Separation of chitosomes and secretory vesicles from the ?slime? variant of Neurospora crassa

1987

Cells from the “slime” variant of Neurospora crassa were broken in isotonic conditions by use of triethanolamine buffer plus EDTA. After removal of large membranous structures by low-speed centrifugation, chitosomes and secretory vesicles were separated by means of gel filtration, precipitation of membranous contaminants with Concanavalin A, and centrifugation in sucrose or glycerol gradients. Polypeptidic composition of fractions enriched in secretory vesicles or chitosomes was found to be distinct. By these criteria we concluded that chitosomes and secretory vesicles represent different populations of microvesicles. Both microvesicular populations appeared free of endoplasmic reticulum an…

Self-assembly properties of the proteinaceous coat secreted by the ?slime? variant of Neurospora crassa

1989

The proteinaceous extracellular material (PEM) synthesized by the cells of the ‘slime” strain of Neurospora crassa (see Martinez et al. 1989) was solubilized by treatment with urea or guanidine. Removal of these chemicals by dialysis, caused reassembly of the solubilized proteins into material with the same microscopic appearance as the original PEM. Polypeptide patterns from both native and reassembled structures were identical. Dialysis-mediated reassembly of the solubilized proteins appeared to be dependent on both concentration of the soluble macromolecules and time. Gel chromatography of PEM solubilized with different agents revealed two discrete populations of complexes with molecular…

Direct Toll-Like Receptor-Mediated Stimulation of Hematopoietic Stem and Progenitor Cells Occurs In Vivo and Promotes Differentiation Toward Macropha…

2012

Abstract As Toll-like receptors (TLRs) are expressed by hematopoietic stem and progenitor cells (HSPCs), they may play a role in hematopoiesis in response to pathogens during infection. We show here that TLR2, TLR4, and TLR9 agonists (tripalmitoyl-S-glyceryl-L-Cys-Ser-(Lys)4 [Pam3CSK4], lipopolysaccharide [LPS], and CpG oligodeoxynucleotide [ODN]) induce the in vitro differentiation of purified murine lineage negative cells (Lin−) as well as HSPCs (identified as Lin− c-Kit+ Sca-1+ IL-7Rα− [LKS] cells) toward macrophages (Mph), through a myeloid differentiation factor 88 (MyD88)-dependent pathway. In order to investigate the possible direct interaction of soluble microorganism-associated mol…

Binding of extracellular matrix proteins to Aspergillus fumigatus conidia

1996

As detected by confocal immunofluorescence microscopy, binding of fibronectin and laminin appeared to be associated with the protrusions present on the outer cell wall layer of resting Aspergillus fumigatus conidia. Flow cytometry confirmed that binding of laminin to conidia was dose dependent and saturable. Laminin binding was virtually eliminated in trypsin-treated organisms, thus suggesting the protein nature of the binding site. Conidia were also able to specifically adhere to laminin immobilized on microtiter plates. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting (immunoblotting) with laminin and antilaminin antibody of whole conidial homogenates allowed…

Influence of aging on murine neutrophil and macrophage function against Candida albicans.

2008

Previous work by our group showed that aged C57BL/6 mice develop an altered innate and adaptive immune response to Candida albicans and are more susceptible to systemic primary candidiasis. In this work, we used young (2-3 months old) and aged (18-20 months old) C57BL/6 mice to study in vitro the influence of aging on (1) the fungicidal activity of neutrophils and macrophages, (2) the production of cytokines by resident peritoneal macrophages in response to C. albicans, and (3) cell surface Toll-like receptor (TLR) 2 expression on resident peritoneal macrophages. Our results indicate that murine phagocytes have a fungicidal activity well preserved with aging. In vitro production of proinfla…

Candida and Candidiasis: The Cell Wall as a Potential Molecular Target for Antifungal Therapy

2004

The fungal species Candida albicans is an opportunistic pathogen, which causes serious infections in humans, particularly in immunocompromised patients. Depending on the underlying host defect, C. albicans causes a variety of infections, ranging from superficial mucocutaneous candidiasis to life-threatening disseminated infections. Both the limited spectrum of antifungal drugs currently in clinical use and the emergence of resistances make necessary the development of new effective antifungal drugs with minimal side effects; however, such a research is limited by the small number of specific target sites identified to date. The cell wall is a fungal specific dynamic structure essential to a…

