The Sec1/Munc18 (SM) protein Vps45 is involved in iron uptake, mitochondrial function and virulence in the pathogenic fungus Cryptococcus neoformans

ABSTRACTThe battle for iron between invading microorganisms and mammalian hosts is a pivotal determinant of the outcome of infection. The pathogenic fungus,Cryptococcus neoformans, employs multiple mechanisms to compete for iron during cryptococcosis, a disease primarily of immunocompromised hosts. In this study, we examined the role of endocytic trafficking in iron uptake by characterizing a mutant defective in the Sec1/Munc18 (SM) protein Vps45. This protein is known to regulate the machinery for vesicle trafficking and fusion via interactions with SNARE proteins. As expected, avps45deletion mutant was impaired in endocytosis and showed sensitivity to trafficking inhibitors. The mutant also showed poor growth on iron-limited media and a defect in transporting the Cfo1 ferroxidase of the high-affinity iron uptake system from the plasma membrane to the vacuole. Remarkably, we made the novel observation that Vps45 also contributes to mitochondrial function in that a Vps45-Gfp fusion protein associated with mitotracker, and avps45mutant showed enhanced sensitivity to inhibitors of electron transport complexes as well as changes in mitochondrial membrane potential. Consistent with mitochondrial function, thevps45mutant was impaired in calcium homeostasis. To assess the relevance of these defects for virulence, we examined cell surface properties of thevps45mutant and found increased sensitivity to agents that challenge cell wall integrity and antifungal drugs. A change in cell wall properties was consistent with our observation of altered capsule polysaccharide attachment, and with attenuated virulence in a mouse model of cryptococcosis. Overall, our studies reveal a novel role for Vps45-mediated trafficking for iron uptake, mitochondrial function and virulence.

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The vacuolar sorting protein Vps45 links iron uptake, mitochondrial function and virulence in the pathogenic fungus Cryptococcus neoformans.

10.26226/morressier.5ac39997d462b8028d89a0c5 ◽

2018 ◽

Author(s):

MÉLISSA Caza

Keyword(s):

Cryptococcus Neoformans ◽

Mitochondrial Function ◽

Iron Uptake ◽

Pathogenic Fungus ◽

Vacuolar Sorting

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Expression Patterns in Reductive Iron Assimilation and Functional Consequences during Phagocytosis of Lichtheimia corymbifera, an Emerging Cause of Mucormycosis

Journal of Fungi ◽

10.3390/jof7040272 ◽

2021 ◽

Vol 7 (4) ◽

pp. 272

Author(s):

Felicia Adelina Stanford ◽

Nina Matthias ◽

Zoltán Cseresnyés ◽

Marc Thilo Figge ◽

Mohamed I. Abdelwahab Hassan ◽

...

Keyword(s):

Gene Expression ◽

Iron Uptake ◽

Intracellular Transport ◽

Pathogenic Fungus ◽

Expression Patterns ◽

Iron Depletion ◽

Iron Assimilation ◽

Human Pathogenic Fungus ◽

Hyphal Formation ◽

Yeast Mutants

Iron is an essential micronutrient for most organisms and fungi are no exception. Iron uptake by fungi is facilitated by receptor-mediated internalization of siderophores, heme and reductive iron assimilation (RIA). The RIA employs three protein groups: (i) the ferric reductases (Fre5 proteins), (ii) the multicopper ferroxidases (Fet3) and (iii) the high-affinity iron permeases (Ftr1). Phenotyping under different iron concentrations revealed detrimental effects on spore swelling and hyphal formation under iron depletion, but yeast-like morphology under iron excess. Since access to iron is limited during pathogenesis, pathogens are placed under stress due to nutrient limitations. To combat this, gene duplication and differential gene expression of key iron uptake genes are utilized to acquire iron against the deleterious effects of iron depletion. In the genome of the human pathogenic fungus L. corymbifera, three, four and three copies were identified for FRE5, FTR1 and FET3 genes, respectively. As in other fungi, FET3 and FTR1 are syntenic and co-expressed in L. corymbifera. Expression of FRE5, FTR1 and FET3 genes is highly up-regulated during iron limitation (Fe-), but lower during iron excess (Fe+). Fe- dependent upregulation of gene expression takes place in LcFRE5 II and III, LcFTR1 I and II, as well as LcFET3 I and II suggesting a functional role in pathogenesis. The syntenic LcFTR1 I–LcFET3 I gene pair is co-expressed during germination, whereas LcFTR1 II- LcFET3 II is co-expressed during hyphal proliferation. LcFTR1 I, II and IV were overexpressed in Saccharomyces cerevisiae to represent high and moderate expression of intracellular transport of Fe3+, respectively. Challenge of macrophages with the yeast mutants revealed no obvious role for LcFTR1 I, but possible functions of LcFTR1 II and IVs in recognition by macrophages. RIA expression pattern was used for a new model of interaction between L. corymbifera and macrophages.

