Cell-wall dyes interfere with Cryptococcus neoformans melanin deposition.
Ontology highlight
ABSTRACT:
SUBMITTER: Perez-Dulzaides R
PROVIDER: S-EPMC6152417 | biostudies-literature | 2018 Aug
REPOSITORIES: biostudies-literature
Publications
Cell-wall dyes interfere with Cryptococcus neoformans melanin deposition.
Perez-Dulzaides Ricardo R Camacho Emma E Cordero Radames J B RJB Casadevall Arturo A
Microbiology (Reading, England) 20180625 8
Melanization is an intrinsic characteristic of many fungal species, but details of this process are poorly understood because melanins are notoriously difficult pigments to study. While studying the binding of cell-wall dyes, Eosin Y or Uvitex, to melanized and non-melanized Cryptococcus neoformans cells we noted that melanization leads to reduced fluorescence intensity, suggesting that melanin interfered with dye binding to the cell wall. The growth of C. neoformans in melanizing conditions wit ...[more]
Similar Datasets
Project description:Cryptococcus neoformans is an environmental fungus that belongs to the phylum Basidiomycetes and is a major pathogen in immunocompromised patients. The ability of C. neoformans to produce melanin pigments represents its second most important virulence factor, after the presence of a polysaccharide capsule. Both the capsule and melanin are closely associated with the fungal cell wall, a complex structure that is essential for maintaining cell morphology and viability under conditions of stress. The amino sugar N-acetylglucosamine (GlcNAc) is a key constituent of the cell-wall chitin and is used for both N-linked glycosylation and GPI anchor synthesis. Recent studies have suggested additional roles for GlcNAc as an activator and mediator of cellular signalling in fungal and plant cells. Furthermore, chitin and chitosan polysaccharides interact with melanin pigments in the cell wall and have been found to be essential for melanization. Despite the importance of melanin, its molecular structure remains unresolved; however, we previously obtained critical insights using advanced nuclear magnetic resonance (NMR) and imaging techniques. In this study, we investigated the effect of GlcNAc supplementation on cryptococcal cell-wall composition and melanization. C. neoformans was able to metabolize GlcNAc as a sole source of carbon and nitrogen, indicating a capacity to use a component of a highly abundant polymer in the biospherenutritionally. C. neoformans cells grown with GlcNAc manifested changes in the chitosan cell-wall content, cell-wall thickness and capsule size. Supplementing cultures with isotopically 15N-labelled GlcNAc demonstrated that the exogenous monomer serves as a building block for chitin/chitosan and is incorporated into the cell wall. The altered chitin-to-chitosan ratio had no negative effects on the mother-daughter cell separation; growth with GlcNAc affected the fungal cell-wall scaffold, resulting in increased melanin deposition and assembly. In summary, GlcNAc supplementation had pleiotropic effects on cell-wall and melanin architectures, and thus established its capacity to perturb these structures, a property that could prove useful for metabolic tracking studies.
| S-EPMC5775898 | biostudies-literature
Project description:Melanins are synthesized macromolecules that are found in all biological kingdoms. These pigments have a myriad of roles that range from microbial virulence to key components of the innate immune response in invertebrates. Melanins also exhibit unique properties with potential applications in physics and material sciences, ranging from electrical batteries to novel therapeutics. In the fungi, melanins, such as eumelanins, are components of the cell wall that provide protection against biotic and abiotic elements. Elucidation of the smallest fungal cell wall-associated melanin unit that serves as a building block is critical to understand the architecture of these polymers, its interaction with surrounding components, and their functional versatility. In this study, we used isopycnic gradient sedimentation, NMR, EPR, high-resolution microscopy, and proteomics to analyze the melanin in the cell wall of the human pathogenic fungus Cryptococcus neoformans We observed that melanin is assembled into the cryptococcal cell wall in spherical structures ?200 nm in diameter, termed melanin granules, which are in turn composed of nanospheres ?30 nm in diameter, termed fungal melanosomes. We noted that melanin granules are closely associated with proteins that may play critical roles in the fungal melanogenesis and the supramolecular structure of this polymer. Using this structural information, we propose a model for C. neoformans' melanization that is similar to the process used in animal melanization and is consistent with the phylogenetic relatedness of the fungal and animal kingdoms.
