) results in ergosterol 3β-D-glucoside accumulation, renders Cn non-pathogenic, and immunizes mice against secondary infections by wild-type Cn, even in condition of CD4+ T cell deficiency. Here, we disclose two distinct chemical classes that inhibit Sgl1 function in vitro and in Cn cells. Pharmacological inhibition of Sgl1 phenocopies a growth defect of the Cn Δsgl1 mutant and prevents dissemination of wild-type Cn to the brain in a mouse model of infection. Crystal structures of Sgl1 alone and with inhibitors explain Sgl1’s substrate specificity and enable the rational design of antifungal agents targeting Sgl1.

scholarly journals Structural analysis of fungal pathogenicity-related casein kinase α subunit, Cka1, in the human fungal pathogen Cryptococcus neoformans

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Belinda X. Ong ◽  
Youngki Yoo ◽  
Myeong Gil Han ◽  
Jun Bae Park ◽  
Myung Kyung Choi ◽  
...  
Keyword(s):  
Cryptococcus Neoformans ◽  
Pathogenic Fungus ◽  
Kinase Assay ◽  
Α Subunit ◽  
Dynamic Architecture ◽  
Human Fungal Pathogen ◽  
Life Threatening ◽  
The Difference ◽  
Anti Fungal

Abstract CK2α is a constitutively active and highly conserved serine/threonine protein kinase that is involved in the regulation of key cellular metabolic pathways and associated with a variety of tumours and cancers. The most well-known CK2α inhibitor is the human clinical trial candidate CX-4945, which has recently shown to exhibit not only anti-cancer, but also anti-fungal properties. This prompted us to work on the CK2α orthologue, Cka1, from the pathogenic fungus Cryptococcus neoformans, which causes life-threatening systemic cryptococcosis and meningoencephalitis mainly in immunocompromised individuals. At present, treatment of cryptococcosis remains a challenge due to limited anti-cryptococcal therapeutic strategies. Hence, expanding therapeutic options for the treatment of the disease is highly clinically relevant. Herein, we report the structures of Cka1-AMPPNP-Mg2+ (2.40 Å) and Cka1-CX-4945 (2.09 Å). Structural comparisons of Cka1-AMPPNP-Mg2+ with other orthologues revealed the dynamic architecture of the N-lobe across species. This may explain for the difference in binding affinities and deviations in protein-inhibitor interactions between Cka1-CX-4945 and human CK2α-CX-4945. Supporting it, in vitro kinase assay demonstrated that CX-4945 inhibited human CK2α much more efficiently than Cka1. Our results provide structural insights into the design of more selective inhibitors against Cka1.

scholarly journals Identification of ENA1 as a Virulence Gene of the Human Pathogenic Fungus Cryptococcus neoformans through Signature-Tagged Insertional Mutagenesis

2009 ◽  
Vol 8 (3) ◽  
pp. 315-326 ◽  
Author(s):  
Alexander Idnurm ◽  
Felicia J. Walton ◽  
Anna Floyd ◽  
Jennifer L. Reedy ◽  
Joseph Heitman
Keyword(s):  
Cryptococcus Neoformans ◽  
Fungal Pathogens ◽  
Insertional Mutagenesis ◽  
Pathogenic Fungus ◽  
Mammalian Host ◽  
New Genes ◽  
Strain Collection ◽  
Mutant Strains ◽  
Human Pathogenic Fungus

ABSTRACT A library of more than 4,500 signature-tagged insertion mutants of the human pathogenic fungus Cryptococcus neoformans was generated, and a subset was screened in a murine inhalation model to identify genes required for virulence. New genes that regulate aspects of C. neoformans virulence were also identified by screening the entire library for in vitro phenotypes related to the ability to cause disease, including melanin production, growth at high temperature, and growth under conditions of nutrient limitation. A screen of 10% of the strain collection in mice identified an avirulent mutant strain with an insertion in the ENA1 gene, which is predicted to encode a fungus-specific sodium or potassium P-type ATPase. The results of the deletion of the gene and complementation experiments confirmed its key role in mammalian virulence. ena1 mutant strains exhibited no change in sensitivity to high salt concentrations but were sensitive to alkaline pH conditions, providing evidence that the fungus may have to survive at elevated pH during infection of the mammalian host. The mutation of the well-characterized virulence factor calcineurin (CNA1) also rendered C. neoformans strains sensitive to elevated pH. ENA1 transcripts in wild-type and cna1 mutant strains were upregulated in response to high pH, and cna1 ena1 double mutant strains exhibited increased sensitivity to elevated pH, indicating that at least two pathways in the fungus mediate survival under alkaline conditions. Signature-tagged mutagenesis is an effective strategy for the discovery of new virulence genes in fungal pathogens of animals.

