β2 Integrin-Mediated Susceptibility to Paracoccidioides brasiliensis Experimental Infection in Mice

The earliest interaction between macrophages and Paracoccidioides brasiliensis is particularly important in paracoccidioidomycosis (PCM) progression, and surface proteins play a central role in this process. The present study investigated the contribution of β2 integrin in P. brasiliensis-macrophage interaction and PCM progression. We infected β2-low expression (CD18low) and wild type (WT) mice with P. brasiliensis 18. Disease progression was evaluated for fungal burden, lung granulomatous lesions, nitrate levels, and serum antibody production. Besides, the in vitro capacity of macrophages to internalize and kill fungal yeasts was investigated. Our results revealed that CD18low mice infected with Pb18 survived during the time analyzed; their lungs showed fewer granulomas, a lower fungal load, lower levels of nitrate, and production of high levels of IgG1 in comparison to WT animals. Our results revealed that in vitro macrophages from CD18low mice slowly internalized yeast cells, showing a lower fungal burden compared to WT cells. The migration capacity of macrophages was compromised and showed a higher intensity in the lysosome signal when compared with WT mice. Our data suggest that β2 integrins play an important role in fungal survival inside macrophages, and once phagocytosed, the macrophage may serve as a protective environment for P. brasiliensis.

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The Monoclonal Antibody against the Major Diagnostic Antigen of Paracoccidioides brasiliensis Mediates Immune Protection in Infected BALB/c Mice Challenged Intratracheally with the Fungus

Infection and Immunity ◽

10.1128/iai.00349-08 ◽

2008 ◽

Vol 76 (7) ◽

pp. 3321-3328 ◽

Cited By ~ 44

Author(s):

R. Buissa-Filho ◽

R. Puccia ◽

A. F. Marques ◽

F. A. Pinto ◽

J. E. Muñoz ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Vaccine Development ◽

Paracoccidioides Brasiliensis ◽

Passive Immunization ◽

Yeast Cells ◽

No Production ◽

Fungal Burden ◽

Diagnostic Antigen

ABSTRACT The protective role of specific antibodies against Paracoccidioides brasiliensis is controversial. In the present study, we analyzed the effects of monoclonal antibodies on the major diagnostic antigen (gp43) using in vitro and in vivo P. brasiliensis infection models. The passive administration of some monoclonal antibodies (MAbs) before and after intratracheal or intravenous infections led to a reduced fungal burden and decreased pulmonary inflammation. The protection mediated by MAb 3E, the most efficient MAb in the reduction of fungal burden, was associated with the enhanced phagocytosis of P. brasiliensis yeast cells by J774.16, MH-S, or primary macrophages. The ingestion of opsonized yeast cells led to an increase in NO production by macrophages. Passive immunization with MAb 3E induced enhanced levels of gamma interferon in the lungs of infected mice. The reactivity of MAb 3E against a panel of gp43-derived peptides suggested that the sequence NHVRIPIGWAV contains the binding epitope. The present work shows that some but not all MAbs against gp43 can reduce the fungal burden and identifies a new peptide candidate for vaccine development.

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Paracoccidioides brasiliensis 14-3-3 protein is important for virulence in a murine model

Medical Mycology ◽

10.1093/mmy/myy112 ◽

2018 ◽

Vol 57 (7) ◽

pp. 900-904

Author(s):

Caroline Maria Marcos ◽

Haroldo Cesar de Oliveira ◽

Patricia Akemi Assato ◽

Cleverton Roberto de Andrade ◽

Ana Marisa Fusco-Almeida ◽

...

Keyword(s):

Virulence Factor ◽

Murine Model ◽

Pulmonary Infection ◽

Paracoccidioides Brasiliensis ◽

Yeast Cells ◽

Fungal Burden ◽

Important Virulence Factor

AbstractThe Paracoccidioides brasiliensis strain downregulated the expression of adhesin Pb14-3-3 (Pb14-3-3 aRNA) was evaluated in a murine model of paracoccidioidomycosis (PCM). Pb14-3-3 aRNA displays attenuated virulence and triggered the formation of fewer granulomas by lowering the fungal burden in the lungs. Additionally, the Pb14-3-3 aRNA showed more elongated yeast cells and less ability to induce pneumocytes apoptosis in vitro. Our results show that 14-3-3 is an important virulence factor in P. brasiliensis-induced pulmonary infection.

