scholarly journals Vesicular Trans-Cell Wall Transport in Fungi: A Mechanism for the Delivery of Virulence-Associated Macromolecules?

2008 ◽  
Vol 2 ◽  
pp. LPI.S1000 ◽  
Author(s):  
Marcio L. Rodrigues ◽  
Leonardo Nimrichter ◽  
Debora L. Oliveira ◽  
Joshua D. Nosanchuk ◽  
Arturo Casadevall
Keyword(s):  
Cell Wall ◽  
Extracellular Vesicles ◽  
Fungal Pathogens ◽  
Histoplasma Capsulatum ◽  
Biological Significance ◽  
Sporothrix Schenckii ◽  
Compositional Analysis ◽  
Fungal Cell ◽  
Yeast Saccharomyces Cerevisiae ◽  
Cellular Origin

Fungal cells are encaged in rigid, complex cell walls. Until recently, there was remarkably little information regarding the trans-fungal cell wall transfer of intracellular macromolecules to the extracellular space. Recently, several studies have begun to elucidate the mechanisms that fungal cells utilize to secrete a wide variety of macromolecules through the cell wall. The combined use of transmission electron microscopy, serology, biochemistry, proteomics and lipidomics have revealed that the fungal pathogens Cryptococcus neoformans, Histoplasma capsulatum, Candida albicans, Candida parapsilosis and Sporothrix schenckii, as well as the model yeast Saccharomyces cerevisiae, each produces extracellular vesicles that carry lipids, proteins, polysaccharides and pigment-like structures of unquestionable biological significance. Compositional analysis of the C. neoformans and H. capsulatum extracellular vesicles suggests that they may function as ‘virulence bags’, with the potential to modulate the host-pathogen interaction in favor of the fungus. The cellular origin of the extracellular vesicles remains unknown, but morphological and biochemical features indicate that they are similar to the well-described mammalian exosomes.

scholarly journals Yeast Mnn9 is both a priming glycosyltransferase and an allosteric activator of mannan biosynthesis

Open Biology ◽  
2013 ◽  
Vol 3 (9) ◽  
pp. 130022 ◽  
Author(s):  
Alexander Striebeck ◽  
David A. Robinson ◽  
Alexander W. Schüttelkopf ◽  
Daan M. F. van Aalten
Keyword(s):  
Cell Wall ◽  
Fungal Pathogens ◽  
Enzyme Assay ◽  
Antibiotic Sensitivity ◽  
Site Directed Mutagenesis ◽  
Fungal Cell ◽  
Pol I ◽  
Conserved Amino Acids ◽  
Allosteric Activator ◽  
Mannan Synthesis

The fungal cell possesses an essential carbohydrate cell wall. The outer layer, mannan, is formed by mannoproteins carrying highly mannosylated O - and N -linked glycans. Yeast mannan biosynthesis is initiated by a Golgi-located complex (M-Pol I) of two GT-62 mannosyltransferases, Mnn9p and Van1p, that are conserved in fungal pathogens. Saccharomyces cerevisiae and Candida albicans mnn9 knockouts show an aberrant cell wall and increased antibiotic sensitivity, suggesting the enzyme is a potential drug target. Here, we present the structure of Sc Mnn9 in complex with GDP and Mn 2+ , defining the fold and catalytic machinery of the GT-62 family. Compared with distantly related GT-78/GT-15 enzymes, Sc Mnn9 carries an unusual extension. Using a novel enzyme assay and site-directed mutagenesis, we identify conserved amino acids essential for Sc Mnn9 ‘priming’ α-1,6-mannosyltransferase activity. Strikingly, both the presence of the Sc Mnn9 protein and its product, but not Sc Mnn9 catalytic activity, are required to activate subsequent Sc Van1 processive α-1,6-mannosyltransferase activity in the M-Pol I complex. These results reveal the molecular basis of mannan synthesis and will aid development of inhibitors targeting this process.

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.

scholarly journals Characterization of extracellular vesicles produced by Aspergillus fumigatus protoplasts

2020 ◽  
Author(s):  
Juliana Rizzo ◽  
Thibault Chaze ◽  
Kildare Miranda ◽  
Robert W. Roberson ◽  
Olivier Gorgette ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Cell Wall ◽  
Aspergillus Fumigatus ◽  
Extracellular Vesicles ◽  
Fungal Pathogen ◽  
Fungal Pathogens ◽  
Sugar Metabolism ◽  
Biologically Active ◽  
Thick Cell Wall ◽  
A Cell

