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

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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Identification of an Aminothiazole with Antifungal Activity against Intracellular Histoplasma capsulatum

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00459-13 ◽

2013 ◽

Vol 57 (9) ◽

pp. 4349-4359 ◽

Cited By ~ 13

Author(s):

Jessica A. Edwards ◽

Megan M. Kemski ◽

Chad A. Rappleye

Keyword(s):

Drug Targets ◽

Fungal Pathogens ◽

Histoplasma Capsulatum ◽

Host Cells ◽

Yeast Cells ◽

Aromatic Substituent ◽

Content Type ◽

Fungistatic Activity ◽

Phenotypic Screen ◽

Cell Components

ABSTRACTAs eukaryotes, fungi possess relatively few molecules sufficiently unique from mammalian cell components to be used as drug targets. Consequently, most current antifungals have significant host cell toxicity. Primary fungal pathogens (e.g.,Histoplasma) are of particular concern, as few antifungals are effective in treating them. To identify additional antifungal candidates for the treatment of histoplasmosis, we developed a high-throughput platform for monitoringHistoplasmagrowth and employed it in a phenotypic screen of 3,600 commercially available compounds. Seven hit compounds that inhibitedHistoplasmayeast growth were identified. Compound 41F5 has fungistatic activity againstHistoplasmayeast at micromolar concentrations, with a 50% inhibitory concentration (IC50) of 0.87 μM, and has the greatest selectivity for yeast (at least 62-fold) relative to host cells. Structurally, 41F5 consists of an aminothiazole core with an alicyclic substituent at the 2-position and an aromatic substituent at the 5-position. 41F5 inhibitsHistoplasmagrowth in liquid culture and similarly inhibits yeast cells within macrophages, the actual host environment of this fungal pathogen during infection. Importantly, 41F5 protects infected host cells fromHistoplasma-induced macrophage death, making this aminothiazole hit compound an excellent candidate for development as an antifungal forHistoplasmainfections.

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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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Morphogenetic Circuitry Regulating Growth and Development in the Dimorphic Pathogen Penicillium marneffei

Eukaryotic Cell ◽

10.1128/ec.00234-12 ◽

2012 ◽

Vol 12 (2) ◽

pp. 154-160 ◽

Cited By ~ 28

Author(s):

Kylie J. Boyce ◽

Alex Andrianopoulos

Keyword(s):

Fungal Pathogens ◽

Pathogenic Fungus ◽

Hyphal Growth ◽

Penicillium Marneffei ◽

Yeast Cells ◽

Asexual Development ◽

Infectious Agents ◽

Temperature Dependent ◽

Content Type ◽

Human Pathogenic Fungus

ABSTRACTPenicillium marneffeiis an emerging human-pathogenic fungus endemic to Southeast Asia. Like a number of other fungal pathogens,P. marneffeiexhibits temperature-dependent dimorphic growth and grows in two distinct cellular morphologies, hyphae at 25°C and yeast cells at 37°C. Hyphae can differentiate to produce the infectious agents, asexual spores (conidia), which are inhaled into the host lung, where they are phagocytosed by pulmonary alveolar macrophages. Within macrophages, conidia germinate into unicellular yeast cells, which divide by fission. This minireview focuses on the current understanding of the genes required for the morphogenetic control of conidial germination, hyphal growth, asexual development, and yeast morphogenesis inP. marneffei.

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The Yeast-Phase Virulence Requirement for α-Glucan Synthase Differs among Histoplasma capsulatum Chemotypes

Eukaryotic Cell ◽

10.1128/ec.00214-10 ◽

2010 ◽

Vol 10 (1) ◽

pp. 87-97 ◽

Cited By ~ 52

Author(s):

Jessica A. Edwards ◽

Elizabeth A. Alore ◽

Chad A. Rappleye

Keyword(s):

