Extracellular Vesicles From Sporothrix brasiliensis Yeast Cells Increases Fungicidal Activity in Macrophages

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.

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Proteomic Analysis of the Responses of Candida albicans during Infection of Galleria mellonella Larvae

Journal of Fungi ◽

10.3390/jof5010007 ◽

2019 ◽

Vol 5 (1) ◽

pp. 7 ◽

Cited By ~ 7

Author(s):

Gerard Sheehan ◽

Kevin Kavanagh

Keyword(s):

Protein Synthesis ◽

Candida Albicans ◽

Fungicidal Activity ◽

Galleria Mellonella ◽

Ex Vivo ◽

Yeast Cells ◽

Cellular Respiration ◽

Biological Processes ◽

Larval Hemolymph ◽

Proteomic Responses

This study assessed the development of disseminated candidiasis within Galleria mellonella larvae and characterized the proteomic responses of Candida albicans to larval hemolymph. Infection of larvae with an inoculum of 1 × 106 yeast cells reduced larval viability 24 (53.33 ± 3.33%), 48 (33.33 ± 3.33%) and 72 (6.66 ± 3.33%) h post infection. C. albicans infection quickly disseminated from the site of inoculation and the presence of yeast and hyphal forms were found in nodules extracted from infected larvae at 6 and 24 h. A range of proteins secreted during infection of G. mellonella by C. albicans were detected in larval hemolymph and these were enriched for biological processes such as interaction with host and pathogenesis. The candicidal activity of hemolymph after immediate incubation of yeast cells resulted in a decrease in yeast cell viability (0.23 ± 0.03 × 106 yeast cells/mL), p < 0.05) as compared to control (0.99 ± 0.01 × 106 yeast cells/mL). C. albicans responded to incubation in hemolymph ex vivo by the induction of an oxidative stress response, a decrease in proteins associated with protein synthesis and an increase in glycolytic proteins. The results presented here indicate that C. albicans can overcome the fungicidal activity of hemolymph by altering protein synthesis and cellular respiration, and commence invasion and dissemination throughout the host.

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Complement-Mediated Differential Immune Response of Human Macrophages to Sporothrix Species Through Interaction With Their Cell Wall Peptidorhamnomannans

Frontiers in Immunology ◽

10.3389/fimmu.2021.749074 ◽

2021 ◽

Vol 12 ◽

Author(s):

Gabriela W. P. Neves ◽

Sarah Sze Wah Wong ◽

Vishukumar Aimanianda ◽

Catherine Simenel ◽

J. Iñaki Guijarro ◽

...

Keyword(s):

Immune Response ◽

Complement System ◽

Sporothrix Schenckii ◽

Direct Interaction ◽

Pathogenic Fungi ◽

Human Monocyte ◽

Yeast Cells ◽

Complement Protein ◽

Sporothrix Brasiliensis ◽

Complement Receptor 3

In this study, the human immune response mechanisms against Sporothrix brasiliensis and Sporothrix schenckii, two causative agents of human and animal sporotrichosis, were investigated. The interaction of S. brasiliensis and S. schenckii with human monocyte-derived macrophages (hMDMs) was shown to be dependent on the thermolabile serum complement protein C3, which facilitated the phagocytosis of Sporothrix yeast cells through opsonization. The peptidorhamnomannan (PRM) component of the cell walls of these two Sporothrix yeasts was found to be one of their surfaces exposed pathogen-associated molecular pattern (PAMP), leading to activation of the complement system and deposition of C3b on the Sporothrix yeast surfaces. PRM also showed direct interaction with CD11b, the specific component of the complement receptor-3 (CR3). Furthermore, the blockade of CR3 specifically impacted the interleukin (IL)-1β secretion by hMDM in response to both S. brasiliensis and S. schenckii, suggesting that the host complement system plays an essential role in the inflammatory immune response against these Sporothrix species. Nevertheless, the structural differences in the PRMs of the two Sporothrix species, as revealed by NMR, were related to the differences observed in the host complement activation pathways. Together, this work reports a new PAMP of the cell surface of pathogenic fungi playing a role through the activation of complement system and via CR3 receptor mediating an inflammatory response to Sporothrix species.