AbstractExtracellular vesicles (EVs) are outer membranous compartments produced by yeast and mycelial forms of several fungal species. One of the difficulties to perceive the role of EVs during the fungal life is the fact that an active secretion of these EVs has not been clearly demonstrated in situ due to the presence of a thick cell wall. One alternative to have a better access to these vesicles is to use protoplasts. This approach has been investigated here with Aspergillus fumigatus, one of the most common opportunistic fungal pathogens worldwide. Analysis of regenerating protoplasts by scanning electron microscopy and fluorescence microscopy indicated the occurrence of outer membrane projections in association with surface components and the release of particles with properties resembling those of fungal EVs. EVs in culture supernatants were characterized by transmission electron microscopy and nanoparticle tracking analysis. Proteomic and glycome analysis of EVs revealed the presence of a complex array of enzymes related to lipid / sugar metabolism, pathogenic processes, and cell wall biosynthesis. Our data indicate that i) EV production is a common feature of different morphological stages of this major fungal pathogen, and ii) protoplastic EVs are a promising tool to undertake studies of vesicle functions in fungal cells.IMPORTANCEFungal cells use extracellular vesicles (EVs) to export biologically active molecules to the outer space. Since fungal cells are encaged in a thick cell wall, it is reasonable to expect that this structure might impact the vesicle-mediated molecular export. In this study, we used protoplasts of Aspergillus fumigatus, a major fungal pathogen, as a model to evaluate EV production in the absence of a cell wall. Our results demonstrated that wall-less A. fumigatus exports plasma membrane-derived EVs containing a complex combination of proteins and glycans. Our study is the first to characterize fungal EVs in the absence of a cell wall. Our results suggest that protoplasts are a promising model for functional studies of fungal vesicles.

scholarly journals Drug Interaction Studies of a Glucan Synthase Inhibitor (LY 303366) and a Chitin Synthase Inhibitor (Nikkomycin Z) for Inhibition and Killing of Fungal Pathogens

2000 ◽  
Vol 44 (9) ◽  
pp. 2547-2548 ◽  
Author(s):  
David A. Stevens
Keyword(s):  
Cell Wall ◽  
Fungal Pathogens ◽  
Enzyme Inhibitors ◽  
Chitin Synthase ◽  
Synergistic Activity ◽  
Fungal Cell ◽  
Glucan Synthase ◽  
Nikkomycin Z ◽  
Synthase Inhibitor

ABSTRACT The interaction between inhibitors of components of the fungal cell wall, glucan and chitin, was studied in vitro with the respective synthase enzyme inhibitors LY 303366 and nikkomycin Z. WithAspergillus fumigatus synergy was noted for inhibition and killing, and synergistic activity was also noted for some isolates of other species presently regarded as difficult to treat.

scholarly journals The GPI-modified proteins Pga59 and Pga62 of Candida albicans are required for cell wall integrity

Microbiology ◽  
2009 ◽  
Vol 155 (6) ◽  
pp. 2004-2020 ◽  
Author(s):  
Emilia Moreno-Ruiz ◽  
Giuseppe Ortu ◽  
Piet W. J. de Groot ◽  
Fabien Cottier ◽  
Céline Loussert ◽  
...  
Keyword(s):  
Cell Wall ◽  
Candida Albicans ◽  
Fungal Pathogens ◽  
High Sensitivity ◽  
Wall Structure ◽  
Minor Role ◽  
Cell Wall Proteins ◽  
Calcofluor White ◽  
Fungal Cell ◽  
A Minor

The fungal cell wall is essential in maintaining cellular integrity and plays key roles in the interplay between fungal pathogens and their hosts. The PGA59 and PGA62 genes encode two short and related glycosylphosphatidylinositol-anchored cell wall proteins and their expression has been previously shown to be strongly upregulated when the human pathogen Candida albicans grows as biofilms. Using GFP fusion proteins, we have shown that Pga59 and Pga62 are cell-wall-located, N- and O-glycosylated proteins. The characterization of C. albicans pga59Δ/pga59Δ, pga62Δ/pga62Δ and pga59Δ/pga59Δ pga62Δ/pga62Δ mutants suggested a minor role of these two proteins in hyphal morphogenesis and that they are not critical to biofilm formation. Importantly, the sensitivity to different cell-wall-perturbing agents was altered in these mutants. In particular, simultaneous inactivation of PGA59 and PGA62 resulted in high sensitivity to Calcofluor white, Congo red and nikkomicin Z and in resistance to caspofungin. Furthermore, cell wall composition and observation by transmission electron microscopy indicated an altered cell wall structure in the mutant strains. Collectively, these data suggest that the cell wall proteins Pga59 and Pga62 contribute to cell wall stability and structure.