| S-EPMC6615676 | biostudies-literature
Project description:Cryptococcus neoformans and Cryptococcus gattii are two species complexes in the large fungal genus Cryptococcus and are responsible for potentially lethal disseminated infections. These two complexes share several phenotypic traits, such as production of the protective compound melanin. In C. neoformans, the pigment associates with key cellular constituents that are essential for melanin deposition within the cell wall. Consequently, melanization is modulated by changes in cell-wall composition or ultrastructure. However, whether similar factors influence melanization in C. gattii is unknown. Herein, we used transmission EM, biochemical assays, and solid-state NMR spectroscopy of representative isolates and "leaky melanin" mutant strains from each species complex to examine the compositional and structural factors governing cell-wall pigment deposition in C. neoformans and C. gattii. The principal findings were the following. 1) C. gattii R265 had an exceptionally high chitosan content compared with C. neoformans H99; a rich chitosan composition promoted homogeneous melanin distribution throughout the cell wall but did not increase the propensity of pigment deposition. 2) Strains from both species manifesting the leaky melanin phenotype had reduced chitosan content, which was compensated for by the production of lipids and other nonpolysaccharide constituents that depended on the species or mutation. 3) Changes in the relative rigidity of cell-wall chitin were associated with aberrant pigment retention, implicating cell-wall flexibility as an independent variable in cryptococcal melanin assembly. Overall, our results indicate that cell-wall composition and molecular architecture are critical factors for the anchoring and arrangement of melanin pigments in both C. neoformans and C. gattii species complexes.
| S-EPMC7029119 | biostudies-literature
Project description:Melanin is an antioxidant polyphenol pigment required for the pathogenicity of many fungal pathogens, but comprehensive regulatory mechanisms remain unidentified. In this study, we systematically analyzed melanin-regulating signaling pathways in Cryptococcus neoformans and identified four melanin-regulating core transcription factors (TFs), Bzp4, Usv101, Mbs1, and Hob1, required for induction of the laccase gene (LAC1). Bzp4, Usv101, and Mbs1 independently regulate LAC1 induction, whereas Hob1 controls Bzp4 and Usv101 expression. Both Bzp4 and Usv101 are localized in the cytoplasm under nutrient-rich conditions (i.e., in the presence of yeast extract-peptone-dextrose [YPD] medium) but translocate into the nucleus upon nutrient starvation (i.e., in the presence of yeast nitrogen base [YNB] medium without glucose), and Mbs1 is constitutively localized in the nucleus. Notably, the cAMP pathway is not involved in regulation of the four TFs, but the high-osmolarity glycerol response (HOG) pathway negatively regulates induction of BZP4 and LAC1 Next, we searched for potential kinases upstream of the core TFs and identified nine core kinases; their deletion led to defective melanin production and LAC1 induction. Deletion of GSK3 or KIC1 abolished induction of LAC1 and BZP4 and perturbed nuclear translocation of Bzp4. Notably, Gsk3 also regulated expression of HOB1, USV101, and MBS1, indicating that it is a critical melanin-regulating kinase. Finally, an RNA sequencing-based transcriptome analysis of the wild-type strain and of bzp4?, usv101?, hob1?, and mbs1? strains under nutrient-rich and nutrient-starved conditions revealed that the melanin-regulating core TFs govern redundant and distinct classes of genes involved in a variety of biological processes.IMPORTANCE Melanins are dark green, brown, or black pigments that serve as antioxidant, reactive oxygen species (ROS) scavengers that protect fungal pathogens from radiation and host immune responses. Cryptococcus neoformans, the major etiological agent of fungal meningoencephalitis, also utilizes melanin as a key virulence factor. In this basidiomycete pathogen, melanin production is regulated by the cAMP and high-osmolarity glycerol response (HOG) pathways, and yet its complex signaling networks remain poorly described. In this study, we uncovered novel melanin synthesis regulatory networks consisting of core transcription factors (TFs), including Bzp4, Usv101, Hob1, and Mbs1, and core kinases Gsk3 and Kic1. These networks were identified through coupling systematic analyses of the expression and epistatic relationships of TF and kinase mutant libraries in the presence of diverse melanin substrates with transcriptome profiling of the core TF mutants. Thus, this report provides comprehensive insight into the melanin-regulating pathways in C. neoformans and other fungal pathogens.
| S-EPMC6775464 | biostudies-literature
Project description:Cryptococcus neoformans produces 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.
| S-EPMC4248683 | biostudies-literature
Project description:Melanin 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 yielded more melanin than expected if only the preferred constituent dopamine had been incorporated, 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.
