scholarly journals Role for Chitin and Chitooligomers in the Capsular Architecture of Cryptococcus neoformans

2009 ◽  
Vol 8 (10) ◽  
pp. 1543-1553 ◽  
Author(s):  
Fernanda L. Fonseca ◽  
Leonardo Nimrichter ◽  
Radames J. B. Cordero ◽  
Susana Frases ◽  
Jessica Rodrigues ◽  
...  
Keyword(s):  
Cell Wall ◽  
Cryptococcus Neoformans ◽  
Pathogenic Fungus ◽  
Monosaccharide Composition ◽  
Altered Expression ◽  
Functional Activities ◽  
Differential Reactivity ◽  
Human Pathogenic Fungus

ABSTRACT Molecules composed of β-1,4-linked N-acetylglucosamine (GlcNAc) and deacetylated glucosamine units play key roles as surface constituents of the human pathogenic fungus Cryptococcus neoformans. GlcNAc is the monomeric unit of chitin and chitooligomers, which participate in the connection of capsular polysaccharides to the cryptococcal cell wall. In the present study, we evaluated the role of GlcNAc-containing structures in the assembly of the cryptococcal capsule. The in vivo expression of chitooligomers in C. neoformans varied depending on the infected tissue, as inferred from the differential reactivity of yeast forms to the wheat germ agglutinin (WGA) in infected brain and lungs of rats. Chromatographic and dynamic light-scattering analyses demonstrated that glucuronoxylomannan (GXM), the major cryptococcal capsular component, interacts with chitin and chitooligomers. When added to C. neoformans cultures, chitooligomers formed soluble complexes with GXM and interfered in capsular assembly, as manifested by aberrant capsules with defective connections with the cell wall and no reactivity with a monoclonal antibody to GXM. Cultivation of C. neoformans in the presence of an inhibitor of glucosamine 6-phosphate synthase resulted in altered expression of cell wall chitin. These cells formed capsules that were loosely connected to the cryptococcal wall and contained fibers with decreased diameters and altered monosaccharide composition. These results contribute to our understanding of the role played by chitin and chitooligosaccharides on the cryptococcal capsular structure, broadening the functional activities attributed to GlcNAc-containing structures in this biological system.

scholarly journals Vesicle-associated melanization in Cryptococcus neoformans

Microbiology ◽  
2009 ◽  
Vol 155 (12) ◽  
pp. 3860-3867 ◽  
Author(s):  
Helene C. Eisenman ◽  
Susana Frases ◽  
André M. Nicola ◽  
Marcio L. Rodrigues ◽  
Arturo Casadevall
Keyword(s):  
Cell Wall ◽  
Cryptococcus Neoformans ◽  
Extracellular Vesicles ◽  
Lipid A ◽  
Pathogenic Fungus ◽  
Pathogenic Fungi ◽  
Spherical Particles ◽  
Human Pathogenic Fungus ◽  
Kinetics Of

Recently, several pathogenic fungi were shown to produce extracellular vesicles that contain various components associated with virulence. In the human pathogenic fungus Cryptococcus neoformans, these components included laccase, an enzyme that catalyses melanin synthesis. Spherical melanin granules have been observed in the cell wall of C. neoformans. Given that melanin granules have dimensions that are comparable to those of extracellular vesicles, and that metazoan organisms produce melanin in vesicular structures known as melanosomes, we investigated the role of vesicles in cryptococcal melanization. Extracellular vesicles melanized when incubated with the melanin precursor l-3,4-dihydroxyphenylalanine (l-DOPA). The kinetics of substrate incorporation into cells and vesicles was analysed using radiolabelled l-DOPA. The results indicated that substrate incorporation was different for cells and isolated vesicles. Acid-generated melanin ghosts stained with lipophilic dyes, implying the presence of associated lipid. A model for C. neoformans melanization is proposed that accounts for these observations and provides a mechanism for the assembly of melanin into relatively uniform spherical particles stacked in an orderly arrangement in the cell wall.

scholarly journals Cryptococcus neoformans melanization incorporates multiple catecholamines to produce polytypic melanin

2021 ◽  
Author(s):  
Rosanna P. Baker ◽  
Christine Chrissian ◽  
Ruth E. Stark ◽  
Arturo Casadevall
Keyword(s):  
Cryptococcus Neoformans ◽  
Brain Tissue ◽  
Pathogenic Fungus ◽  
Structural Complexity ◽  
Pathogenic Fungi ◽  
Human Infection ◽  
Melanin Pigment ◽  
Practical Standpoint ◽  
Human Pathogenic Fungus