scholarly journals Insights Into Histoplasma capsulatum Behavior on Zinc Deprivation

2020 ◽  
Vol 10 ◽  
Author(s):  
Leandro do Prado Assunção ◽  
Dayane Moraes ◽  
Lucas Weba Soares ◽  
Mirelle Garcia Silva-Bailão ◽  
Janaina Gomes de Siqueira ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Cell Wall ◽  
Histoplasma Capsulatum ◽  
Zinc Homeostasis ◽  
Metal Availability ◽  
Fungal Cell ◽  
Tropical Regions ◽  
Macrophage Activity ◽  
Zip Family ◽  
Zn Availability

Histoplasma capsulatum is a thermodimorphic fungus that causes histoplasmosis, a mycosis of global incidence. The disease is prevalent in temperate and tropical regions such as North America, South America, Europe, and Asia. It is known that during infection macrophages restrict Zn availability to H. capsulatum as a microbicidal mechanism. In this way the present work aimed to study the response of H. capsulatum to zinc deprivation. In silico analyses showed that H. capsulatum has eight genes related to zinc homeostasis ranging from transcription factors to CDF and ZIP family transporters. The transcriptional levels of ZAP1, ZRT1, and ZRT2 were induced under zinc-limiting conditions. The decrease in Zn availability increases fungicidal macrophage activity. Proteomics analysis during zinc deprivation at 24 and 48 h showed 265 proteins differentially expressed at 24 h and 68 at 48 h. Proteins related to energy production pathways, oxidative stress, and cell wall remodeling were regulated. The data also suggested that low metal availability increases the chitin and glycan content in fungal cell wall that results in smoother cell surface. Metal restriction also induces oxidative stress triggered, at least in part, by reduction in pyridoxin synthesis.

scholarly journals The Pathogenic Fungus Paracoccidioides brasiliensis Exports Extracellular Vesicles Containing Highly Immunogenic α-Galactosyl Epitopes

2011 ◽  
Vol 10 (3) ◽  
pp. 343-351 ◽  
Author(s):  
Milene C. Vallejo ◽  
Alisson L. Matsuo ◽  
Luciane Ganiko ◽  
Lia C. Soares Medeiros ◽  
Kildare Miranda ◽  
...  
Keyword(s):  
Extracellular Vesicles ◽  
Fungal Pathogens ◽  
Histoplasma Capsulatum ◽  
Paracoccidioides Brasiliensis ◽  
Pathogenic Fungus ◽  
Yeast Cells ◽  
Enzyme Linked Immunosorbent Assay ◽  
Dimorphic Fungus ◽  
Content Type

ABSTRACTExosome-like vesicles containing virulence factors, enzymes, and antigens have recently been characterized in fungal pathogens, such asCryptococcus neoformansandHistoplasma capsulatum. Here, we describe extracellular vesicles carrying highly immunogenic α-linked galactopyranosyl (α-Gal) epitopes inParacoccidioides brasiliensis. P. brasiliensisis a dimorphic fungus that causes human paracoccidioidomycosis (PCM). For vesicle preparations, cell-free supernatant fluids from yeast cells cultivated in Ham's defined medium-glucose were concentrated in an Amicon ultrafiltration system and ultracentrifuged at 100,000 ×g. P. brasiliensisantigens were present in preparations from phylogenetically distinct isolates Pb18 and Pb3, as observed in immunoblots revealed with sera from PCM patients. In an enzyme-linked immunosorbent assay (ELISA), vesicle components containing α-Gal epitopes reacted strongly with anti-α-Gal antibodies isolated from both Chagas' disease and PCM patients, withMarasmius oreadesagglutinin (MOA) (a lectin that recognizes terminal α-Gal), but only faintly with natural anti-α-Gal. Reactivity was inhibited after treatment with α-galactosidase. Vesicle preparations analyzed by electron microscopy showed vesicular structures of 20 to 200 nm that were labeled both on the surface and in the lumen with MOA. InP. brasiliensiscells, components carrying α-Gal epitopes were found distributed on the cell wall, following a punctuated confocal pattern, and inside large intracellular vacuoles. Lipid-free vesicle fractions reacted with anti-α-Gal in ELISA only when not digested with α-galactosidase, while reactivity with glycoproteins was reduced after β-elimination, which is indicative of partial O-linked chain localization. Our findings open new areas to explore in terms of host-parasite relationships in PCM and the role playedin vivoby vesicle components and α-galactosyl epitopes.

scholarly journals Involvement of Fungal Cell Wall Components in Adhesion of Sporothrix schenckii to Human Fibronectin