Analysis of the polypeptide composition of the cell walls of Neurospora crassa. Similarities with the proteinaceous material secreted by the slime va…

1991

The polypeptide composition of cell walls from the wild-type strain of Neurospora crassa is compared with that of the proteinaceous extracellular coat (PEM) secreted by the slime strain of this fungus. Analyses included determination of the polypeptide pattern by polyacrylamide gel electrophoresis and blotting followed by staining with Concanavalin A and antibodies raised against the overall antigenic components present in either (whole cell walls or PEM) structure. A complex protein assortment was found associated to the walls of the wild type strain. The similarities observed between the polypeptide patterns of the cell walls and PEM, in addition to the immunological cross-reactivity exhi…

MyD88 is dispensable for resistance toParacoccidioides brasiliensisin a murine model of blood-borne disseminated infection

2008

We have studied the role of MyD88, an adaptor protein of Toll-like receptors (TLRs), in murine defenses against Paracoccidioides brasiliensis in a model of blood-borne disseminated infection. Wild-type (WT) and MyD88-deficient mice infected intravenously with P. brasiliensis yeast cells showed an equivalent fungal burden, as well as similar levels of proinflammatory IL-1beta, IL-6, IL-12p70, tumor necrosis factor (TNF)-alpha and MIP-2, T-helper type 1 (Th1) (IFN-gamma) and Th2 cytokines (IL-4) in tissue homogenates. In vitro production of TNF-alpha, IFN-gamma and IL-12p70, by antigen-stimulated splenocytes from infected animals, was also similar in both types of mice; this production of Th1…

Changes in the cell wall glycoprotein composition of Candida albicans associated to the inhibition of germ tube formation by EDTA.

1994

Hyphal development in Candida albicans was blocked by EDTA. This effect was not due to a general growth inhibition since the chelator did not affect protein and DNA synthesis. Recovery of mycelial growth was observed when EDTA-grown cells were incubated at 37 degrees C in EDTA-free medium. High-molecular-weight mannoproteins (HMWM) that are mycelium-specific wall components, and particularly a 260-kDa species (HMWM-260), were absent in the wall of cells grown under germination conditions in the presence of EDTA. Synthesis of the HMWM-260 species was not inhibited but its incorporation (secretion) into the wall structure seemed to be blocked in EDTA-treated cells.

The Candida albicans cell wall-associated glyceraldehyde-3-phosphate dehydrogenase activity increases in response to starvation and temperature upshi…

2002

We have determined the effect of environmental factors (mild thermal upshift and starvation) on the Candida albicans cell wall-associated glyceraldehyde-3-phosphate dehydrogenase (cwGAPDH) activity. Temperature upshift (from 28 to 37 degrees C) and/ or starvation (at 28 or 37 degrees C in water) of exponentially growing yeast cells caused an increase in cwGAPDH activity (3 to 5-, and 7 to 8-fold, respectively). This increase in activity did not correlate with an increase in the amount of cwGAPDH protein present, as determined by flow cytometry, immunoelectron microscopy and Western-blotting. These results indicate that thermal upshift and starvation cause an activation of the cwGAPDH in C. …

Binding of human fibronectin to Aspergillus fumigatus conidia.

1996

Aspergillus fumigatus conidia exhibited the ability to bind purified human fibronectin, whereas mycelial forms did not bind the ligand, as detected by an indirect immunofluorescence assay with an antifibronectin polyclonal antibody after incubation of the cells with fibronectin. Flow cytometry confirmed that binding of the ligand to conidia was dose dependent and saturable. Pretreatment of the cells with trypsin markedly reduced binding, which suggested a protein nature for the binding sites present at the surface of conidia. Intact conidia were also able to adhere to fibronectin or antifibronectin antibodies, a significant reduction (from 88 to 92%) in the binding of conidia was noticed, t…

About the role of TLR2 and TLR4 in cytokine secretion by murine macrophages in response to Candida albicans.