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Susceptibilities of Cryptococcus neoformans Strains to Platelet Binding In Vivo and to the Fungicidal Activity of Thrombin-Induced Platelet Microbicidal Proteins In Vitro

Infection and Immunity ◽

10.1128/iai.69.2.1221-1225.2001 ◽

2001 ◽

Vol 69 (2) ◽

pp. 1221-1225 ◽

Cited By ~ 3

Author(s):

S. Nail ◽

R. Robert ◽

F. Dromer ◽

A. Marot-Leblond ◽

J. M. Senet

Keyword(s):

Cryptococcus Neoformans ◽

Microscopic Observation ◽

Fungicidal Activity ◽

Blood Platelets ◽

Yeast Cells ◽

Blood Samples ◽

Platelet Binding

ABSTRACT In this study we investigated the interactions between capsular and acapsular strains of Cryptococcus neoformans and blood platelets. In vivo microscopic observation of blood samples from mice inoculated with C. neoformans yeast cells demonstrated that encapsulated and nonencapsulated yeast cells disappeared quickly from the bloodstream and that platelets were attached solely to yeast cells of the nonencapsulated strains. In vitro we observed that only the acapsular strains were susceptible to the fungicidal activity of thrombin-induced platelet microbicidal proteins.

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Extracellular Vesicles from Sporothrix Yeast Cells

Fungal Extracellular Vesicles - Current Topics in Microbiology and Immunology ◽

10.1007/978-3-030-83391-6_4 ◽

2021 ◽

pp. 35-44

Author(s):

Marcelo Augusto Kazuo Ikeda ◽

Karen Spadari Ferreira

Keyword(s):

Extracellular Vesicles ◽

Yeast Cells

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Extracellular vesicles regulate yeast growth, biofilm formation, and yeast-to-hypha differentiation in Candida albicans

10.1101/2021.01.21.427696 ◽

2021 ◽

Author(s):

Leandro Honorato ◽

Joana Feital Demetrio ◽

Cameron C. Ellis ◽

Alicia Piffer ◽

Yan Pereira ◽

...

Keyword(s):

Candida Albicans ◽

Extracellular Vesicles ◽

Biofilm Formation ◽

Galleria Mellonella ◽

Morphological Changes ◽

Inhibitory Effect ◽

Yeast Cells ◽

Mass Spectrometry Analysis ◽

Yeast Growth

AbstractThe ability to undergo morphological changes during adaptation to distinct environments is exploited by Candida albicans and has a direct impact on virulence. In this study, we investigated the influence of fungal extracellular vesicles (EVs) during yeast growth, biofilm formation, and morphogenesis in C. albicans. Addition of C. albicans EVs (Ca EVs) to the culture medium positively affected yeast growth. Using crystal violet staining and scanning electron microscopy (SEM), we demonstrated that Ca EVs inhibited biofilm formation by C. albicans in vitro. By time-lapse microscopy and SEM, we showed that Ca EV-treatment stops filamentation promoting pseudohyphae formation with multiple sites for yeast budding. The ability of Ca EVs to regulate dimorphism was further compared to EVs isolated from different C. albicans strains, Saccharomyces cerevisiae, and Histoplasma capsulatum. Ca EVs from distinct strains robustly inhibited yeast-to-hyphae differentiation with morphological changes occurring in less than 4 hours. A minor inhibitory effect was promoted by EVs from S. cerevisiae and H. capsulatum only after 24 hours of incubation. The inhibitory effect of Ca EVs was promoted by a combination of lipid compounds identified by gas chromatography-tandem mass spectrometry analysis as sesquiterpenes, diterpenes, and fatty acids. Remarkably, Ca EVs were also able to reverse filamentation, transforming hyphal growth to yeast forms. Transcriptomic analysis demonstrated that treatment with Ca EVs modified the expression of more than 300 genes. The most effectively upregulated pathways were related to DNA metabolism. The downregulated genes were mostly associated with extracellular and adhesion proteins. Finally, yeast cells treated with Ca EVs for 24 hours lost their agar invasive ability and were avirulent when inoculated in Galleria mellonella larvae. In summary, our results indicate that fungal EVs can profoundly modify C. albicans growth and regulate yeast-to-hypha differentiation inhibiting biofilm formation and virulence.

