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 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 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 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 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 Induction of Fusarium lytic Enzymes by Extracts from Resistant and Susceptible Cultivars of Pea (Pisum sativum L.)

Pathogens ◽  
2020 ◽  
Vol 9 (11) ◽  
pp. 976
Author(s):  
Lakshmipriya Perincherry ◽  
Chaima Ajmi ◽  
Souheib Oueslati ◽  
Agnieszka Waśkiewicz ◽  
Łukasz Stępień
Keyword(s):  
Cell Wall ◽  
Plant Extracts ◽  
Plant Cell Wall ◽  
Pathogenic Fungi ◽  
Human Beings ◽  
Cell Wall Degrading Enzymes ◽  
Lytic Enzymes ◽  
Mycelium Growth ◽  
Oat Bran

Being pathogenic fungi, Fusarium produce various extracellular cell wall-degrading enzymes (CWDEs) that degrade the polysaccharides in the plant cell wall. They also produce mycotoxins that contaminate grains, thereby posing a serious threat to animals and human beings. Exposure to mycotoxins occurs through ingestion of contaminated grains, inhalation and through skin absorption, thereby causing mycotoxicoses. The toxins weaken the host plant, allowing the pathogen to invade successfully, with the efficiency varying from strain to strain and depending on the plant infected. Fusariumoxysporum predominantly produces moniliformin and cyclodepsipeptides, whereas F. proliferatum produces fumonisins. The aim of the study was to understand the role of various substrates and pea plant extracts in inducing the production of CWDEs and mycotoxins. Additionally, to monitor the differences in their levels when susceptible and resistant pea plant extracts were supplemented. The cultures of F. proliferatum and F. oxysporum strains were supplemented with various potential inducers of CWDEs. During the initial days after the addition of substrates, the fungus cocultivated with pea extracts and other carbon substrates showed increased activities of β-glucosidase, xylanase, exo-1,4-glucanase and lipase. The highest inhibition of mycelium growth (57%) was found in the cultures of F. proliferatum strain PEA1 upon the addition of cv. Sokolik extract. The lowest fumonisin content was exhibited by the cultures with the pea extracts and oat bran added, and this can be related to the secondary metabolites and antioxidants present in these substrates.

scholarly journals Nutritional Conditions Modulate C. neoformans Extracellular Vesicles’ Capacity to Elicit Host Immune Response

2020 ◽  
Vol 8 (11) ◽  
pp. 1815
Author(s):  
Clara Luna Marina ◽  
Pedro Henrique Bürgel ◽  
Daniel Paiva Agostinho ◽  
Daniel Zamith-Miranda ◽  
Lucas de Oliveira Las-Casas ◽  
...  
Keyword(s):  
Extracellular Vesicles ◽  
Pathogenic Fungus ◽  
Nutritional Condition ◽  
Nutritional Conditions ◽  
Rich Media ◽  
Nutritional Medium ◽  
Human Pathogenic Fungus ◽  
Inflammatory Pattern ◽  
The Rich ◽  
Poor Nutritional Condition

Cryptococcus neoformans is a human pathogenic fungus that mainly afflicts immunocompromised patients. One of its virulence strategies is the production of extracellular vesicles (EVs), containing cargo with immunomodulatory properties. We evaluated EV’s characteristics produced by capsular and acapsular strains of C. neoformans (B3501 and ΔCap67, respectively) growing in nutritionally poor or rich media and co-cultures with bone marrow-derived macrophages or dendritic cells from C57BL/6 mice. EVs produced under a poor nutritional condition displayed a larger hydrodynamic size, contained more virulence compounds, and induced a more robust inflammatory pattern than those produced in a rich nutritional medium, independently of strain. We treated infected mice with EVs produced in the rich medium, and the EVs inhibited more genes related to the inflammasome than untreated infected mice. These findings suggest that the EVs participate in the pathogenic processes that result in the dissemination of C. neoformans. Thus, these results highlight the versatility of EVs’ properties during infection by C. neoformans in different tissues and support ongoing efforts to harness EVs to prevent and treat cryptococcosis.

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 Interaction of Cryptococcus neoformans Extracellular Vesicles with the Cell Wall

2014 ◽  
Vol 13 (12) ◽  
pp. 1484-1493 ◽  
Author(s):  
Julie M. Wolf ◽  
Javier Espadas-Moreno ◽  
Jose L. Luque-Garcia ◽  
Arturo Casadevall
Keyword(s):  
Cell Wall ◽  
Plasma Membrane ◽  
Cryptococcus Neoformans ◽  
Extracellular Vesicles ◽  
Protein Composition ◽  
Multivesicular Body ◽  
Dense Matrix ◽  
Fungal Cell ◽  
Content Type ◽  
Pass Through

ABSTRACTCryptococcus neoformansproduces extracellular vesicles containing a variety of cargo, including virulence factors. To become extracellular, these vesicles not only must be released from the plasma membrane but also must pass through the dense matrix of the cell wall. The greatest unknown in the area of fungal vesicles is the mechanism by which these vesicles are released to the extracellular space given the presence of the fungal cell wall. Here we used electron microscopy techniques to image the interactions of vesicles with the cell wall. Our goal was to define the ultrastructural morphology of the process to gain insights into the mechanisms involved. We describe single and multiple vesicle-leaving events, which we hypothesized were due to plasma membrane and multivesicular body vesicle origins, respectively. We further utilized melanized cells to “trap” vesicles and visualize those passing through the cell wall. Vesicle size differed depending on whether vesicles left the cytoplasm in single versus multiple release events. Furthermore, we analyzed different vesicle populations for vesicle dimensions and protein composition. Proteomic analysis tripled the number of proteins known to be associated with vesicles. Despite separation of vesicles into batches differing in size, we did not identify major differences in protein composition. In summary, our results indicate that vesicles are generated by more than one mechanism, that vesicles exit the cell by traversing the cell wall, and that vesicle populations exist as a continuum with regard to size and protein composition.

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