Cell Wall ◽

Histoplasma Capsulatum ◽

Yeast Cells ◽

Dependent Manner ◽

Glucan Synthase ◽

Content Type ◽

I Factor ◽

Yeast Phase

ABSTRACTHistoplasma capsulatumstrains can be classified into two chemotypes based on cell wall composition. The cell wall of chemotype II yeast contains a layer of α-(1,3)-glucan that masks immunostimulatory β-(1,3)-glucans from detection by the Dectin-1 receptor on host phagocytes. This α-(1,3)-glucan cell wall component is essential for chemotype IIHistoplasmavirulence. In contrast, chemotype I yeast cells lack α-(1,3)-glucanin vitro, yet they remain fully virulentin vivo. Analysis of the chemotype I α-glucan synthase (AGS1) locus revealed a 2.7-kb insertion in the promoter region that diminishesAGS1expression. Nonetheless,AGS1mRNA can be detected during respiratory infection with chemotype I yeast, suggesting that α-(1,3)-glucan could be produced duringin vivogrowth despite its absencein vitro. To directly test whetherAGS1contributes to chemotype I strain virulence, we preventedAGS1function by RNA interference and by insertional mutation. Loss ofAGS1function in chemotype I does not impair the cytotoxicity ofags1(−) mutant yeast to cultured macrophages, nor does it affect the intracellular growth of yeast. In a murine model of histoplasmosis, theags1(−) chemotype I mutant strains show no defect in lung infection or in extrapulmonary dissemination. Together, these studies demonstrate thatAGS1expression is dispensable for chemotype I yeast virulence, in contrast to the case for chemotype II yeast. Despite the absence of cell wall α-(1,3)-glucan, chemotype I yeast can avoid detection by Dectin-1 in a growth stage-dependent manner. This suggests the production of a uniqueHistoplasmachemotype I factor that, at least partially, circumvents the α-(1,3)-glucan requirement for yeast virulence.

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Protective Response in Experimental Paracoccidioidomycosis Elicited by Extracellular Vesicles Containing Antigens of Paracoccidioides brasiliensis

Cells ◽

10.3390/cells10071813 ◽

2021 ◽

Vol 10 (7) ◽

pp. 1813

Author(s):

Ludmila Matos Baltazar ◽

Gabriela Fior Ribeiro ◽

Gustavo J. Freitas ◽

Celso Martins Queiroz-Junior ◽

Caio Tavares Fagundes ◽

...

Keyword(s):

Extracellular Vesicles ◽

Treatment Effectiveness ◽

Ex Vivo ◽

Paracoccidioides Brasiliensis ◽

Systemic Disease ◽

Host Immune System ◽

Antigen Delivery ◽

Activated T Lymphocytes ◽

Cell Mobilization

Paracoccidioidomycosis (PCM) is a systemic disease caused by Paracoccidioides spp. PCM is endemic in Latin America and most cases are registered in Brazil. This mycosis affects mainly the lungs, but can also spread to other tissues and organs, including the liver. Several approaches have been investigated to improve treatment effectiveness and protection against the disease. Extracellular vesicles (EVs) are good antigen delivery vehicles. The present work aims to investigate the use of EVs derived from Paracoccidioides brasiliensis as an immunization tool in a murine model of PCM. For this, male C57BL/6 were immunized with two doses of EVs plus adjuvant and then infected with P. brasiliensis. EV immunization induced IgM and IgG in vivo and cytokine production by splenocytes ex vivo. Further, immunization with EVs had a positive effect on mice infected with P. brasiliensis, as it induced activated T lymphocytes and NKT cell mobilization to the infected lungs, improved production of proinflammatory cytokines and the histopathological profile, and reduced fungal burden. Therefore, the present study shows a new role for P. brasiliensis EVs in the presence of adjuvant as modulators of the host immune system, suggesting their utility as immunizing agents.

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Dynamics ofMycobacterium tuberculosisAg85B Revealed by a Sensitive Enzyme-Linked Immunosorbent Assay

mBio ◽

10.1128/mbio.00611-19 ◽

2019 ◽

Vol 10 (2) ◽

Cited By ~ 2

Author(s):

Joel D. Ernst ◽

Amber Cornelius ◽

Miriam Bolz

Keyword(s):