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The influence of carbohydrates in the interaction of Paracoccidioides brasiliensis with CCL-6 cells in vitro

Revista da Sociedade Brasileira de Medicina Tropical ◽

10.1590/s0037-86822012000600016 ◽

2012 ◽

Vol 45 (6) ◽

pp. 739-744 ◽

Cited By ~ 2

Author(s):

Francisco Laurindo da Silva ◽

Raphael Sanzio Pimenta ◽

Juliana Fonseca Moreira da Silva ◽

Déborah Aparecida Negrão Corrêa ◽

Ary Corrêa Junior

Keyword(s):

Epithelial Cell ◽

Paracoccidioides Brasiliensis ◽

Yeast Cells ◽

Epithelial Cell Line ◽

Con A ◽

Culture Methods ◽

Disease Treatment ◽

Adhesion Behavior ◽

And Control

INTRODUCTION: Little is known about the early events in the interaction between Paracoccidioides brasiliensis and its host. To understand the effect of carbohydrates in the interaction between the fungus and epithelial cell in culture, we analyzed the influence of different carbohydrate solutions on the adhesion of P. brasiliensis yeast cells to CCL-6 cells in culture. METHODS: Fungal cells were cultivated with the epithelial cell line, and different concentrations of D-fucose, N-acetyl-glucosamine, D-mannose, D-glucosamine, D-galactosamine, sorbitol and fructose were added at the beginning of the experiment. Six hours after the treatment, the cells were fixed and observed by light microscopy. The number of P. brasiliensis cells that were adhered to the CCL-6 monolayer was estimated. RESULTS: The number of adhesion events was diminished following treatments with D-fucose, N-acetyl-glucosamine, D-mannose, D-glucosamine and D-galactosamine as compared to the untreated controls. Sorbitol and fructose-treated cells had the same adhesion behavior as the observed in the control. P. brasiliensis propagules were treated with fluorescent lectins. The FITC-labeled lectins WGA and Con-A bound to P. brasiliensis yeast cells, while SBA and PNA did not. CONCLUSIONS: The perceptual of adhesion between P. brasiliensis and CCL-6 cells decreased with the use of D-mannose, N-acetyl-glucosamine and D-glucosamine. The assay using FITC-labeled lectins suggests the presence of N-acetyl-glucosamine, α-mannose and α-glucose on the P. brasiliensis cell surface. An enhanced knowledge of the mediators of adhesion on P. brasiliensis could be useful in the future for the development of more efficient and less harmful methods for disease treatment and control.

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Immune response to stage-specific surface antigens of the parasitic nematode Trichinella spiralis.

Journal of Experimental Medicine ◽

10.1084/jem.154.1.210 ◽

1981 ◽

Vol 154 (1) ◽

pp. 210-215 ◽

Cited By ~ 54

Author(s):

M Philipp ◽

P M Taylor ◽

R M Parkhouse ◽

B M Ogilvie

Keyword(s):

Primary Infection ◽

Time Course ◽

Parasitic Nematode ◽

Trichinella Spiralis ◽

Serum Antibody ◽

Surface Antigens ◽

Surface Proteins ◽

Infective Larvae ◽

Intestinal Worms

Rats were infected with the nematode Trichinella spiralis and the primary serum antibody response to antigenic surface proteins of infective larvae, intestinal worms, and newborn larvae was studies. 1 wk after infection, the sera contained antibodies to surface antigens of both infective larvae and intestinal worms. These early sera, however, failed to react with newborn larvae surface antigens. In addition, adsorption of sera with living intestinal worms or infective larvae removed antibodies to surface antigens of the homologous stage only. Finally, the time-course of appearance of antibodies that mediate eosinophil adherence to the surface of each stage of the parasite. We concluded that in a primary infection in rats, the surface proteins of T. spiralis used in this study are antigenically stage specific. Furthermore, they could be targets for the stage-specific, antibody-dependent eosinophil-mediated destruction of this parasite, known to occur in vitro.

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Absence of Interleukin-4 Determines Less Severe Pulmonary Paracoccidioidomycosis Associated with Impaired Th2 Response

Infection and Immunity ◽

10.1128/iai.72.4.2369-2378.2004 ◽

2004 ◽

Vol 72 (4) ◽

pp. 2369-2378 ◽

Cited By ~ 29

Author(s):

Adriana Pina ◽

Rita C. Valente-Ferreira ◽

Eugênia E. W. Molinari-Madlum ◽

Celidéia A. C. Vaz ◽

Alexandre C. Keller ◽

...