NADPH Cytochrome P-450 Oxidoreductase and Susceptibility to Ketoconazole

1998 ◽  
Vol 42 (7) ◽  
pp. 1756-1761 ◽  
Author(s):  
K. Venkateswarlu ◽  
Diane E. Kelly ◽  
Nigel J. Manning ◽  
Steven L. Kelly
Keyword(s):  
Ergosterol Biosynthesis ◽  
Squalene Epoxidase ◽  
Wild Type ◽  
Gene Encoding ◽  
Nadph Cytochrome ◽  
Cytochrome P 450 ◽  
Different Levels ◽  
Donor System

ABSTRACT The phenotype of a strain of Saccharomyces cerevisiaecontaining a disruption of the gene encoding NADPH cytochrome P-450 oxidoreductase (CPR) was quantified biochemically and microbiologically, as were those of various transformants of this strain after expression of native CPR, cytochrome P-45051 (CYP51), and a fusion protein of CYP51-CPR (FUS). Only a 4-fold decrease in ergosterol biosynthesis was observed for the cpr strain, but ketoconazole sensitivity increased 200-fold, indicating hypersensitivity to the alternative electron donor system incpr strains. Both phenotypes could be reversed in transformants expressing the CPR and FUS, indicating the availability of the CPR in FUS as well as the expressed native CPR for monoxygenase-associated reactions. The complementation of function was observed both in vitro and in vivo for the monoxygenases squalene epoxidase, CYP51, and CYP61 in the ergosterol biosynthesis pathway with which CPR is coupled. Overexpression of CYP51 and FUS produced different levels of ketoconazole resistance in wild-type cells, indicating that the availability of CPR may limit the potential of overproduction of CYP51 as a mechanism of resistance to azole antifungal agents.

scholarly journals In Vitro Antifungal Activities of Inhibitors of Phospholipases from the Fungal Pathogen Cryptococcus neoformans

2004 ◽  
Vol 48 (5) ◽  
pp. 1561-1569 ◽  
Author(s):  
Ranjini Ganendren ◽  
Fred Widmer ◽  
Vatsala Singhal ◽  
Christabel Wilson ◽  
Tania Sorrell ◽  
...  
Keyword(s):  
Cryptococcus Neoformans ◽  
Fungal Pathogen ◽  
Antifungal Agents ◽  
Pathogenic Fungus ◽  
Fungal Enzymes ◽  
Compound A ◽  
Phospholipase B ◽  
The One ◽  
Dioctadecyldimethylammonium Bromide

ABSTRACT Secreted phospholipase B is a proven virulence factor for the pathogenic fungus Cryptococcus neoformans and exhibits three phospholipase activities in the one protein. These are phospholipase B (PLB), lysophospholipase (LPL), and lysophospholipase transacylase (LPTA). Our aim was to investigate the feasibility of using this enzyme as a target for antifungal therapy. We determined in C. neoformans var. grubii strain H99 that 82% of PLB activity was secreted but that 64% of LPL activity and 70% of LPTA activity were cell associated. Cell-associated activities (cytosolic and membrane) were further characterized, since it is likely that any fungicidal effect would depend on inhibition of these enzymes. Four commercially available compounds with structural similarities to phospholipid substrates were tested as inhibitors. These were alexidine dihydrochloride (compound A), dioctadecyldimethylammonium bromide (compound O), 1,12 bis-(tributylphosphonium)dodecane dibromide (compound P), and decamethonium dibromide (compound D). The best phospholipase inhibitors (compounds A and P) were also the most potent antifungal agents by the standard broth microdilution test. Compound A was highly selective for secreted and cell-associated PLB activities and showed no inhibition of mammalian phospholipase A 2 at 0.25 μM. Compound O, which was specific for secretory and cytosolic LPL and LPTA and membrane-associated PLB, was not antifungal. We conclude that inhibitors of cryptococcal phospholipases can be selective for fungal enzymes and intrinsically antifungal. They also provide tools for assessing the relative importance of the various enzyme activities in virulence. Our results enable further rational structure-function studies to validate the use of phospholipases as antifungal targets.