AbstractMelanin is a major virulence factor in pathogenic fungi that enhances the ability of fungal cells to resist immune clearance. Cryptococcus neoformans is an important human pathogenic fungus that synthesizes melanin from exogenous tissue catecholamine precursors during infection, but the type of melanin made in cryptococcal meningoencephalitis is unknown. We analyzed the efficacy of various catecholamines found in brain tissue in supporting melanization using animal brain tissue and synthetic catecholamine mixtures reflecting brain tissue proportions. Solid-state NMR spectra of the melanin pigment produced from such mixtures displayed larger melanin contributions than expected from the most robust dopamine constituent, suggesting uptake of additional catecholamines. Probing the biosynthesis of melanin using radiolabeled catecholamines revealed that C. neoformans melanization simultaneously incorporated more than one catecholamine, implying that the pigment was polytypic in nature. Nonetheless, melanin derived from individual or mixed catecholamines had comparable ability to protect C. neoformans against ultraviolet light and oxidants. Our results indicate that melanin produced during infection differs depending on the catecholamine composition of tissue and that melanin pigment synthesized in vivo is likely to accrue from the polymerization of a mixture of precursors. From a practical standpoint our results strongly suggest that using dopamine as a polymerization precursor is capable of producing melanin pigment comparable to that produced during infection. On a more fundamental level our findings uncover additional structural complexity for natural cryptococcal melanin by demonstrating that pigment produced during human infection is likely to be composed of polymerized moieties derived from chemically different precursors.

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

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.

scholarly journals The Role of Dimorphism Regulating Histidine Kinase (Drk1) in the Pathogenic Fungus Paracoccidioides brasiliensis Cell Wall

2021 ◽  
Vol 7 (12) ◽  
pp. 1014
Author(s):  
Marina Valente Navarro ◽  
Yasmin Nascimento de Barros ◽  
Wilson Dias Segura ◽  
Alison Felipe Alencar Chaves ◽  
Grasielle Pereira Jannuzzi ◽  
...  
Keyword(s):  
Cell Wall ◽  
Histidine Kinase ◽  
Mammalian Cells ◽  
Paracoccidioides Brasiliensis ◽  
Pathogenic Fungus ◽  
Fungal Resistance ◽  
Phagocytic Index ◽  
Mammalian Host ◽  
Control Group

Dimorphic fungi of the Paracoccidioides genus are the causative agents of paracoccidioidomycosis (PCM), an endemic disease in Latin America with a high incidence in Brazil. This pathogen presents as infective mycelium at 25 °C in the soil, reverting to its pathogenic form when inhaled by the mammalian host (37 °C). Among these dimorphic fungal species, dimorphism regulating histidine kinase (Drk1) plays an essential role in the morphological transition. These kinases are present in bacteria and fungi but absent in mammalian cells and are important virulence and cellular survival regulators. Hence, the purpose of this study was to investigate the role of PbDrk1 in the cell wall modulation of P. brasiliensis. We observed that PbDrk1 participates in fungal resistance to different cell wall-disturbing agents by reducing viability after treatment with iDrk1. To verify the role of PbDRK1 in cell wall morphogenesis, qPCR results showed that samples previously exposed to iDrk1 presented higher expression levels of several genes related to cell wall modulation. One of them was FKS1, a β-glucan synthase that showed a 3.6-fold increase. Furthermore, confocal microscopy analysis and flow cytometry showed higher β-glucan exposure on the cell surface of P. brasiliensis after incubation with iDrk1. Accordingly, through phagocytosis assays, a significantly higher phagocytic index was observed in yeasts treated with iDrk1 than the control group, demonstrating the role of PbDrk1 in cell wall modulation, which then becomes a relevant target to be investigated. In parallel, the immune response profile showed increased levels of proinflammatory cytokines. Finally, our data strongly suggest that PbDrk1 modulates cell wall component expression, among which we can identify β-glucan. Understanding this signalling pathway may be of great value for identifying targets of antifungal molecular activity since HKs are not present in mammals.

scholarly journals Laccase Expression in Murine Pulmonary Cryptococcus neoformans Infection