Keyword(s):

Paracoccidioides Brasiliensis ◽

Fungal Growth ◽

Protective Role ◽

Yeast Cells ◽

Interleukin 4 ◽

Delayed Type Hypersensitivity ◽

Th2 Response ◽

Leukocyte Influx ◽

Ifn Γ

ABSTRACT Host resistance to paracoccidiodomycosis, the main deep mycosis in Latin America, is mainly due to cellular immunity and gamma interferon (IFN-γ) production. To assess the role of interleukin-4 (IL-4), a Th2-inducing cytokine, pulmonary paracoccidioidomycosis was studied in IL-4-deficient (IL-4−/−) and wild-type (WT) C57BL/6 mice at the innate and acquired phases of immune response. Forty-eight hours after infection, equivalent numbers of viable Paracoccidioides brasiliensis yeast cells were recovered from the lungs of IL-4−/− and WT mice intratracheally infected with one million fungal cells. Alveolar macrophages from infected IL-4−/− mice controlled in vitro fungal growth more efficiently than macrophages from WT mice and secreted higher levels of nitric oxide. Compared with WT mice, IL-4−/− animals presented increased levels of pulmonary IFN-γ and augmented polymorphonuclear leukocyte influx to the lungs. Decreased pulmonary fungal loads were characterized in deficient mice at week 2 postinfection, concomitant with diminished presence of IL-10. At week 8, lower numbers of yeasts were recovered from lungs and liver of IL-4−/− mice associated with increased production of IFN-γ but impaired synthesis of IL-5 and IL-10. However, a clear shift to a Th1 pattern was not characterized, since IL-4−/− mice did not alter delayed-type hypersensitivity anergy or IL-2 levels. In addition, IL-4 deficiency resulted in significantly reduced levels of pulmonary IL-12, granulocyte-macrophage colony-stimulating factor, IL-3, monocyte chemotactic protein 1, and specific antibody isotypes. In IL-4−/− mice, well-organized granulomas restraining fungal cells replaced the more extensive lesions containing high numbers of fungi and inflammatory leukocytes developed by IL-4-sufficient mice. These results clearly showed that genetically determined deficiency of IL-4 can exert a protective role in pulmonary paracoccidioidomycosis.

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Depletion of Neutrophils Exacerbates the Early Inflammatory Immune Response in Lungs of Mice Infected withParacoccidioides brasiliensis

Mediators of Inflammation ◽

10.1155/2016/3183285 ◽

2016 ◽

Vol 2016 ◽

pp. 1-17 ◽

Cited By ~ 9

Author(s):

Paula Andrea Pino-Tamayo ◽

Juan David Puerta-Arias ◽

Damaris Lopera ◽

Martha Eugenia Urán-Jiménez ◽

Ángel González

Keyword(s):

Monoclonal Antibody ◽

Immune Response ◽

Inflammatory Response ◽

Paracoccidioides Brasiliensis ◽

Yeast Cells ◽

Histopathological Analysis ◽

Acute Inflammatory Response ◽

Fungal Load ◽

Inflammatory Immune Response

Neutrophils predominate during the acute phase of theParacoccidioides brasiliensisinfection. Herein, we determined the role of the neutrophil during the early stages of experimental pulmonary paracoccidioidomycosis using a monoclonal antibody (mAb) specific for neutrophils. Male BALB/c mice were inoculated intranasally with1.5×106or2×106P. brasiliensisyeast cells. The mAb was administered 24 h before infection, followed by doses every 48 h until mice were sacrificed. Survival time was evaluated and mice were sacrificed at 48 h and 96 h after inoculation to assess cellularity, fungal load, cytokine/chemokine levels, and histopathological analysis. Neutrophils from mAb-treated mice were efficiently depleted (99.04%). Eighty percent of the mice treated with the mAb and infected with1.5×106yeast cells died during the first two weeks after infection. When mice were treated and infected with2×106yeast cells, 100% of them succumbed by the first week after infection. During the acute inflammatory response significant increases in numbers of eosinophils, fungal load and levels of proinflammatory cytokines/chemokines were observed in the mAb-treated mice. We also confirmed that neutrophils are an important source of IFN-γand IL-17. These results indicate that neutrophils are essential for protection as well as being important for regulating the early inflammatory immune response in experimental pulmonary paracoccidioidomycosis.

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Detection of Antibodies against Paracoccidioides brasiliensis Melanin inIn VitroandIn VivoStudies during Infection

Clinical and Vaccine Immunology ◽

10.1128/cvi.05099-11 ◽

2011 ◽

Vol 18 (10) ◽

pp. 1680-1688 ◽

Cited By ~ 14

Author(s):

Martha E. Urán ◽

Joshua D. Nosanchuk ◽

Angela Restrepo ◽

Andrew J. Hamilton ◽

Beatriz L. Gómez ◽

...