scholarly journals A Unique α-1,3 Mannosyltransferase of the Pathogenic Fungus Cryptococcus neoformans

1999 ◽  
Vol 181 (17) ◽  
pp. 5482-5488 ◽  
Author(s):  
Tamara L. Doering
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Drug Discovery ◽  
Cryptococcus Neoformans ◽  
Glucuronic Acid ◽  
Divalent Cations ◽  
Pathogenic Fungus ◽  
Product Formation ◽  
Ph Optimum ◽  
Major Virulence Factor

ABSTRACT The major virulence factor of the pathogenic fungusCryptococcus neoformans is an extensive polysaccharide capsule which surrounds the cell. Almost 90% of the capsule is composed of a partially acetylated linear α-1,3-linked mannan substituted with d-xylose and d-glucuronic acid. A novel mannosyltransferase with specificity appropriate for a role in the synthesis of this glucuronoxylomannan is active in cryptococcal membranes. This membrane-associated activity transfers mannose in vitro from GDP-mannose to an α-1,3-dimannoside acceptor, forming a second α-1,3 linkage. Product formation by the transferase is dependent on protein, time, temperature, divalent cations, and each substrate. It is not affected by amphomycin or tunicamycin but is inhibited by GDP and mannose-1-phosphate. The described activity is not detectable in the model yeast Saccharomyces cerevisiae, consistent with the absence of a similar polysaccharide structure in that organism. A second mannosyltransferase from C. neoformans membranes adds mannose in α-1,2 linkage to the same dimannoside acceptor. The two activities differ in pH optimum and cation preference. While the α-1,2 transferase does not have specificity appropriate for a role in glucuronoxylomannan synthesis, it may participate in production of mannoprotein components of the capsule. This study suggests two new targets for antifungal drug discovery.

scholarly journals Cryptococcus neoformans Is a Facultative Intracellular Pathogen in Murine Pulmonary Infection

2000 ◽  
Vol 68 (7) ◽  
pp. 4225-4237 ◽  
Author(s):  
Marta Feldmesser ◽  
Yvonne Kress ◽  
Phyllis Novikoff ◽  
Arturo Casadevall
Keyword(s):  
Cryptococcus Neoformans ◽  
Pulmonary Infection ◽  
Pathogenic Fungus ◽  
Microbial Pathogens ◽  
Yeast Cells ◽  
Intracellular Pathogen ◽  
Phagocytic Cells ◽  
Macrophage Infection

ABSTRACT To produce chronic infection, microbial pathogens must escape host immune defenses. Infection with the human pathogenic fungusCryptococcus neoformans is typically chronic. To understand the mechanism by which C. neoformans survives in tissue after the infection of immunocompetent hosts, we systematically studied the course of pulmonary infection in mice by electron microscopy. The macrophage was the primary phagocytic cell at all times of infection, but neutrophils also ingested yeast. Alveolar macrophages rapidly internalized yeast cells after intratracheal infection, and intracellular yeast cells were noted at all times of infection from 2 h through 28 days. However, the proportion of yeast cells in the intracellular and extracellular spaces varied with the time of infection. Early in infection, yeast cells were found predominantly in the intracellular compartment. A shift toward extracellular predominance occurred by 24 h that was accompanied by macrophage cytotoxicity and disruption. Later in infection, intracellular persistence in vivo was associated with replication, residence in a membrane-bound phagosome, polysaccharide accumulation inside cells, and cytotoxicity to macrophages, despite phagolysosomal fusion. Many phagocytic vacuoles with intracellular yeast had discontinuous membranes. Macrophage infection resulted in cells with a distinctive appearance characterized by large numbers of vacuoles filled with polysaccharide antigen. Similar results were observed in vitro using a macrophage-like cell line. Our results show that C. neoformans is a facultative intracellular pathogen in vivo. Furthermore, our observations suggest that C. neoformansoccupies a unique niche among the intracellular pathogens whereby survival in phagocytic cells is accompanied by intracellular polysaccharide production.

scholarly journals Cell Wall Targeting of Laccase of Cryptococcus neoformans during Infection of Mice

2006 ◽  
Vol 75 (2) ◽  
pp. 714-722 ◽  
Author(s):  
Scott R. Waterman ◽  
Moshe Hacham ◽  
John Panepinto ◽  
Guowu Hu ◽  
Soowan Shin ◽  
...  
Keyword(s):  
Cell Wall ◽  
Fusion Protein ◽  
Cryptococcus Neoformans ◽  
Fluorescent Protein ◽  
Pathogenic Fungus ◽  
Expression Studies ◽  
Green Fluorescent ◽  
The Brain ◽  
Mouse Lungs