2001 ◽  
Vol 69 (11) ◽  
pp. 6874-6880 ◽  
Author(s):  
Osana C. Lima ◽  
Camila C. Figueiredo ◽  
José O. Previato ◽  
Lucia Mendonça-Previato ◽  
Verônica Morandi ◽  
...  
Keyword(s):  
Cell Wall ◽  
Sporothrix Schenckii ◽  
Matrix Proteins ◽  
Yeast Cells ◽  
Binding Assays ◽  
Fungal Cell ◽  
Host Fungus ◽  
Human Fibronectin ◽  
Dose Dependent ◽  
Cell Surface Glycoconjugates

ABSTRACT Systemic sporotrichosis is an emerging infection potentially fatal for immunocompromised patients. Adhesion to extracellular matrix proteins is thought to play a crucial role in invasive fungal diseases. Here we report studies of the adhesion of Sporothrix schenckii to the extracellular protein fibronectin (Fn). Both yeast cells and conidia of S. schenckii were able to adhere to Fn as detected by enzyme-linked immunosorbent binding assays. Adhesion of yeast cells to Fn is dose dependent and saturable.S. schenckii adheres equally well to 40-kDa and 120-kDa Fn proteolytic fragments. While adhesion to Fn was increased by Ca2+, inhibition assays demonstrated that it was not RGD dependent. A carbohydrate-containing cell wall neutral fraction blocked up to 30% of the observed adherence for the yeast cells. The biochemical nature of this fraction suggests the participation of cell surface glycoconjugates in binding by their carbohydrate or peptide moieties. These results provide new data concerning S. schenckii adhesion mechanisms, which could be important in host-fungus interactions and the establishment of sporotrichosis.

scholarly journals Answers to naysayers regarding microbial extracellular vesicles

2019 ◽  
Vol 47 (4) ◽  
pp. 1005-1012 ◽  
Author(s):  
Carolina Coelho ◽  
Arturo Casadevall
Keyword(s):  
Cell Wall ◽  
Extracellular Vesicles ◽  
Living Cell ◽  
Physiological Function ◽  
Biological Significance ◽  
Gram Negative Bacteria ◽  
Biological Processes ◽  
Gram Positive ◽  
Gram Negative ◽  
Gram Positive Bacteria

Abstract It is now over 30 years since the discovery of extracellular vesicles (EVs) in Gram-negative bacteria. However, for cell-walled microbes such as fungi, mycobacteria and Gram-positive bacteria it was thought that EV release would be impossible, since such structures were not believed to cross the thick cell wall. This notion was disproven 10 years ago with the discovery of EVs in fungi, mycobacteria, and gram-positive bacteria. Today, EVs have been described in practically every species tested, ranging from Fungi through Bacteria and Archaea, suggesting that EVs are a feature of every living cell. However, there continues to be skepticism in some quarters regarding EV release and their biological significance. In this review, we list doubts that have been verbalized to us and provide answers to counter them. In our opinion, there is no doubt as to existence and physiological function of EVs and we take this opportunity to highlight the most pressing topics in our understanding of the biological processes underlying these structures.

scholarly journals Surface Localization of the Yps3p Protein of Histoplasma capsulatum

2005 ◽  
Vol 4 (4) ◽  
pp. 685-693 ◽  
Author(s):  
Megan L. Bohse ◽  
Jon P. Woods
Keyword(s):  
Cell Wall ◽  
Cellular Localization ◽  
Histoplasma Capsulatum ◽  
Flow Cytometric Analysis ◽  
Binding Assays ◽  
Fungal Cell ◽  
Pathogenic Yeast ◽  
Surface Localization ◽  
Carbohydrate Polymer ◽  
Yeast Phase

ABSTRACT The YPS3 gene of Histoplasma capsulatum encodes a protein that is both resident in the cell wall and also released into the culture medium. This protein is produced only during the pathogenic yeast phase of infection and is also expressed differently in H. capsulatum strains that differ in virulence. We investigated the cellular localization of Yps3p. We demonstrated that the cell wall fraction of Yps3p was surface localized in restriction fragment length polymorphism class 2 strains. We also established that Yps3p released into the G217B culture supernatant binds to the surface of strains that do not naturally express the protein. This binding was saturable and occurred within 5 min of exposure and occurred similarly with live and heat-killed H. capsulatum. Flow cytometric analysis of H. capsulatum after enzymatic treatments was consistent with Yps3p binding to chitin, a carbohydrate polymer that is a component of fungal cell walls. Polysaccharide binding assays demonstrated that chitin but not cellulose binds to and extracts Yps3p from culture supernatants.

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