Keyword(s):

Pathogenic Bacteria ◽

Paracoccidioides Brasiliensis ◽

Humoral Response ◽

Yeast Cells ◽

Enzyme Linked Immunosorbent Assay ◽

Microbial Diseases ◽

Content Type ◽

Fungal Structure ◽

Melanin Binding

ABSTRACTSeveral cell wall constituents, including melanins or melanin-like compounds, have been implicated in the pathogenesis of a wide variety of microbial diseases caused by diverse species of pathogenic bacteria, fungi, and helminthes. Among these microorganisms, the dimorphic fungal pathogenParacoccidioides brasiliensisproduces melanin in its conidial and yeast forms. In the present study, melanin particles fromP. brasiliensiswere injected into BALB/c mice in order to produce monoclonal antibodies (MAbs). We identified five immunoglobulin G1 (IgG1) κ-chain and four IgM melanin-binding MAbs. The five IgG1 κ-chain isotypes are the first melanin-binding IgG MAbs ever reported. The nine MAbs labeledP. brasiliensisconidia and yeast cells bothin vitroand in pulmonary tissues. The MAbs cross-reacted with melanin-like purified particles from other fungi and also with commercial melanins, such as synthetic andSepia officinalismelanin. Melanization during paracoccidioidomycosis (PCM) was also further supported by the detection of IgG antibodies reactive to melanin fromP. brasiliensisconidia and yeast in sera and bronchoalveolar lavage fluids fromP. brasiliensis-infected mice, as well as in sera from human patients with PCM. Serum specimens from patients with other mycoses were also tested for melanin-binding antibodies by enzyme-linked immunosorbent assay, and cross-reactivities were detected for melanin particles from different fungal sources. These results suggest that melanin fromP. brasiliensisis an immunologically active fungal structure that activates a strong IgG humoral response in humans and mice.

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Paracoccidioides brasiliensis Enolase Is a Surface Protein That Binds Plasminogen and Mediates Interaction of Yeast Forms with Host Cells

Infection and Immunity ◽

10.1128/iai.00221-10 ◽

2010 ◽

Vol 78 (9) ◽

pp. 4040-4050 ◽

Cited By ~ 72

Author(s):

Sarah Veloso Nogueira ◽

Fernanda L. Fonseca ◽

Marcio L. Rodrigues ◽

Vasanth Mundodi ◽

Erika A. Abi-Chacra ◽

...

Keyword(s):

Epithelial Cells ◽

Paracoccidioides Brasiliensis ◽

Surface Protein ◽

Host Cells ◽

Yeast Cells ◽

Soluble Form ◽

Dimorphic Fungus ◽

Systemic Disorder ◽

Fibronectin Binding Protein

ABSTRACT Paracoccidioidomycosis (PCM), caused by the dimorphic fungus Paracoccidioides brasiliensis, is a disseminated, systemic disorder that involves the lungs and other organs. The ability of the pathogen to interact with host components, including extracellular matrix (ECM) proteins, is essential to further colonization, invasion, and growth. Previously, enolase (EC 4.2.1.11) was characterized as a fibronectin binding protein in P. brasiliensis. Interaction of surface-bound enolase with plasminogen has been incriminated in tissue invasion for pathogenesis in several pathogens. In this paper, enolase was expressed in Escherichia coli as a recombinant glutathione S-transferase (GST) fusion protein (recombinant P. brasiliensis enolase [rPbEno]). The P. brasiliensis native enolase (PbEno) was detected at the fungus surface and cytoplasm by immunofluorescence with an anti-rPbEno antibody. Immobilized purified rPbEno bound plasminogen in a specific, concentration-dependent fashion. Both native enolase and rPbEno activated conversion of plasminogen to plasmin through tissue plasminogen activator. The association between PbEno and plasminogen was lysine dependent. In competition experiments, purified rPbEno, in its soluble form, inhibited plasminogen binding to fixed P. brasiliensis, suggesting that this interaction required surface-localized PbEno. Plasminogen-coated P. brasiliensis yeast cells were capable of degrading purified fibronectin, providing in vitro evidence for the generation of active plasmin on the fungus surface. Exposure of epithelial cells and phagocytes to enolase was associated with an increased expression of surface sites of adhesion. In fact, the association of P. brasiliensis with epithelial cells and phagocytes was increased in the presence of rPbEno. The expression of PbEno was upregulated in yeast cells derived from mouse-infected tissues. These data indicate that surface-associated PbEno may contribute to the pathogenesis of P. brasiliensis.

