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

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.

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Antifungal activity of dendritic cell lysosomal proteins against Cryptococcus neoformans

Scientific Reports ◽

10.1038/s41598-021-92991-6 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Benjamin N. Nelson ◽

Savannah G. Beakley ◽

Sierra Posey ◽

Brittney Conn ◽

Emma Maritz ◽

...

Keyword(s):

Antifungal Activity ◽

Cryptococcus Neoformans ◽

Mammalian Cells ◽

Cysteine Proteases ◽

Dependent Manner ◽

Life Threatening ◽

Opportunistic Fungal Pathogen ◽

Dose Dependent ◽

Lysosomal Proteins

AbstractCryptococcal meningitis is a life-threatening disease among immune compromised individuals that is caused by the opportunistic fungal pathogen Cryptococcus neoformans. Previous studies have shown that the fungus is phagocytosed by dendritic cells (DCs) and trafficked to the lysosome where it is killed by both oxidative and non-oxidative mechanisms. While certain molecules from the lysosome are known to kill or inhibit the growth of C. neoformans, the lysosome is an organelle containing many different proteins and enzymes that are designed to degrade phagocytosed material. We hypothesized that multiple lysosomal components, including cysteine proteases and antimicrobial peptides, could inhibit the growth of C. neoformans. Our study identified the contents of the DC lysosome and examined the anti-cryptococcal properties of different proteins found within the lysosome. Results showed several DC lysosomal proteins affected the growth of C. neoformans in vitro. The proteins that killed or inhibited the fungus did so in a dose-dependent manner. Furthermore, the concentration of protein needed for cryptococcal inhibition was found to be non-cytotoxic to mammalian cells. These data show that many DC lysosomal proteins have antifungal activity and have potential as immune-based therapeutics.

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Structure and inhibition of Cryptococcus neoformans sterylglucosidase to develop antifungal agents

Nature Communications ◽

10.1038/s41467-021-26163-5 ◽

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.

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Pleiotropic Roles of the Msi1-Like Protein Msl1 in Cryptococcus neoformans

Eukaryotic Cell ◽

10.1128/ec.00261-12 ◽

2012 ◽

Vol 11 (12) ◽

pp. 1482-1495 ◽

Cited By ~ 9

Author(s):

Dong-Hoon Yang ◽

Shinae Maeng ◽

Anna K. Strain ◽

Anna Floyd ◽

Kirsten Nielsen ◽

...

Keyword(s):

Cryptococcus Neoformans ◽

Stress Responses ◽

Fungal Pathogens ◽

Independent Manner ◽

Content Type ◽

Antifungal Drug Resistance ◽

Human Fungal Pathogen ◽

Assembly Factor ◽

Life Threatening ◽

Stress Related Genes

ABSTRACT Msi1-like (MSIL) proteins contain WD40 motifs and have a pleiotropic cellular function as negative regulators of the Ras/cyclic AMP (cAMP) pathway and components of chromatin assembly factor 1 (CAF-1), yet they have not been studied in fungal pathogens. Here we identified and characterized an MSIL protein, Msl1, in Cryptococcus neoformans , which causes life-threatening meningoencephalitis in humans. Notably, Msl1 plays pleiotropic roles in C. neoformans in both cAMP-dependent and -independent manners largely independent of Ras. Msl1 negatively controls antioxidant melanin production and sexual differentiation, and this was repressed by the inhibition of the cAMP-signaling pathway. In contrast, Msl1 controls thermotolerance, diverse stress responses, and antifungal drug resistance in a Ras/cAMP-independent manner. Cac2, which is the second CAF-1 component, appears to play both redundant and distinct functions compared to the functions of Msl1. Msl1 is required for the full virulence of C. neoformans . Transcriptome analysis identified a group of Msl1-regulated genes, which include stress-related genes such as HSP12 and HSP78 . In conclusion, this study demonstrates pleiotropic roles of Msl1 in the human fungal pathogen C. neoformans , providing insight into a potential novel antifungal therapeutic target.

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

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.41.7.1465 ◽

1997 ◽

Vol 41 (7) ◽

pp. 1465-1467 ◽

Cited By ~ 7

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.

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Role of the RNA Polymerase α Subunits in MetR-Dependent Activation of metE and metH: Important Residues in the C-Terminal Domain and Orientation Requirements within RNA Polymerase

Journal of Bacteriology ◽

10.1128/jb.182.19.5539-5550.2000 ◽

2000 ◽

Vol 182 (19) ◽

pp. 5539-5550 ◽

Cited By ~ 15

Author(s):

Paula S. Fritsch ◽

Mark L. Urbanowski ◽

George V. Stauffer

Keyword(s):