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Heterogeneity of Size and Distribution of Intramembrane Particles in the Plasma Membrane of Yeast

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100080407 ◽

1977 ◽

Vol 35 ◽

pp. 596-597

Author(s):

E. Keyhani

Keyword(s):

Plasma Membrane ◽

Candida Utilis ◽

Synthetic Medium ◽

Freeze Fracture ◽

Translational Diffusion ◽

Yeast Cells ◽

Distilled Water ◽

Intramembrane Particles ◽

The Matrix ◽

Water Cell

The matrix of biological membranes consists of a lipid bilayer into which proteins or protein aggregates are intercalated. Freeze-fracture techni- ques permit these proteins, perhaps in association with lipids, to be visualized in the hydrophobic regions of the membrane. Thus, numerous intramembrane particles (IMP) have been found on the fracture faces of membranes from a wide variety of cells (1-3). A recognized property of IMP is their tendency to form aggregates in response to changes in experi- mental conditions (4,5), perhaps as a result of translational diffusion through the viscous plane of the membrane. The purpose of this communica- tion is to describe the distribution and size of IMP in the plasma membrane of yeast (Candida utilis).Yeast cells (ATCC 8205) were grown in synthetic medium (6), and then harvested after 16 hours of culture, and washed twice in distilled water. Cell pellets were suspended in growth medium supplemented with 30% glycerol and incubated for 30 minutes at 0°C, centrifuged, and prepared for freeze-fracture, as described earlier (2,3).

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Ultrathin frozen sections of yeast without aldehyde prefixation

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100081152 ◽

1978 ◽

Vol 36 (2) ◽

pp. 58-59

Author(s):

K. J. Böhm ◽

a. E. Unger

Keyword(s):

Aqueous Solutions ◽

Biological Material ◽

Yeast Cells ◽

Frozen Sections ◽

Good Preservation ◽

Ultrathin Frozen Sections

During the last years it was shown that also by means of cryo-ultra-microtomy a good preservation of substructural details of biological material was possible. However the specimen generally was prefixed in these cases with aldehydes.Preparing ultrathin frozen sections of chemically non-prefixed material commonly was linked up to considerable technical and manual expense and the results were not always satisfying. Furthermore, it seems to be impossible to carry out cytochemical investigations by means of treating sections of unfixed biological material with aqueous solutions.We therefore tried to overcome these difficulties by preparing yeast cells (S. cerevisiae) in the following manner:

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Deformation of Yeast Plasma Membrane by Ethidium Bromide

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100103334 ◽

1988 ◽

Vol 46 ◽

pp. 254-255

Author(s):

E. Keyhani

Keyword(s):

Plasma Membrane ◽

Thin Section ◽

Ethidium Bromide ◽

Freeze Fracture ◽

Mutagenic Effect ◽

Yeast Cells ◽

Hydrophobic Region ◽

Thin Section Electron Microscopy ◽

Section Electron ◽

Deep Pits

The mutagenic effect of ethidium bromide on the mitochondrial DNA is well established. Using thin section electron microscopy, it was shown that when yeast cells were grown in the presence of ethidium bromide, besides alterations in the mitochondria, the plasma membrane also showed alterations consisting of 75 to 110 nm-deep pits. Furthermore, ethidium bromide induced an increase in the length and number of endoplasmic reticulum and in the number of intracytoplasmic vesicles.Freeze-fracture, by splitting the hydrophobic region of the membrane, allows the visualization of the surface view of the membrane, and consequently, any alteration induced by ethidium bromide on the membrane can be better examined by this method than by the thin section method.Yeast cells, Candida utilis. were grown in the presence of 35 μM ethidium bromide. Cells were harvested and freeze-fractured according to the procedure previously described.

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