ABSTRACT Laccase is a major virulence factor of the pathogenic fungus Cryptococcus neoformans, which afflicts both immunocompetent and immunocompromised individuals. In the present study, laccase was expressed in C. neoformans lac1Δ cells as a fusion protein with an N-terminal green fluorescent protein (GFP) using C. neoformans codon usage. The fusion protein was robustly localized to the cell wall at physiological pH, but it was mislocalized at low pH. Structural analysis of the laccase identified a C-terminal region unique to C. neoformans, and expression studies showed that the region was required for efficient transport to the cell wall both in vitro and during infection of mouse lungs. During infection of mice, adherence to alveolar macrophages was also associated with a partial mislocalization of GFP-laccase within cytosolic vesicles. In addition, recovery of cryptococcal cells from lungs of two strains of mice (CBA/J and Swiss Albino) later in infection was also associated with cytosolic mislocalization, but cells from the brain showed almost exclusive localization to cell walls, suggesting that there was more efficient cell wall targeting during infection of the brain. These data suggest that host cell antifungal defenses may reduce effective cell wall targeting of laccase during infection of the lung but not during infection of the brain, which may contribute to a more predominant role for the enzyme during infection of the brain.

scholarly journals Reduced accumulation of drug in Candida krusei accounts for itraconazole resistance.

1996 ◽  
Vol 40 (11) ◽  
pp. 2443-2446 ◽  
Author(s):  
K Venkateswarlu ◽  
D W Denning ◽  
N J Manning ◽  
S L Kelly
Keyword(s):  
Acquired Resistance ◽  
Sterol Composition ◽  
Candida Krusei ◽  
Resistant Isolate ◽  
Ergosterol Biosynthesis ◽  
Intrinsic Resistance ◽  
Binding Studies ◽  
Intracellular Content ◽  
Fluconazole Therapy

Due to intrinsic resistance Candida krusei is emerging as a systemic pathogen in AIDS patients undergoing fluconazole therapy, but acquired resistance to itraconazole has not been studied biochemically. We report here studies on the basis for azole resistance and sterol composition in C. krusei. An itraconazole-resistant isolate showed reduced susceptibility to azole drugs in in vitro growth inhibition studies. Accumulation of 14 alpha-methyl-3,6-diol under azole treatment was associated with growth arrest. In vitro ergosterol biosynthesis and type II binding studies suggested no alteration in the affinity to azole drugs of the target enzyme, the cytochrome P-450 sterol 14 alpha-demethylase, in the resistant isolate. Resistance was associated with a decreased intracellular content of drug in the resistant isolate.

Oxygenation of 18-hydroxycorticosterone as the final reaction for aldosterone biosynthesis

1984 ◽  
Vol 107 (3) ◽  
pp. 395-400 ◽  
Author(s):  
Itaru Kojima ◽  
Etsuro Ogata ◽  
Hiroshi Inano ◽  
Bun-ichi Tamaoki
Keyword(s):  
Carbon Monoxide ◽  
Hydroxysteroid Dehydrogenase ◽  
Dependent Manner ◽  
In Vitro Production ◽  
Mitochondrial Preparation ◽  
Final Reaction ◽  
Vitro Production ◽  
Dose Dependent ◽  
Cytochrome P 450

Abstract. Incubation of 18-hydroxycorticosterone with the sonicated mitochondrial preparation of bovine adrenal glomerulosa tissue leads to the production of aldosterone, as measured by radioimmunoassay. The in vitro production of aldosterone from 18-hydroxycorticosterone requires both molecular oxygen and NADPH, and is inhibited by carbon monoxide. Cytochrome P-450 inhibitors such as metyrapone, SU 8000. SU 10603, SKF 525A, amphenone B and spironolactone decrease the biosynthesis of aldosterone from 18-hydroxycorticosterone. These results support the conclusion that the final reaction in aldosterone synthesis from 18-hydroxycorticosterone is catalyzed by an oxygenase, but not by 18-hydroxysteroid dehydrogenase. By the same preparation, the production of [3H]aldosterone but not [3H]18-hydroxycorticosterone from [1,2-3H ]corticosterone is decreased in a dose-dependent manner by addition of non-radioactive 18-hydroxycorticosterone.

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