Protein Secretion ◽

Immunosorbent Assay ◽

Bacterial Population ◽

Enzyme Linked Immunosorbent Assay ◽

Bacterial Protein ◽

Gram Positive ◽

Gram Negative ◽

Content Type ◽

Bacterial Proteins

ABSTRACTSecretion of specific proteins contributes to pathogenesis and immune responses in tuberculosis and other bacterial infections, yet the kinetics of protein secretion and fate of secreted proteinsin vivoare poorly understood. We generated new monoclonal antibodies that recognize theMycobacteriumtuberculosissecreted protein Ag85B and used them to establish and characterize a sensitive enzyme-linked immunosorbent assay (ELISA) to quantitate Ag85B in samples generatedin vitroandin vivo. We found that nutritional or culture conditions had little impact on the secretion of Ag85B and that there is considerable variation in Ag85B secretion by distinct strains in theM. tuberculosiscomplex: compared with the commonly used H37Rv strain (lineage 4),Mycobacteriumafricanum(lineage 6) secretes less Ag85B, and two strains from lineage 2 secrete more Ag85B. We also used the ELISA to determine that the rate of secretion of Ag85B is 10- to 100-fold lower than that of proteins secreted by Gram-negative and Gram-positive bacteria, respectively. ELISA quantitation of Ag85B in lung homogenates ofM. tuberculosisH37Rv-infected mice revealed that although Ag85B accumulates in the lungs as the bacterial population expands, the amount of Ag85B per bacterium decreases nearly 10,000-fold at later stages of infection, coincident with the development of T cell responses and arrest of bacterial population growth. These results indicate that bacterial protein secretionin vivois dynamic and regulated, and quantitation of secreted bacterial proteins can contribute to the understanding of pathogenesis and immunity in tuberculosis and other infections.IMPORTANCEBacterial protein secretion contributes to host-pathogen interactions, yet the process and consequences of bacterial protein secretion during infection are poorly understood. We developed a sensitive ELISA to quantitate a protein (termed Ag85B) secreted byM. tuberculosisand used it to find that Ag85B secretion occurs with slower kinetics than for proteins secreted by Gram-positive and Gram-negative bacteria and that accumulation of Ag85B in the lungs is markedly regulated as a function of the bacterial population density. Our results demonstrate that quantitation of bacterial proteins during infection can reveal novel insights into host-pathogen interactions.

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Dissecting the Structure-Function Relationship of a Fungicidal Peptide Derived from the Constant Region of Human Immunoglobulins

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01753-15 ◽

2016 ◽

Vol 60 (4) ◽

pp. 2435-2442 ◽

Cited By ~ 5

Author(s):

Tecla Ciociola ◽

Thelma A. Pertinhez ◽

Laura Giovati ◽

Martina Sperindè ◽

Walter Magliani ◽

...

Keyword(s):

Self Assembly ◽

Galleria Mellonella ◽

Critical Role ◽

Yeast Cells ◽

Therapeutic Effects ◽

Constant Region ◽

Content Type ◽

Complementarity Determining Regions

ABSTRACTSynthetic peptides encompassing sequences related to the complementarity-determining regions of antibodies or derived from their constant region (Fc peptides) were proven to exert differential antimicrobial, antiviral, antitumor, and/or immunomodulatory activitiesin vitroand/orin vivo, regardless of the specificity and isotype of the parental antibody. Alanine substitution derivatives of these peptides exhibited unaltered, increased, or decreased candidacidal activitiesin vitro. The bioactive IgG-derived Fc N10K peptide (NQVSLTCLVK) spontaneously self-assembles, a feature previously recognized as relevant for the therapeutic activity of another antibody-derived peptide. We evaluated the contribution of each residue to the peptide self-assembling capability by circular-dichroism spectroscopy. The interaction of the N10K peptide and its derivatives withCandida albicanscells was studied by confocal, transmission, and scanning electron microscopy. The apoptosis and autophagy induction profiles in yeast cells treated with the peptides were evaluated by flow cytometry, and the therapeutic efficacy against candidal infection was studied in aGalleria mellonellamodel. Overall, the results indicate a critical role for some residues in the self-assembly process and a correlation of that capability with the candidacidal activities of the peptidesin vitroand their therapeutic effectsin vivo.

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The HIF-1α/LC3-II Axis Impacts Fungal Immunity in Human Macrophages

Infection and Immunity ◽

10.1128/iai.00125-19 ◽

2019 ◽

Vol 87 (7) ◽

Cited By ~ 4

Author(s):

Dirk Friedrich ◽

Dorinja Zapf ◽

Björn Lohse ◽

Roger A. Fecher ◽

George S. Deepe ◽

...

Keyword(s):