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Low Glucose Mediated Fluconazole Tolerance in Cryptococcus neoformans

Journal of Fungi ◽

10.3390/jof7060489 ◽

2021 ◽

Vol 7 (6) ◽

pp. 489

Author(s):

Somanon Bhattacharya ◽

Natalia Kronbauer Oliveira ◽

Anne G. Savitt ◽

Vanessa K. A. Silva ◽

Rachel B. Krausert ◽

...

Keyword(s):

Cryptococcus Neoformans ◽

Mitochondrial Function ◽

Efflux Pump ◽

Nile Red ◽

Fold Increase ◽

Growth Conditions ◽

Chromosome 1 ◽

Normal Glucose ◽

Low Glucose ◽

Synthetic Media

Chronic meningoencephalitis is caused by Cryptococcus neoformans and is treated in many parts of the world with fluconazole (FLC) monotherapy, which is associated with treatment failure and poor outcome. In the host, C. neoformans propagates predominantly under low glucose growth conditions. We investigated whether low glucose, mimicked by growing in synthetic media (SM) with 0.05% glucose (SMlowglu), affects FLC-resistance. A > 4-fold increase in FLC tolerance was observed in seven C. neoformans strains when minimum inhibitory concentration (MIC) was determined in SMlowglu compared to MIC in SM with normal (2%) glucose (SMnlglu). In SMlowglu, C. neoformans cells exhibited upregulation of efflux pump genes AFR1 (8.7-fold) and AFR2 (2.5-fold), as well as decreased accumulation (2.6-fold) of Nile Red, an efflux pump substrate. Elevated intracellular ATP levels (3.2-fold and 3.4-fold), as well as decreased mitochondrial reactive oxygen species levels (12.8-fold and 17-fold), were found in the presence and absence of FLC, indicating that low glucose altered mitochondrial function. Fluorescence microscopy revealed that mitochondria of C. neoformans grown in SMlowglu were fragmented, whereas normal glucose promoted a reticular network of mitochondria. Although mitochondrial membrane potential (MMP) was not markedly affected in SMlowglu, it significantly decreased in the presence of FLC (12.5-fold) in SMnlglu, but remained stable in SMlowglu-growing C. neoformans cells. Our data demonstrate that increased FLC tolerance in low glucose-growing C. neoformans is the result of increased efflux pump activities and altered mitochondrial function, which is more preserved in SMlowglu. This mechanism of resistance is different from FLC heteroresistance, which is associated with aneuploidy of chromosome 1 (Chr1).

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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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The capsule ofCryptococcus neoformansmodulates phagosomal pH through its acid-base properties

10.1101/390328 ◽

2018 ◽

Author(s):

Carlos M. De Leon-Rodriguez ◽

Man Shun Fu ◽

M. Osman Corbali ◽

Radames J.B. Cordero ◽

Arturo Casadevall

Keyword(s):

Cryptococcus Neoformans ◽

Capsular Polysaccharide ◽

Glucuronic Acid ◽

Pathogenic Fungus ◽

Weak Acid ◽

Phagocytic Cells ◽

Acid Base ◽

Encapsulated Cells ◽

Human Pathogenic Fungus ◽

Base Properties

AbstractPhagosomal acidification is a critical cellular mechanism for the inhibition and killing of ingested microbes by phagocytic cells. The acidic environment activates microbicidal proteins and creates an unfavorable environment for the growth of many microbes. Consequently, numerous pathogenic microbes have developed strategies for countering phagosomal acidification through various mechanisms that include interference with phagosome maturation. The human pathogenic fungusCryptococcus neoformansresides in acidic phagosome after macrophage ingestion that actually provides a favorable environment for replication since the fungus replicates faster at acidic pH. We hypothesized that the glucuronic acid residues in the capsular polysaccharide had the capacity to affect phagosome acidity through their acid-base properties. A ratiometric fluorescence comparison of imaged phagosomes containingC. neoformansto those containing beads showed that the latter were significantly more acidic. Similarly, phagosomes containing non-encapsulatedC. neoformanscells were more acidic than those containing encapsulated cells. Acid-base titrations of isolatedC. neoformanspolysaccharide revealed that it behaves as a weak acid with maximal buffering capacity around pH 4-5. We interpret these results as indicating that the glucuronic acid residues in theC. neoformanscapsular polysaccharide can buffer phagosomal acidification. Interference with phagosomal acidification represents a new function for the cryptococcal capsule in virulence and suggests the importance of considering the acid-base properties of microbial capsules in the host-microbe interaction for other microbes with charged residues in their capsules.ImportanceCryptococcus neoformansis the causative agent of cryptococcosis, a devastating fungal disease that affects thousands of individuals worldwide. This fungus has the capacity to survive inside phagocytic cells, which contributes to persistence of infection and dissemination. One of the major mechanisms of host phagocytes is to acidify the phagosomal compartment after ingestion of microbes. This study shows that the capsule ofC. neoformanscan interfere with full phagosomal acidification by serving as a buffer.

