scholarly journals A 32-Kilodalton Hydrolase Plays an Important Role in Paracoccidioides brasiliensis Adherence to Host Cells and Influences Pathogenicity

2010 ◽  
Vol 78 (12) ◽  
pp. 5280-5286 ◽  
Author(s):  
Orville Hernández ◽  
Agostinho J. Almeida ◽  
Angel Gonzalez ◽  
Ana Maria Garcia ◽  
Diana Tamayo ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Paracoccidioides Brasiliensis ◽  
Protein A ◽  
Host Cells ◽  
Yeast Cells ◽  
Dimorphic Fungus ◽  
Morphological Alterations ◽  
Cell Vitality ◽  
Initial Interaction ◽  
Mouse Model Of Infection

ABSTRACT One of the most crucial events during infection with the dimorphic fungus Paracoccidioides brasiliensis is adhesion to pulmonary epithelial cells, a pivotal step in the establishment of disease. In this study, we have evaluated the relevance of a 32-kDa protein, a putative adhesion member of the haloacid dehalogenase (HAD) superfamily of hydrolases, in the virulence of this fungus. Protein sequence analyses have supported the inclusion of PbHad32p as a hydrolase and have revealed a conserved protein only among fungal dimorphic and filamentous pathogens that are closely phylogenetically related. To evaluate its role during the host-pathogen interaction, we have generated mitotically stable P. brasiliensis HAD32 (PbHAD32) antisense RNA (aRNA) strains with consistently reduced gene expression. Knockdown of PbHAD32 did not alter cell vitality or viability but induced morphological alterations in yeast cells. Moreover, yeast cells with reduced PbHAD32 expression were significantly affected in their capacity to adhere to human epithelial cells and presented decreased virulence in a mouse model of infection. These data support the hypothesis that PbHad32p binds to extracellular matrix (ECM) proteins and modulates the initial immune response for evasion of host defenses. Our findings point to PbHAD32 as a novel virulence factor active during the initial interaction with host cells in P. brasiliensis.

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

2010 ◽  
Vol 78 (9) ◽  
pp. 4040-4050 ◽  
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.

scholarly journals Enolase from Paracoccidioides brasiliensis: isolation and identification as a fibronectin-binding protein

2009 ◽  
Vol 58 (6) ◽  
pp. 706-713 ◽  
Author(s):  
Fabiana Cristina Donofrio ◽  
Ana Carolina Alvarez Calil ◽  
Elaine Toscano Miranda ◽  
Ana Marisa Fusco Almeida ◽  
Gil Benard ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Solid Medium ◽  
Binding Protein ◽  
Paracoccidioides Brasiliensis ◽  
Yeast Cells ◽  
Blood Agar ◽  
Isolation And Identification ◽  
Cell Extracts ◽  
Fibronectin Binding Protein ◽  
Fibronectin Binding

Paracoccidioides brasiliensis yeast cells can enter mammalian cells and may manipulate the host cell environment to favour their own growth and survival. Moreover, fibronectin and several other host extracellular matrix proteins are recognized by various components of the yeast cell extracts. The present study was designed to isolate and characterize a fibronectin-binding protein from P. brasiliensis. We also compared P. brasiliensis strain 18, tested before (Pb18a) and after (Pb18b) animal passage, in relation to its adhesion and invasion processes. Extracts from both samples, when cultured on blood agar solid medium, showed higher levels of protein expression than when the same samples were cultured on Fava-Netto solid medium, as demonstrated by two-dimensional electrophoresis and SDS-PAGE. Also, both Pb18a and Pb18b exhibited stronger adhesion to A549 epithelial cells when cultured on blood agar medium than when cultured on Fava-Netto medium. Ligand affinity binding assays revealed a protein of 54 kDa and pI 5.6 in P. brasiliensis cell-free extracts with the properties of a fibronectin-binding adhesin, which was characterized by tryptic digestion and mass spectroscopy as a homologue of enolase from P. brasiliensis. Antibody raised against this 54 kDa protein abolished 80 % of P. brasiliensis adhesion to A549 epithelial cells. Our results demonstrate that P. brasiliensis produces a fibronectin-binding adhesin, irrespective of the culture medium, and that this activity can be inhibited by a specific antibody and is involved in the adhesion of the fungus to pulmonary epithelial cells.

scholarly journals Drk1, a Dimorphism Histidine Kinase, Contributes to Morphology, Virulence, and Stress Adaptation in Paracoccidioides brasiliensis