Mitochondrial Respiration ◽

Histoplasma Capsulatum ◽

Hypoxia Inducible Factor ◽

Peripheral Blood Monocyte ◽

Yeast Cells ◽

Content Type ◽

Human Macrophages ◽

Disseminated Disease ◽

Fungal Immunity ◽

Pathogen Exposure

ABSTRACTThe fungal pathogenHistoplasma capsulatumcauses a spectrum of disease, ranging from local pulmonary infection to disseminated disease. The organism seeks residence in macrophages, which are permissive for its survival. Hypoxia-inducible factor 1α (HIF-1α), a principal regulator of innate immunity to pathogens, is necessary for macrophage-mediated immunity toH. capsulatumin mice. In the present study, we analyzed the effect of HIF-1α in human macrophages infected with this fungus. HIF-1α stabilization was detected in peripheral blood monocyte-derived macrophages at 2 to 24 h after infection with viable yeast cells. Further, host mitochondrial respiration and glycolysis were enhanced. In contrast, heat-killed yeasts induced early, but not later, stabilization of HIF-1α. Since the absence of HIF-1α is detrimental to host control of infection, we asked if large amounts of HIF-1α protein, exceeding those induced byH. capsulatum, altered macrophage responses to this pathogen. Exposure of infected macrophages to an HIF-1α stabilizer significantly reduced recovery ofH. capsulatumfrom macrophages and produced a decrement in mitochondrial respiration and glycolysis compared to those of controls. We observed recruitment of the autophagy-related protein LC3-II to the phagosome, whereas enhancing HIF-1α reduced phagosomal decoration. This finding suggested thatH. capsulatumexploited an autophagic process to survive. In support of this assertion, inhibition of autophagy activated macrophages to limit intracellular growth ofH. capsulatum. Thus, enhancement of HIF-1α creates a hostile environment for yeast cells in human macrophages by interrupting the ability of the pathogen to provoke host cell autophagy.

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THEORETICAL APPROACH ON TARGETING PLANT FUNGAL PATHOGENIC PROTEINS AGAINST NATURALLY ISOLATED COMPOUNDS FROM CHITINIPHILUS SHINANONENSIS

Asian Journal of Pharmaceutical and Clinical Research ◽

10.22159/ajpcr.2019.v12i12.35639 ◽

2019 ◽

pp. 138-142

Author(s):

KRITHIKA S ◽

CHELLARAM C

Keyword(s):

Drug Targets ◽

Fungal Pathogens ◽

Interaction Analysis ◽

Pathogenic Fungus ◽

Three Dimensional ◽

Data Bank ◽

Asiatic Acid ◽

Literature Study

Objective: The objective of this study was to find the potency and bioefficacy of Asiatic acid and triterpene against four different plant fungal pathogens using a structure-based drug designing approach. Methods: The pathogenic fungus which causes a dreadful effect on plants is reviewed from literature study, and its three-dimensional structures are retrieved from the protein data bank database. On the other hand, ligands are prepared. Finally, prepared fungal drug targets are docked with naturally isolated compounds using AutoDock tools. Results: Both compounds Asiatic acid and triterpene structures are complementary to bind at the active site of four different drug targets. Comparatively, it is more favorable for Avr2 effector protein from Fusarium oxysporum with Ki value of 126.60 μM, 1.76 μM, and dock score value of −5.32 kcal/mol and −7.85 kcal/mol for Asiatic acid and triterpene, respectively. Thus, interaction analysis was carried out only for these protein-ligand complexes. Conclusion: The computational biology study states that these two compounds can be the lead candidate for treating disease caused by plant fungal pathogen F. oxysporum. However, further study has to be done in vitro and in vivo to prove its same efficacy.

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Peroxisomal and Mitochondrial β-Oxidation Pathways Influence the Virulence of the Pathogenic Fungus Cryptococcus neoformans

Eukaryotic Cell ◽

10.1128/ec.00128-12 ◽

2012 ◽

Vol 11 (8) ◽

pp. 1042-1054 ◽

Cited By ~ 34

Author(s):

Matthias Kretschmer ◽

Joyce Wang ◽

James W. Kronstad

Keyword(s):

Carbon Source ◽

Cryptococcus Neoformans ◽

Virulence Factor ◽

Fungal Pathogens ◽

Pathogenic Fungus ◽

Subsequent Work ◽

Content Type ◽

Capsule Production ◽

Human Pathogenic Fungus ◽

Host Tissues

ABSTRACTAn understanding of the connections between metabolism and elaboration of virulence factors during host colonization by the human-pathogenic fungusCryptococcus neoformansis important for developing antifungal therapies. Lipids are abundant in host tissues, and fungal pathogens in the phylum basidiomycota possess both peroxisomal and mitochondrial β-oxidation pathways to utilize this potential carbon source. In addition, lipids are important signaling molecules in both fungi and mammals. In this report, we demonstrate that defects in the peroxisomal and mitochondrial β-oxidation pathways influence the growth ofC. neoformanson fatty acids as well as the virulence of the fungus in a mouse inhalation model of cryptococcosis. Disease attenuation may be due to the cumulative influence of altered carbon source acquisition or processing, interference with secretion, changes in cell wall integrity, and an observed defect in capsule production for the peroxisomal mutant. Altered capsule elaboration in the context of a β-oxidation defect was unexpected but is particularly important because this trait is a major virulence factor forC. neoformans. Additionally, analysis of mutants in the peroxisomal pathway revealed a growth-promoting activity forC. neoformans, and subsequent work identified oleic acid and biotin as candidates for such factors. Overall, this study reveals that β-oxidation influences virulence inC. neoformansby multiple mechanisms that likely include contributions to carbon source acquisition and virulence factor elaboration.

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