2005 ◽  
Vol 73 (5) ◽  
pp. 3124-3127 ◽  
Author(s):  
Javier Garcia-Rivera ◽  
Stephanie C. Tucker ◽  
Marta Feldmesser ◽  
Peter R. Williamson ◽  
Arturo Casadevall
Keyword(s):  
Electron Microscopy ◽  
Cell Wall ◽  
Cryptococcus Neoformans ◽  
Lung Tissue ◽  
Immunogold Electron Microscopy ◽  
Cell Cytoplasm ◽  
Fungal Cell ◽  
Time Of Infection

ABSTRACT Cryptococcus neoformans laccase expression during murine infection was investigated in lung tissue by immunohistochemistry and immunogold electron microscopy. Laccase was detected in the fungal cell cytoplasm, cell wall, and capsule in vivo. The amount of laccase found in different sites varied as a function of the time of infection.

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

2008 ◽  
Vol 77 (1) ◽  
pp. 128-140 ◽  
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.

scholarly journals Peroxisomal and Mitochondrial β-Oxidation Pathways Influence the Virulence of the Pathogenic Fungus Cryptococcus neoformans

2012 ◽  
Vol 11 (8) ◽  
pp. 1042-1054 ◽  
Author(s):  
Matthias Kretschmer ◽  
Joyce Wang ◽  
James W. Kronstad
Keyword(s):  
Carbon Source ◽  
Cryptococcus Neoformans ◽  
Virulence Factor ◽  
Fungal Pathogens ◽  
Pathogenic Fungus ◽  
Subsequent Work ◽  
Content Type ◽  
Capsule Production ◽  
Human Pathogenic Fungus ◽  
Host Tissues

ABSTRACTAn understanding of the connections between metabolism and elaboration of virulence factors during host colonization by the human-pathogenic fungusCryptococcus neoformansis important for developing antifungal therapies. Lipids are abundant in host tissues, and fungal pathogens in the phylum basidiomycota possess both peroxisomal and mitochondrial β-oxidation pathways to utilize this potential carbon source. In addition, lipids are important signaling molecules in both fungi and mammals. In this report, we demonstrate that defects in the peroxisomal and mitochondrial β-oxidation pathways influence the growth ofC. neoformanson fatty acids as well as the virulence of the fungus in a mouse inhalation model of cryptococcosis. Disease attenuation may be due to the cumulative influence of altered carbon source acquisition or processing, interference with secretion, changes in cell wall integrity, and an observed defect in capsule production for the peroxisomal mutant. Altered capsule elaboration in the context of a β-oxidation defect was unexpected but is particularly important because this trait is a major virulence factor forC. neoformans. Additionally, analysis of mutants in the peroxisomal pathway revealed a growth-promoting activity forC. neoformans, and subsequent work identified oleic acid and biotin as candidates for such factors. Overall, this study reveals that β-oxidation influences virulence inC. neoformansby multiple mechanisms that likely include contributions to carbon source acquisition and virulence factor elaboration.

scholarly journals Knockdown of cathepsin D in zebrafish fertilized eggs determines congenital myopathy

2013 ◽  
Vol 33 (2) ◽  
Author(s):  
Carlo Follo ◽  
Matteo Ozzano ◽  
Claudia Montalenti ◽  
Massimo Mattia Santoro ◽  
Ciro Isidoro
Keyword(s):  
Cathepsin D ◽  
Muscle Development ◽  
Congenital Myopathy ◽  
Muscle Fibres ◽  
Lysosomal Hydrolase ◽  
Congenital Myopathies ◽  
Altered Expression ◽  
Vertebrate Model

CD (cathepsin D) is a ubiquitous lysosomal hydrolase involved in a variety of pathophysiological functions, including protein turnover, activation of pro-hormones, cell death and embryo development. CD-mediated proteolysis plays a pivotal role in tissue and organ homoeostasis. Altered expression and compartmentalization of CD have been observed in diseased muscle fibres. Whether CD is actively involved in muscle development, homoeostasis and dystrophy remains to be demonstrated. Zebrafish (Danio rerio) is emerging as a valuable ‘in vivo’ vertebrate model for muscular degeneration and congenital myopathies. In this work, we report on the perturbance of the somitic musculature development in zebrafish larvae caused by MPO (morpholino)-mediated silencing of CD in oocytes at the time of fertilization. Restoring CD expression, using an MPO-non-matching mutated mRNA, partially rescued the normal phenotype, confirming the indispensable role of CD in the correct development and integrity of the somitic musculature. This is the first report showing a congenital myopathy caused by CD deficiency in a vertebrate experimental animal model.

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.

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