2021 ◽  
Vol 7 (10) ◽  
pp. 852
Author(s):  
Caroline Maria Marcos ◽  
Haroldo Cesar de Oliveira ◽  
Patrícia Akemi Assato ◽  
Rafael Fernando Castelli ◽  
Ana Marisa Fusco-Almeida ◽  
...  
Keyword(s):  
Cell Wall ◽  
Latin American ◽  
Histidine Kinase ◽  
Paracoccidioides Brasiliensis ◽  
Yeast Cells ◽  
Lower Amount ◽  
Stress Adaptation ◽  
Infection Model ◽  
Dimorphic Fungus ◽  
Virulence Attenuation

P. brasiliensis is a thermally dimorphic fungus belonging to Paracoccidioides complex, causative of a systemic, endemic mycosis limited to Latin American countries. Signal transduction pathways related to important aspects as surviving, proliferation according to the biological niches are linked to the fungal pathogenicity in many species, but its elucidation in P. brasiliensis remains poorly explored. As Drk1, a hybrid histidine kinase, plays regulators functions in other dimorphic fungi species, mainly in dimorphism and virulence, here we investigated its importance in P. brasilensis. We, therefore generated the respective recombinant protein, anti-PbDrk1 polyclonal antibody and a silenced strain. The Drk1 protein shows a random distribution including cell wall location that change its pattern during osmotic stress condition; moreover the P. brasiliensis treatment with anti-PbDrk1 antibody, which does not modify the fungus’s viability, resulted in decreased virulence in G. mellonella model and reduced interaction with pneumocytes. Down-regulating PbDRK1 yielded phenotypic alterations such as yeast cells with more elongated morphology, virulence attenuation in G. mellonella infection model, lower amount of chitin content, increased resistance to osmotic and cell wall stresses, and also caspofungin, and finally increased sensitivity to itraconazole. These observations highlight the importance of PbDrk1 to P. brasiliensis virulence, stress adaptation, morphology, and cell wall organization, and therefore it an interesting target that could help develop new antifungals.

scholarly journals DOSE RESPONSE EFFECT OF Paracoccidioides brasiliensis IN AN EXPERIMENTAL MODEL OF ARTHRITIS

2014 ◽  
Vol 56 (3) ◽  
pp. 259-264 ◽  
Author(s):  
Eduardo Alexandre Loth ◽  
Samia Khalil Biazim ◽  
José Henrique Fermino Ferreira dos Santos ◽  
Rosana Puccia ◽  
Rosimeire Costa Brancalhão ◽  
...  
Keyword(s):  
Experimental Model ◽  
Dose Response ◽  
Paracoccidioides Brasiliensis ◽  
Yeast Cells ◽  
Knee Joints ◽  
Dimorphic Fungus ◽  
Response Effect ◽  
Systemic Dissemination ◽  
Yeast Phase ◽  
Dose Response Effect

Paracoccidioidomycosis (PCM) is caused by the dimorphic fungus Paracoccidioides brasiliensis (Pb) and corresponds to prevalent systemic mycosis in Latin America. The aim of the present work was to evaluate the dose response effect of the fungal yeast phase for the standardization of an experimental model of septic arthritis. The experiments were performed with groups of 14 rats that received doses of 103, 104 or 105 P. brasiliensis (Pb18) cells. The fungi were injected in 50 µL of phosphate-buffered saline (PBS) directly into the knee joints of the animals. The following parameters were analyzed in this work: the formation of swelling in knees infused with yeast cells and the radiological and anatomopathological alterations, besides antibody titer by ELISA. After 15 days of infection, signs of inflammation were evident. At 45 days, some features of damage and necrosis were observed in the articular cartilage. The systemic dissemination of the fungus was observed in 11% of the inoculated animals, and it was concluded that the experimental model is able to mimic articular PCM in humans and that the dose of 105 yeast cells can be used as standard in this model.

scholarly journals Glyceraldehyde-3-Phosphate Dehydrogenase of Paracoccidioides brasiliensis Is a Cell Surface Protein Involved in Fungal Adhesion to Extracellular Matrix Proteins and Interaction with Cells

2006 ◽  
Vol 74 (1) ◽  
pp. 382-389 ◽  
Author(s):  
Mônica Santiago Barbosa ◽  
Sônia Nair Báo ◽  
Patrícia Ferrari Andreotti ◽  
Fabrícia P. de Faria ◽  
Maria Sueli S. Felipe ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Cell Wall ◽  
Western Blot ◽  
Polyclonal Antibody ◽  
Type I Collagen ◽  
Paracoccidioides Brasiliensis ◽  
Host Cells ◽  
Yeast Cells ◽  
Type I ◽  
Extracellular Matrix Components