| S-EPMC8760516 | biostudies-literature
Project description:The cryptococcal capsule is a critical virulence factor of an important pathogen, but little is known about how it is associated with the cell or released into the environment. Two mutants lacking PBX1 and PBX2 were found to shed reduced amounts of the capsule polysaccharide glucuronoxylomannan (GXM). Nuclear magnetic resonance, composition, and physical analyses showed that the shed material was of normal mass but was slightly enriched in xylose. In contrast to previous reports, this material contained no glucose. Notably, the capsule fibers of pbx? mutant cells grown under capsule-inducing conditions were present at a lower than usual density and were loosely attached to the cell wall. Mutant cell walls were also defective, as indicated by phenotypes including abnormal cell morphology, reduced mating filamentation, and altered cell integrity. All observed phenotypes were shared between the two mutants and exacerbated in a double mutant. Consistent with a role in surface glycan synthesis, the Pbx proteins localized to detergent-resistant membrane domains. These results, together with the sequence motifs in the Pbx proteins, suggest that Pbx1 and Pbx2 are redundant proteins that act in remodeling the cell wall to maintain normal cell morphology and precursor availability for other glycan synthetic processes. Their absence results in aberrant cell wall growth and metabolic imbalance, which together impact cell wall and capsule synthesis, cell morphology, and capsule association. The surface changes also lead to increased engulfment by host phagocytes, consistent with the lack of virulence of pbx mutants in animal models.
| S-EPMC4060484 | biostudies-literature
Project description: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 lac1Delta 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.
| S-EPMC1828480 | biostudies-literature
Project description:Random insertional mutagenesis screens are important tools in microbial genetics studies. Investigators in fungal systems have used the plant pathogen Agrobacterium tumefaciens to create tagged, random mutations for genetic screens in their fungal species of interest through a unique process of trans-kingdom cellular transconjugation. However, identifying the locations of insertion has traditionally required tedious PCR-based methods, limiting the effective throughput of this system. We have developed an efficient genomic sequencing and analysis method (AIM-Seq) to facilitate identification of randomly generated genomic insertions in microorganisms. AIM-Seq combines batch sampling, whole genome sequencing, and a novel bioinformatics pipeline, AIM-HII, to rapidly identify sites of genomic insertion. We have specifically applied this technique to Agrobacterium-mediated transconjugation in the human fungal pathogen Cryptococcus neoformans. With this approach, we have screened a library of C. neoformans cell wall mutants, selecting twenty-seven mutants of interest for analysis by AIM-Seq. We identified thirty-five putative genomic insertions in known and previously unknown regulators of cell wall processes in this pathogenic fungus. We confirmed the relevance of a subset of these by creating independent mutant strains and analyzing resulting cell wall phenotypes. Through our sequence-based analysis of these mutations, we observed "typical" insertions of the Agrobacterium transfer DNA as well as atypical insertion events, including large deletions and chromosomal rearrangements. Initially applied to C. neoformans, this mutant analysis tool can be applied to a wide range of experimental systems and methods of mutagenesis, facilitating future microbial genetic screens.
| S-EPMC4693612 | biostudies-literature
Project description:Melanin is an antioxidant polyphenol pigment, which is required for the pathogenicity of many human and plant fungal pathogens. Nevertheless, its comprehensive regulatory mechanism still remain elusive. In this study, we systematically analyzed melanin-regulating signaling pathways in Cryptococcus neoformans. Here we identified four core melanin-regulating TFs, Bzp4, Usv101, Mbs1, and Hob1, which are required for induction of the laccase gene (LAC1). Bzp4, Usv101 and Mbs1 independently regulate LAC1 induction while Hob1 controls Bzp4 and Usv101 expression. Both Bzp4 and Usv101 are translocated from the cytoplasm into the nucleus upon nutrient starved condition, while Mbs1 is constitutively localized in the nucleus. Notably, the cAMP pathway is not involved in regulation of the four TFs, while the HOG pathway negatively regulates induction of BZP4 and LAC1. As potential kinases upstream of the core TFs, we identified the nine core kinases, whose deletion leads to defective melanin production and LAC1 induction. Deletion of GSK3 or KIC1 abolishes induction of LAC1 and BZP4, and also perturbs nuclear translocation of Bzp4. Notably, Gsk3 also regulates the expression of HOB1, USV101, and MBS1, indicating that it is a critical melanin-regulating kinase. Finally, the RNA-sequencing based transcriptome analysis of the wild-type and bzp4Δ, usv101Δ, hob1Δ, and mbs1Δ strains under nutrient rich and starved conditions reveals that the core melanin-regulating TFs govern redundant and distinct classes of genes, which are involved in a variety of biological processes.
2019-05-30 | GSE131891 | GEO