2006

Dear Editor, In a recent issue of FEMS Immunology and Medical Microbiology , Blasi (2005) studied the biological role of Toll-like receptor (TLR)2 and TLR4 in the effector and secretory responses of murine macrophages to the fungal species Candida albicans . In their article, the authors conclude that the secretory response to C. albicans appears to be TLR4- but not TLR2-dependent. In our opinion this statement is misleading as the results reported do not support this conclusion and, therefore, we wish to comment on this issue. There is evidence indicating that TLR2 is the main receptor involved in triggering cytokine production by murine macrophages in response to C. albicans . Phospholipo…

Wall mannoproteins in cells from colonial phenotypic variants of Candida albicans.

1990

Candida albicans ATCC 26555 switched at high frequency (10(-1) to 10(-3)) between several phenotypes identified by colony morphology on a defined mineral amino-acid-containing agar medium supplemented with arginine and zinc (LAZ medium). When cells taken from colonies exhibiting distinct morphologies were plated directly onto LAZ agar, spontaneous conversion to all the variant phenotypes occurred at combined frequencies of 2.1 x 10(-1) to 9.5 x 10(-3). However, when cells taken from the different colonial phenotypes were plated directly onto an undefined medium (yeast extract/peptone/dextrose; YPD medium), or first incubated in liquid YPD medium and then cloned on YPD agar, all colonies obs…

Extracellular vesicles do not mediate the anti-inflammatory actions of mouse-derived adipose tissue mesenchymal stem cells secretome

2021

Este artículo se encuentra disponible en la siguiente URL: https://www.mdpi.com/1422-0067/22/3/1375 Este artículo pertenece al número especial "The Role of Mesenchymal Stem Cells on Inflammatory and Fibrotic Diseases". Adipose tissue represents an abundant source of mesenchymal stem cells (MSC) for therapeutic purposes. Previous studies have demonstrated the anti-inflammatory potential of adipose tissue-derived MSC (ASC). Extracellular vesicles (EV) present in the conditioned medium (CM) have been shown to mediate the cytoprotective effects of human ASC secretome. Nevertheless, the role of EV in the anti-inflammatory effects of mouse-derived ASC is not known. The current study has investiga…

The Cell Wall-Associated Glyceraldehyde-3-Phosphate Dehydrogenase of Candida albicans Is Also a Fibronectin and Laminin Binding Protein

1998

ABSTRACT By immunoelectron microscopy with a polyclonal antibody against the cytosolic glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Candida albicans (anti-GAPDH PAb), the protein was clearly detected at the outer surface of the cell wall, particularly on blastoconidia, as well as in the cytoplasm. Intact blastoconidia were able to adhere to fibronectin and laminin immobilized on microtiter plates, and this adhesion was markedly reduced by both the anti-GAPDH PAb and soluble GAPDH from Saccharomyces cerevisiae . In addition, semiquantitative flow cytometry analysis with the anti-GAPDH PAb showed a decrease in antibody binding to cells in the presence of soluble fib…

Role of IFN-gamma in immune responses to Candida albicans infections

2014

Candida albicans is the most frequent etiologic agent that causes opportunistic fungal infections called candidiasis, a disease whose systemic manifestation could prove fatal and whose incidence is increasing as a result of an expanding immunocompromised population. Here we review the role of interferon-gamma (IFN-γ) in host protection against invasive candidiasis. This cytokine plays an essential role in both the innate and adaptive arms of the immune response to candidiasis. We focus on recent progress on host-pathogen interactions leading to the production of IFN-γ by host cells. IFN-γ is produced by CD4 Th1, CD8, γδ T, and natural killer (NK) cells, essentially in response to both IL-12…

Cell wall protein and glycoprotein constituents of Aspergillus fumigatus that bind to polystyrene may be responsible for the cell surface hydrophobic…

1996

Cell surface hydrophobicity (CSH) of Aspergillus fumigatus grown both in complex medium (yeast extract/peptone/dextrose; YPD) and minimal (Vogel's N) medium was monitored by assessing attachment of polystyrene microspheres to the cell surface. It was found that mature mycelium was hydrophobic. Treatment of intact mycelium with beta-mercaptoethanol (beta ME) abolished binding of the microspheres to hyphal elements, and coating of the microspheres with beta ME extracts from mycelium inhibited their attachment to intact mycelial cells. A. fumigatus mycelium was tagged in vivo with biotin and treated with beta ME. The beta ME extracts were analysed by SDS-PAGE and Western blotting with both per…

The Yeast External Invertase as a Reporter to Study Regulation ofCandida Albicans Promoter Sequences inSaccharomyces Cerevisiae