ABSTRACT The pathogenic fungus Paracoccidioides brasiliensis causes paracoccidioidomycosis, a pulmonary mycosis acquired by inhalation of fungal airborne propagules, which may disseminate to several organs and tissues, leading to a severe form of the disease. Adhesion to and invasion of host cells are essential steps involved in the infection and dissemination of pathogens. Furthermore, pathogens use their surface molecules to bind to host extracellular matrix components to establish infection. Here, we report the characterization of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of P. brasiliensis as an adhesin, which can be related to fungus adhesion and invasion. The P. brasiliensis GAPDH was overexpressed in Escherichia coli, and polyclonal antibody against this protein was obtained. By immunoelectron microscopy and Western blot analysis, GAPDH was detected in the cytoplasm and the cell wall of the yeast phase of P. brasiliensis. The recombinant GAPDH was found to bind to fibronectin, laminin, and type I collagen in ligand far-Western blot assays. Of special note, the treatment of P. brasiliensis yeast cells with anti-GAPDH polyclonal antibody and the incubation of pneumocytes with the recombinant protein promoted inhibition of adherence and internalization of P. brasiliensis to those in vitro-cultured cells. These observations indicate that the cell wall-associated form of the GAPDH in P. brasiliensis could be involved in mediating binding of fungal cells to fibronectin, type I collagen, and laminin, thus contributing to the adhesion of the microorganism to host tissues and to the dissemination of infection.

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 Eimeria acervulina Microneme Protein 3 Inhibits Apoptosis of the Chicken Duodenal Epithelial Cell by Targeting the Casitas B-Lineage Lymphoma Protein

2021 ◽  
Vol 8 ◽  
Author(s):  
Pu Wang ◽  
Yukun Jia ◽  
Yue Han ◽  
Weirong Wang ◽  
Yiran Zhu ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Protein A ◽  
Host Cells ◽  
Intestinal Epithelial ◽  
Infected Chicken ◽  
Eimeria Acervulina ◽  
Host Cell Apoptosis ◽  
B Lineage ◽  
Cbl Protein ◽  
Microneme Protein

Eimeria acervulina (E. acervulina) causes coccidiosis in poultry which persists as economic pain worldwide. Most damage to the intestinal mucosa results from apoptosis of the infected intestinal epithelial cells. The Microneme protein 3 (MIC3) protein is a key virulence factor in some parasites involved in host cell apoptosis inhibition. Here, we studied whether and how MIC3 affects the apoptosis in E. acervulina infected chicken duodenal epithelial cells. Through flow cytometry (FCM), we found that the presence of merozoites and the overexpression of MIC3 significantly decreased apoptosis and the activity of caspase-3 in chicken duodenal epithelial cells at 4, 6, and 8 h post merozoite infection (P < 0.01). Silencing the Casitas B-lineage lymphoma (CBL) protein, a host receptor for MIC3 with shRNA was shown to promote apoptosis in the chicken duodenal epithelial cells. The early apoptotic rate of host cells in the lentiviral-MIC3 group was significantly lower than that in the lentiviral-MIC3 + shRNA CBL group at 4 h after MIC3 expression (P < 0.01), and it was moderately decreased in the lentiviral-MIC3 + shRNA CBL group compared with that in the shRNA CBL group. Our data indicated that MIC3 inhibited early apoptosis of E. acervulina infected chicken duodenal epithelial cells by targeting host receptor-CBL protein. These findings unveiled one of the mechanisms of how intracellular parasites affect the apoptosis of infected host cells, which provided a deeper understanding of their pathogenesis.

scholarly journals MyD88 Signaling Is Required for Efficient Innate and Adaptive Immune Responses to Paracoccidioides brasiliensis Infection

2011 ◽  
Vol 79 (6) ◽  
pp. 2470-2480 ◽  
Author(s):  
Flávio V. Loures ◽  
Adriana Pina ◽  
Maíra Felonato ◽  
Claudia Feriotti ◽  
Eliseu F. de Araújo ◽  
...  
Keyword(s):  
Immune Responses ◽  
Paracoccidioides Brasiliensis ◽  
Fungal Growth ◽  
Yeast Cells ◽  
Adaptive Immune Responses ◽  
Content Type ◽  
Adaptive Immune ◽  
Initial Interaction ◽  
Low Levels ◽  
Myd88 Signaling