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Metabolomic alterations associated with copper stress in Cryptococcus neoformans

Future Microbiology ◽

10.2217/fmb-2020-0290 ◽

2021 ◽

Author(s):

Tianshu Sun ◽

Xiaogang Li ◽

Wei Song ◽

Shuying Yu ◽

Linqi Wang ◽

...

Keyword(s):

Cryptococcus Neoformans ◽

Metabolic Profile ◽

Pathogenic Fungus ◽

Copper Toxicity ◽

Enrichment Analysis ◽

Copper Stress ◽

Pathway Enrichment Analysis ◽

Flight Mass Spectrometry ◽

Opportunistic Pathogenic ◽

Better Than

Background: Copper stress is an effective host strategy in resisting the opportunistic pathogenic fungus Cryptococcus neoformans. We studied metabolic changes in C. neoformans under copper stress. Materials & methods: Wild-type and metallothionein-null C. neoformans were treated with copper on agar containing glucose, glycerol or ethanol as the carbon source and their metabolites were analyzed by untarget metabolomics strategy using gas chromatography coupled with time of flight mass spectrometry. Results: The metabolic profile of C. neoformans varied in the presence and absence of copper. Pathway enrichment analysis showed that the differentially abundant metabolites were related to amino acid and carbohydrate metabolism. C. neoformans grown on glycerol or ethanol resisted copper toxicity better than C. neoformans grown on glucose. Conclusion: Copper stress alters the metabolic profile of C. neoformans.

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

Scientific Reports ◽

10.1038/s41598-019-50678-z ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 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.

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Loss of Allergen 1 Confers a Hypervirulent Phenotype That Resembles Mucoid Switch Variants of Cryptococcus neoformans

Infection and Immunity ◽

10.1128/iai.01079-08 ◽

2008 ◽

Vol 77 (1) ◽

pp. 128-140 ◽

Cited By ~ 19

Author(s):

Neena Jain ◽

Li Li ◽

Ye-Ping Hsueh ◽

Abraham Guerrero ◽

Joseph Heitman ◽

...

Keyword(s):

Intracranial Pressure ◽

Cryptococcus Neoformans ◽

Gene Function ◽

Pathogenic Fungus ◽

Opportunistic Pathogen ◽

Phenotypic Switching ◽

Elevated Intracranial Pressure ◽

Macrophage Phagocytosis ◽

Switch Variant

ABSTRACT Microbial survival in a host is usually dependent on the ability of a pathogen to undergo changes that promote escape from host defense mechanisms. The human-pathogenic fungus Cryptococcus neoformans undergoes phenotypic switching in vivo that promotes persistence in tissue. By microarray and real-time PCR analyses, the allergen 1 gene (ALL1) was found to be downregulated in the hypervirulent mucoid switch variant, both during logarithmic growth and during intracellular growth in macrophages. The ALL1 gene encodes a small cytoplasmic protein that is involved in capsule formation. Growth of an all1Δ gene deletion mutant was normal. Similar to cells of the mucoid switch variant, all1Δ cells produced a larger polysaccharide capsule than cells of the smooth parent and the complemented strain produced, and the enlarged capsule inhibited macrophage phagocytosis. The mutant exhibited a modest defect in capsule induction compared to all of the other variants. In animal models the phenotype of the all1Δ mutant mimicked the hypervirulent phenotype of the mucoid switch variant, which is characterized by decreased host survival and elevated intracranial pressure. Decreased survival is likely the result of both an ineffective cell-mediated immune response and impaired phagocytosis by macrophages. Consequently, we concluded that, unlike loss of most virulence-associated genes, where loss of gene function results in attenuated virulence, loss of the ALL1 gene enhances virulence by altering the host-pathogen interaction and thereby impairing clearance. Our data identified the first cryptococcal gene associated with elevated intracranial pressure and support the hypothesis that an environmental opportunistic pathogen has modified its virulence in vivo by epigenetic downregulation of gene function.

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