Rna Polymerase ◽

Random Mutagenesis ◽

Complex Surface ◽

Α Subunit ◽

C Terminus ◽

Distinct Cluster ◽

In Vitro Reconstitution ◽

The Difference ◽

Α Subunits

ABSTRACT Many transcription factors activate by directly interacting with RNA polymerase (RNAP). The C terminus of the RNAP α subunit (αCTD) is a common target of activators. We used both random mutagenesis and alanine scanning to identify αCTD residues that are crucial for MetR-dependent activation of metE and metH. We found that these residues localize to two distinct faces of the αCTD. The first is a complex surface consisting of residues important for α-DNA interactions, activation of both genes (residues 263, 293, and 320), and activation of either metE only (residues 260, 276, 302, 306, 309, and 322) or metH only (residues 258, 264, 290, 294, and 295). The second is a distinct cluster of residues important for metE activation only (residues 285, 289, 313, and 314). We propose that a difference in the location of the MetR binding site for activation at these two promoters accounts for the differences in the residues of α required for MetR-dependent activation. We have designed an in vitro reconstitution-purification protocol that allows us to specifically orient wild-type or mutant α subunits to either the β-associated or the β′-associated position within RNAP (comprising α2, β, β′, and ς subunits). In vitro transcriptions using oriented α RNAP indicate that a single αCTD on either the β- or the β′-associated α subunit is sufficient for MetR activation of metE, while MetR interacts preferentially with the αCTD on the β-associated α subunit at metH. We propose that the different αCTD requirements at these two promoters are due to a combination of the difference in the location of the activation site and limits on the rotational flexibility of the αCTD.

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

Eukaryotic Cell ◽

10.1128/ec.00375-08 ◽

2009 ◽

Vol 8 (3) ◽

pp. 315-326 ◽

Cited By ~ 63

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.

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Fitness distribution and transgressive segregation across 40 environments in a hybrid progeny population of the human-pathogenic yeast Cryptococcus neoformans

Genome ◽

10.1139/g08-004 ◽

2008 ◽

Vol 51 (4) ◽

pp. 272-281 ◽

Cited By ~ 20

Author(s):

Morvarid Shahid ◽

Susan Han ◽

Heather Yoell ◽

Jianping Xu

Keyword(s):

Cryptococcus Neoformans ◽

Biological Effects ◽

Pathogenic Fungus ◽

Transgressive Segregation ◽

Variable Number ◽

Hybrid Progeny ◽

Pathogenic Yeast ◽

Human Fungal Pathogen ◽

Different Temperatures ◽

Human Pathogenic Fungus

The opportunistic human fungal pathogen Cryptococcus neoformans includes two varieties, C. neoformans var. grubii and C. neoformans var. neoformans, which correspond to serotypes A and D, respectively. Recent population genetic studies revealed that multiple natural hybridizations have occurred recently between these two divergent lineages. However, the biological effects of such hybridizations are little understood. In this study, we used colony size as a proxy for vegetative fitness to examine the phenotypic effects of hybridization between these two lineages in a laboratory cross. Two genetically diverged parental strains that differed in their growth at different temperatures and on different media as well as in their susceptibility to the common antifungal drug fluconazole were chosen. A total of 269 progeny were obtained and their vegetative growth was determined in 40 environments that differed in nutrients, temperature, and fluconazole concentration. Our analyses indicated little evidence for outbreeding depression or heterosis in the average vegetative fitness of the hybrid progeny population. The progeny, each of the three environmental variables, and their two-way, three-way, and four-way interactions all contributed significantly to the overall vegetative fitness variation. Interestingly, a variable number of progeny displayed evidence of transgressive segregation in vegetative fitness among the tested environments. Our study suggests that hybridization could play a significant role in the phenotypic evolution of this important human-pathogenic fungus.

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The Pathogenic Fungus Cryptococcus neoformans Expresses Two Functional GDP-Mannose Transporters with Distinct Expression Patterns and Roles in Capsule Synthesis

Eukaryotic Cell ◽

10.1128/ec.00015-07 ◽

2007 ◽

Vol 6 (5) ◽

pp. 776-785 ◽

Cited By ~ 36

Author(s):

Tricia R. Cottrell ◽

Cara L. Griffith ◽

Hong Liu ◽

Ashley A. Nenninger ◽

Tamara L. Doering

Keyword(s):