2008

In vitro differentiation of murine hematopoietic progenitor cells toward the myeloid lineage occurs in response to Staphylococcus aureus and yeast sp…

2013

We have studied the effect of inactivated microbial stimuli (Candida albicans, Candida glabrata, Saccharomyces boulardii, and Staphylococcus aureus) on the in vitro differentiation of lineage negative (Lin−) hematopoietic progenitor mouse cells. Purified Lin− progenitors were co-cultured for 7 days with the stimuli, and cell differentiation was determined by flow cytometry analysis. All the stimuli assayed caused differentiation toward the myeloid lineage. S. boulardii and particularly C. glabrata were the stimuli that induced in a minor extent differentiation of Lin− cells, as the major population of differentiated cells corresponded to monocytes, whereas C. albicans and S. aureus induced …

Starvation and temperature upshift cause an increase in the enzymatically active cell wall-associated glyceraldehyde-3-phosphate dehydrogenase protei…

2003

The cell wall-associated glyceraldehyde-3-phosphate dehydrogenase (cwGAPDH) activity in Saccharomyces cerevisiae increases (two- to 10-fold, depending on the strain) in response to starvation and temperature upshift. Assays using transformants carrying pTDH, a yeast centromer derivative plasmid containing the Candida albicans TDH3 gene (encoding GAPDH) fused in frame with the yeast SUC2-coding region for internal invertase, showed that starvation and/or temperature upshift result in a similar increase in both cwGAPDH and cell wall-associated invertase activities. In addition, this incorporation of GAPDH protein into the cell wall in response to stress does not require (i) de novo protein sy…

Characterization of a proteinaceous extracellular coat synthesized by the ?slime? variant of Neurospora crassa

1989

Cells of the “slime” strain of Neurospora crassa synthesize a coherent extracellular material which remains attached to the cell surface, but is released into the liquid medium by shaking. The material was purified and studied by different criteria. By electron microscopy it appears as long wavy sheets which strongly bind concanavalin A, but not wheat germ agglutinin, and maintain their integrity in the absence of structural polysaccharides. Analysis of the purified material revealed that it was free of contaminating membranes; it contained more than 70% protein, 1% neutral sugars (glucose, mannose, fucose and galactose), less than 2% lipids and ca. 4% not-characterized hexosaminelike compo…

Study of the activation of TLR receptors in neurospheres from glioblastoma cells in vitro

2018

IFN-? in Candida albicans infections

2009

The dimorphic fungus Candida albicans is the most frequent etiologic agent that causes opportunistic infections called candidiasis, a disease whose systemic manifestation could prove fatal and whose incidence is increasing as a result of an expanding immunocompromised population. Here we review the role of interferon-gamma (IFN-gamma) in the host protection against invasive candidiasis. This cytokine plays an essential role in both the innate and adaptive arms of the immune response to candidiasis. We focus on recent progress on host-pathogen interactions at the molecular level, leading to the production of IFN-gamma by host cells. IFN-gamma is produced by CD4 Th1, CD8, gamma delta T, and n…

The glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase of Candida albicans is a surface antigen.

1997

A lambda gt11 cDNA library from Candida albicans ATCC 26555 was screened by using pooled sera from two patients with systemic candidiasis and five neutropenic patients with high levels of anti-C. albicans immunoglobulin M antibodies. Seven clones were isolated from 60,000 recombinant phages. The most reactive one contained a 0.9-kb cDNA encoding a polypeptide immunoreactive only with sera from patients with systemic candidiasis. The whole gene was isolated from a genomic library by using the cDNA as a probe. The nucleotide sequence of the coding region showed homology (78 to 79%) to the Saccharomyces cerevisiae TDH1 to TDH3 genes coding for glyceraldehyde-3-phosphate dehydrogenase (GAPDH), …

High vancomycin MICs within the susceptible range in Staphylococcus aureus bacteraemia isolates are associated with increased cell wall thickness and…

2016

Vancomycin minimum inhibitory concentrations (MICs) at the upper end of the susceptible range for Staphylococcus aureus have been associated with poor clinical outcomes of bloodstream infections. We tested the hypothesis that high vancomycin MICs in S. aureus bacteraemia isolates are associated with increased cell wall thickness and suboptimal bacterial internalisation or lysis by human phagocytes. In total, 95 isolates were evaluated. Original vancomycin MICs were determined by Etest. The susceptibility of S. aureus isolates to killing by phagocytes was assessed in a human whole blood assay. Internalisation of bacterial cells by phagocytes was investigated by flow cytometry. Cell wall thic…

Antigenic cell wall mannoproteins in Candida albicans isolates and in other Candida species.