ABSTRACTThe mechanisms that govern the initial interaction betweenParacoccidioides brasiliensis, a primary dimorphic fungal pathogen, and cells of the innate immunity need to be clarified. Our previous studies showed that Toll-like receptor 2 (TLR2) and TLR4 regulate the initial interaction of fungal cells with macrophages and the pattern of adaptive immunity that further develops. The aim of the present investigation was to assess the role of MyD88, an adaptor molecule used by TLRs to activate genes of the inflammatory response in pulmonary paracoccidioidomycosis. Studies were performed with normal and MyD88−/−C57BL/6 mice intratracheally infected withP. brasiliensisyeast cells. MyD88−/−macrophages displayed impaired interaction with fungal yeast cells and produced low levels of IL-12, MCP-1, and nitric oxide, thus allowing increased fungal growth. Compared with wild-type (WT) mice, MyD88−/−mice developed a more severe infection of the lungs and had marked dissemination of fungal cells to the liver and spleen. MyD88−/−mice presented low levels of Th1, Th2, and Th17 cytokines, suppressed lymphoproliferation, and impaired influx of inflammatory cells to the lungs, and this group of cells comprised lower numbers of neutrophils, activated macrophages, and T cells. Nonorganized, coalescent granulomas, which contained high numbers of fungal cells, characterized the severe lesions of MyD88−/−mice; the lesions replaced extensive areas of several organs. Therefore, MyD88−/−mice were unable to control fungal growth and showed a significantly decreased survival time. In conclusion, our findings demonstrate that MyD88 signaling is important in the activation of fungicidal mechanisms and the induction of protective innate and adaptive immune responses againstP. brasiliensis.

scholarly journals Characterization of a novel yeast phase-specific antigen expressed during in vitro thermal phase transition of Talaromyces marneffei

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Kritsada Pruksaphon ◽  
Mc Millan Nicol Ching ◽  
Joshua D. Nosanchuk ◽  
Anna Kaltsas ◽  
Kavi Ratanabanangkoon ◽  
...  
Keyword(s):  
Phase Transition ◽  
Therapeutic Potential ◽  
Specific Antigen ◽  
Human Monocyte ◽  
Host Cells ◽  
Yeast Cells ◽  
Fungal Pathogenesis ◽  
Dimorphic Fungus ◽  
Talaromyces Marneffei

AbstractTalaromyces marneffei is a dimorphic fungus that has emerged as an opportunistic pathogen particularly in individuals with HIV/AIDS. Since its dimorphism has been associated with its virulence, the transition from mold to yeast-like cells might be important for fungal pathogenesis, including its survival inside of phagocytic host cells. We investigated the expression of yeast antigen of T.marneffei using a yeast-specific monoclonal antibody (MAb) 4D1 during phase transition. We found that MAb 4D1 recognizes and binds to antigenic epitopes on the surface of yeast cells. Antibody to antigenic determinant binding was associated with time of exposure, mold to yeast conversion, and mammalian temperature. We also demonstrated that MAb 4D1 binds to and recognizes conidia to yeast cells’ transition inside of a human monocyte-like THP-1 cells line. Our studies are important because we demonstrated that MAb 4D1 can be used as a tool to study T.marneffei virulence, furthering the understanding of the therapeutic potential of passive immunity in this fungal pathogenesis.

scholarly journals Type IV Pili PromoteClostridium difficileAdherence and Persistence in a Mouse Model of Infection

2018 ◽  
Vol 86 (5) ◽  
Author(s):  
Robert W. McKee ◽  
Naira Aleksanyan ◽  
Elizabeth M. Garrett ◽  
Rita Tamayo
Keyword(s):  
Epithelial Cells ◽  
Mouse Model ◽  
Type Iv Pili ◽  
Host Cells ◽  
Content Type ◽  
Type Iv ◽  
Flagellum Biosynthesis ◽  
Mouse Model Of Infection ◽  
Null Mutants

ABSTRACTCyclic diguanylate (c-di-GMP) is a second messenger that regulates the transition from motile to sessile lifestyles in numerous bacteria and controls virulence factor production in a variety of pathogens. InClostridium difficile, c-di-GMP negatively regulates flagellum biosynthesis and swimming motility and promotes the production of type IV pili (TFP), biofilm formation, and surface motilityin vitro. Flagella have been identified as colonization factors inC. difficile, but the role of TFP in adherence to host cells and in colonization of the mammalian gut is unknown. Here we show that c-di-GMP promotes adherence to epithelial cellsin vitro, which can be partly attributed to the loss of flagella. Using TFP-null mutants, we demonstrate that adherence to epithelial cells is partially mediated by TFP and that this TFP-mediated adherence requires c-di-GMP regulation. In a mouse model of colonization, the TFP-null mutants initially colonized the intestine as well as the parental strain but were cleared more quickly. Moreover, compared to the parent strain,C. difficilestrains lacking TFP were particularly deficient in association with the cecal mucosa. Together these data indicate that TFP and their positive regulation by c-di-GMP promote attachment ofC. difficileto the intestinal epithelium and contribute to persistence ofC. difficilein the host intestine.

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