Cryptococcus Neoformans ◽

Fungal Pathogen ◽

Pathogenic Fungus ◽

Expression Patterns ◽

Growth Conditions ◽

Phosphomannose Isomerase ◽

Microarray Experiments ◽

Life Threatening ◽

Nucleotide Sugars ◽

Intracellular Organelles

ABSTRACT Cryptococcus neoformans is a fungal pathogen that is responsible for life-threatening disease, particularly in the context of compromised immunity. This organism makes extensive use of mannose in constructing its cell wall, glycoproteins, and glycolipids. Mannose also comprises up to two-thirds of the main cryptococcal virulence factor, a polysaccharide capsule that surrounds the cell. The glycosyltransfer reactions that generate cellular carbohydrate structures usually require activated donors such as nucleotide sugars. GDP-mannose, the mannose donor, is produced in the cytosol by the sequential actions of phosphomannose isomerase, phosphomannomutase, and GDP-mannose pyrophosphorylase. However, most mannose-containing glycoconjugates are synthesized within intracellular organelles. This topological separation necessitates a specific transport mechanism to move this key precursor across biological membranes to the appropriate site for biosynthetic reactions. We have discovered two GDP-mannose transporters in C. neoformans, in contrast to the single such protein reported previously for other fungi. Biochemical studies of each protein expressed in Saccharomyces cerevisiae show that both are functional, with similar kinetics and substrate specificities. Microarray experiments indicate that the two proteins Gmt1 and Gmt2 are transcribed with distinct patterns of expression in response to variations in growth conditions. Additionally, deletion of the GMT1 gene yields cells with small capsules and a defect in capsule induction, while deletion of GMT2 does not alter the capsule. We suggest that C. neoformans produces two GDP-mannose transporters to satisfy its enormous need for mannose utilization in glycan synthesis. Furthermore, we propose that the two proteins have distinct biological roles. This is supported by the different expression patterns of GMT1 and GMT2 in response to environmental stimuli and the dissimilar phenotypes that result when each gene is deleted.

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Efficacy of Amphotericin B in Combination with Flucytosine against Flucytosine-Susceptible or Flucytosine-Resistant Isolates of Cryptococcus neoformans during Disseminated Murine Cryptococcosis

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.50.1.113-120.2006 ◽

2006 ◽

Vol 50 (1) ◽

pp. 113-120 ◽

Cited By ~ 46

Author(s):

Patrick Schwarz ◽

Françoise Dromer ◽

Olivier Lortholary ◽

Eric Dannaoui

Keyword(s):

Cryptococcus Neoformans ◽

Amphotericin B ◽

Resistant Isolate ◽

Fungal Burden ◽

Cryptococcal Infection ◽

Susceptible Isolate ◽

The Difference ◽

The Brain ◽

Better Than

ABSTRACT Whether or not flucytosine should be administered to patients infected with Cryptococcus neoformans isolates found to be resistant to flucytosine in vitro remains a controversial issue. Thus, the efficacy of amphotericin B and flucytosine in combination was investigated by mortality and fungal burden studies in a murine model of disseminated cryptococcosis using two clinical isolates of Cryptococcus neoformans, one susceptible and one resistant (i.e., 64 μg/ml) to flucytosine. Amphotericin B was given intraperitoneally at 0.25 or 0.5 mg/kg/day, while flucytosine was given at 100 or 250 mg/kg/day orally. Treatment was started 24 h or day 6 after inoculation and continued for 5 days in fungal burden and mortality studies, respectively. The combination of amphotericin B at 0.5 mg/kg/day and flucytosine at 250 mg/kg/day was significantly more effective than monotherapies for reducing fungal burden in brain, spleen, and lungs after infection by the flucytosine-susceptible isolate and in brain and spleen for the flucytosine-resistant isolate. For the flucytosine-resistant isolate, the combination of amphotericin B at 0.5 mg/kg/day with flucytosine at 100 mg/kg/day was significantly better than monotherapies for reducing the fungal burden in the brain. Survival obtained after the combination of amphotericin B at 0.5 mg/kg/day and flucytosine at 250 mg/kg/day increased compared to that obtained with monotherapies for both isolates, but the difference was statistically significant only for the flucytosine-susceptible isolate. Antagonism was never observed. This study demonstrates the beneficial effect of the addition of flucytosine to amphotericin B against experimental disseminated cryptococcal infection even when the C. neoformans isolate is resistant to flucytosine.

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Identification of bridgin, an unconventional linker, connects the outer kinetochore to centromeric chromatin

10.1101/816199 ◽

2019 ◽

Author(s):

Shreyas Sridhar ◽

Tetsuya Hori ◽

Reiko Nakagawa ◽

Tatsuo Fukagawa ◽

Kaustuv Sanyal

Keyword(s):

Cryptococcus Neoformans ◽

Fungal Pathogen ◽

Centromeric Chromatin ◽

Microtubule Binding ◽

Human Fungal Pathogen ◽

Functional Analyses

ABSTRACTThe microtubule-binding outer kinetochore is linked to centromeric chromatin through the inner kinetochore CENP-CMif2, CENP-TCnn1, and CENP-UAme1 pathways. These are the only known kinetochore linker proteins across eukaryotes. Linker proteins are structurally less conserved than their outer kinetochore counterparts. Here, we demonstrate the recurrent loss of most inner kinetochore CCAN, including certain linker proteins during evolution in the fungal phylum of Basidiomycota. By studying the kinetochore interactome, a previously undescribed linker protein, bridgin was identified in the basidiomycete Cryptococcus neoformans, a human fungal pathogen. In vivo and in vitro functional analyses of bridgin reveal that it binds to the outer kinetochore and centromere chromatin simultaneously to ensure accurate kinetochore-microtubule attachments. Unlike known linker proteins, bridgin is recruited by the outer kinetochore. Homologs of bridgin were identified outside fungi. These results showcase a divergent strategy, with a more ancient origin than fungi, to link the outer kinetochore to centromeric chromatin.

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