1991

Polyclonal antibodies (pAbs) and monoclonal antibodies (mAbs), raised against mannoprotein components from Candida albicans ATCC 26555 (serotype A) blastoconidia and mycelial cell walls, were used to investigate antigenic similarities among wall mannoproteins from other C. albicans serotype A and B strains, and from C. tropicalis and C. guilliermondii. Radioactively labelled walls isolated from cells grown at either 28 degrees C or 37 degrees C were digested with a beta-glucanase complex (Zymolyase 20T) to release cell-wall-bound mannoproteins. Numerous molecular species with different electrophoretic mobilities were released from the various isolates. Differences appeared to be related to …

Toll-like receptor 4 defective mice carrying point or null mutations do not show increased susceptibility toCandida albicansin a model of hematogenou…

2006

We have studied the role of TLR4 in murine defenses against Candida albicans in two TLR4-defective mouse strains: C3H/HeJ mice which have defective TLR4 signaling, and TLR4-/- knockout mice. Both TLR4-defective mice strains experimentally infected with virulent C. albicans cells showed no significant difference in survival as compared with their respective controls. Recruitment of neutrophils to the peritoneal cavity of i.p. infected mice was not affected in TLR4-/-animals, but significantly enhanced in C3H/HeJ mice, compared with their control mice. In vitro production of TNF-alpha by macrophages from both types of TLR4-defective mice, in response to yeasts and hyphae of C. albicans, was n…

Signalling through TLR2/MyD88 induces differentiation of murine bone marrow stem and progenitor cells to functional phagocytes in response to Candida…

2009

Summary We have previously demonstrated that inactivated yeasts and hyphae of Candida albicans induce in vitro the proliferation of murine haematopoietic stem and progenitor cells (HSPCs, sorted as LKS cells: Lin - c-Kit + Sca-1 + ) as well as their differentia- tion to lineage-positive cells, through a MyD88- dependent pathway. In this work, we have found that this process is mainly mediated by TLR2, and that expanding cells express myeloid and not lym- phoid markers. Incubation of long-term repopulat- ing HSCs (Lin - CD105 + and Sca-1 + ) with C. albicans yeasts resulted in their proliferation and up regu- lation of the common myeloid progenitors (CMPs) markers, CD34 and FcgRII/III, by a …

Molecular cloning and characterization of theCandida albicansUBI3 gene coding for a ubiquitin-hybrid protein

2000

Using a polyubiquitin cDNA as a probe, we have isolated a clone (pPR3, a pEMBLYe23 derivative plasmid) containing the Candida albicans UBI3 gene coding for a fusion protein. This protein is formed by one ubiquitin subunit fused, at its C-terminus, to an unrelated peptide which is similar to the ribosomal protein encoded by the 3' tail of the Saccharomyces cerevisiae UBI3 gene. Southern blot analysis of chromosomal DNA probed with the 3' non-ubiquitin tail of UBI3 indicated that only one homologous gene is present in the C. albicans genome. Heterelogous expression of pPR3 in a S. cerevisiae ubi3 mutant strain complements the mutant phenotype (slow growth) conferred by the ubi3 defect; this p…

Dectin-1 mediatesin vitrophagocytosis ofCandida albicansyeast cells by retinal microglia: Figure 1

2011

We have investigated the expression of TLR2 and Dectin-1 in retinal microglia and their involvement in Candida albicans phagocytosis using a cytometric approach. The expression of both receptors has been demonstrated in CD11b+ retinal cells. Phagocytosis of pHrodo-labelled C. albicans yeasts by microglial CD11b+ cells of C57BL/6 mice was inhibited both by the Dectin-1 antagonist laminarin and anti-Dectin-1 antibodies, whereas phagocytosis of yeasts by retinal microglia of TLR2 KO mice was unaffected. These data indicate that phagocytosis of C. albicans yeasts by retinal microglia is mediated by Dectin-1, whereas TLR2 does not play a significant role in this process.