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Detection of Melanin-Like Pigments in the Dimorphic Fungal Pathogen Paracoccidioides brasiliensis In Vitro and during Infection

Infection and Immunity ◽

10.1128/iai.69.9.5760-5767.2001 ◽

2001 ◽

Vol 69 (9) ◽

pp. 5760-5767 ◽

Cited By ~ 86

Author(s):

Beatriz L. Gómez ◽

Joshua D. Nosanchuk ◽

Soraya Dı́ez ◽

Sirida Youngchim ◽

Philip Aisen ◽

...

Keyword(s):

Fungal Pathogen ◽

Proteolytic Enzymes ◽

Paracoccidioides Brasiliensis ◽

Polyacrylamide Gel Electrophoresis ◽

Yeast Cells ◽

Resonance Spectroscopy ◽

Microbial Infections ◽

Melanin Binding

ABSTRACT Melanins are implicated in the pathogenesis of several human diseases, including some microbial infections. In this study, we analyzed whether the conidia and the yeasts of the thermally dimorphic fungal pathogen Paracoccidioides brasiliensis produce melanin or melanin-like compounds in vitro and during infection. Growth of P. brasiliensis mycelia on water agar alone produced pigmented conidia, and growth of yeasts in minimal medium withl-3,4-dihydroxyphenylalanine (l-DOPA) produced pigmented cells. Digestion of the pigmented conidia and yeasts with proteolytic enzymes, denaturant, and hot concentrated acid yielded dark particles that were the same size and shape as their propagules. Immunofluorescence analysis demonstrated reactivity of a melanin-binding monoclonal antibody (MAb) with the pigmented conidia, yeasts, and particles. Electron spin resonance spectroscopy identified the yeast-derived particles produced in vitro when P. brasiliensis was grown in l-DOPA medium as a melanin-like compound. Nonreducing polyacrylamide gel electrophoresis of cytoplasmic yeast extract revealed a protein that catalyzed melanin synthesis from l-DOPA. The melanin binding MAb reacted with yeast cells in tissue from mice infected with P. brasiliensis. Finally digestion of infected tissue liberated particles reactive to the melanin binding MAb that had the typical morphology of P. brasiliensis yeasts. These data strongly suggest that P. brasiliensis propagules, both conidia and yeast cells, can produce melanin or melanin-like compounds in vitro and in vivo. Based on what is known about the function of melanin in the virulence of other fungi, this pigment may play a role in the pathogenesis of paracoccidioidomycosis.

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The PbMDJ1 Gene Belongs to a Conserved MDJ1/LON Locus in Thermodimorphic Pathogenic Fungi and Encodes a Heat Shock Protein That Localizes to both the Mitochondria and Cell Wall of Paracoccidioides brasiliensis

Eukaryotic Cell ◽

10.1128/ec.5.2.379-390.2006 ◽

2006 ◽

Vol 5 (2) ◽

pp. 379-390 ◽

Cited By ~ 24

Author(s):

Wagner L. Batista ◽

Alisson L. Matsuo ◽

Luciane Ganiko ◽

Tânia F. Barros ◽

Thiago R. Veiga ◽

...

Keyword(s):

Cell Wall ◽

Heat Shock ◽

Cell Surface ◽

Paracoccidioides Brasiliensis ◽

Pathogenic Fungus ◽

Fungal Growth ◽

Pathogenic Fungi ◽

Yeast Cells ◽

Shock Proteins

ABSTRACT J-domain (DnaJ) proteins, of the Hsp40 family, are essential cofactors of their cognate Hsp70 chaperones, besides acting as independent chaperones. In the present study, we have demonstrated the presence of Mdj1, a mitochondrial DnaJ member, not only in the mitochondria, where it is apparently sorted, but also in the cell wall of Paracoccidioides brasiliensis, a thermodimorphic pathogenic fungus. The molecule (PbMdj1) was localized to fungal yeast cells using both confocal and electron microscopy and also flow cytometry. The anti-recombinant PbMdj1 antibodies used in the reactions specifically recognized a single 55-kDa mitochondrial and cell wall (alkaline β-mercaptoethanol extract) component, compatible with the predicted size of the protein devoid of its matrix peptide-targeting signal. Labeling was abundant throughout the cell wall and especially in the budding regions; however, anti-PbMdj1 did not affect fungal growth in the concentrations tested in vitro, possibly due to the poor access of the antibodies to their target in growing cells. Labeled mitochondria stood preferentially close to the plasma membrane, and gold particles were detected in the thin space between them, toward the cell surface. We show that Mdj1 and the mitochondrial proteinase Lon homologues are heat shock proteins in P. brasiliensis and that their gene organizations are conserved among thermodimorphic fungi and Aspergillus, where the genes are adjacent and have a common 5′ region. This is the first time a DnaJ member has been observed on the cell surface, where its function is speculative.

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