Cerebriform colonies of Paracoccidioides brasiliensis isolated from nine-banded armadillos (Dasypus novemcinctus) at room temperature

Twelve isolates of Paracoccidioides brasiliensis generated cerebriform colonies at room temperature on potato glucose agar slants (PDA). These isolates contained abundant chlamydospores and yeast-like cells and are a subset of the 65 isolates obtained from nine-banded armadillos (Dasypus novemcinctus). They grew as a yeast form with typical multiple buddings at 37 degreesC on brain heart infusion agar supplemented with 1% glucose. After replating on PDA and culturing at room temperature for 2 months, the mutants appeared as cottonous colonies, which indicated that the morphological characteristics were unstable.

Glyceraldehyde-3-phosphate dehydrogenase of Paracoccidioides brasiliensis is a cell surface protein involved in fungal adhesion to extracellular matrix proteins and interaction with cells

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.

Prostaglandin E-2 inhibits Paracoccidioides brasiliensis killing by human monocytes

Human monocytes lacked fungicidal activity against high virulence strain of Paracoccidioides brasiliensis, even after IFN-gamma activation. However, monocytes treated with indomethacin exhibited an effective killing against this fungus, suggesting a role of prostaglandin E-2 (PGE(2)) in the inhibition process. Thus, the purpose of this work was to determine whether the effect of PGE2 in fungicidal activity was related with decrease on H2O2 release, the metabolite involved in P. brasiliensis killing, and changes in the levels of TNF-alpha, IL-6 and IL-10. Human monocytes challenged with the fungus produced high PGE(2) levels, which in turn inhibited the fungicidal activity of these cells by reducing H2O2 and TNF-alpha production. (C) 2007 Elsevier Masson SAS. All rights reserved.

Paracoccidioides brasiliensis (Lutz, 1908) isolado por meio de hemocultura em um paciente portadora de símdrome de imunodeficiência adquirida (SIDA)

A case of an AIDS-patient with positive blood culture for Paracoccidioides brasiliensis is reported. The patient was a 29 years old male born in Nova Londrina (Parana State, Brazil) who presented splenomegaly and fever of unknown origin. Three blood cultures were performed, each one of them for aerobic bacteria, mycobacteria and fungi. Cultures for aerobic bacteria and mycobacteria were negative. However, the yeast phase P. brasiliensis was isolated from two cultures in BHI agar, 20 days after inoculation in Negroni medium. The patient was classified in group V according to the Centers for Disease Control (CDC) criteria for AIDS, due to a Pneumocystis carinii pneumonia. Treatment was discontinued due to an individual decision of the patient on the second dose of amphotericin B. This antibiotic was replaced by ketoconazole in the daily dose of 800 mg. The patient died one year after the isolation of P. brasiliensis on blood culture.

SUPPORT OF PARACOCCIDIOIDES-BRASILIENSIS MULTIPLICATION BY HUMAN MONOCYTES OR MACROPHAGES - INHIBITION BY ACTIVATED PHAGOCYTES

The interaction of human monocytes or monocyte-derived macrophages and yeast-form Paracoccidioides brasiliensis was studied in vitro. Yeast cells were readily ingested by adherent monocytes or macrophages. Multiplication of P. brasiliensis, measured by growth as colony forming units (cfu) on a supplemented medium with good plating efficiency, was greater in monocyte co-cultures compared to the number of cfu obtained from complete tissue-culture medium (CTCM). Multiplication increased with time in macrophage cocultures, e.g., from two-six-fold in 24 h to nine-fold in 72 h. Microscopic observations indicated that ingested yeast cells multiplied inside macrophages. When monocytes were treated with supernate cytokines (CK) from concanavalin-A-stimulated mononuclear cells, then co-cultured with P. brasiliensis, multiplication was significantly inhibited compared with control monocyte co-cultures. Treatment of macrophages-derived from monocytes by culture in vitro for 3 days-for a further 3 days with CK resulted in maximal inhibition of multiplication over the subsequent 72 h. Similarly, when monocyte-derived macrophages (after culture for 7 days) were treated for 3 days with recombinant human gamma-interferon (IFN; 300 U/ml) or CK they restricted multiplication of P. brasiliensis by 65% and 95%, respectively, compared with control macrophages, Antibody to IFN abrogated the effect of IFN or CK treatment. These findings show that ingested P. brasiliensis can multiply in human monocytes or macrophages and that this multiplication can be restricted by activated monocytes or macrophages.

Role of Paracoccidioides brasiliensis cell wall fraction containing beta-glucan in tumor necrosis factor-alpha production by human monocytes: correlation with fungicidal activity

The polysaccharide fraction of Paracoccidioides brasiliensis mycelial cell wall (F1 fraction), the active component of which is composed of beta-glucan, was investigated in regard to the activation of human monocytes for fungal killing. The cells were primed with interferon-gamma (IFN-gamma) or F1 (100 and 200 mug ml(-1)) or F1 (100 and 200 mug ml(-1)) plus IFN-gamma for 24 h and then evaluated for H2O2 release. In other experiments, the cells were pretreated with the same stimuli, challenged with a virulent strain of P. brasiliensis and evaluated for fungicidal activity and levels of tumor necrosis factor (TNF-alpha) in the supernatants. F1 increased the levels of H2O2 in a similar manner to IFN-gamma. However, a synergistic effect between these two activators was not detected. on the contrary, a significant fungicidal activity was only obtained after priming with IFN-gamma plus F1. This higher activity was associated with high levels of TNF-alpha in the supernatants of the cocultures. Overall, P. brasiliensis F1 fraction induced human monocytes to release relatively high levels of TNF-alpha, which, in combination with IFN-gamma, is responsible for the activation of human monocytes for effective killing of P. brasiliensis.

Primers for clinical detection of Paracoccidioides brasiliensis

From a 0.72-kb fragment universally generated in Paracoccidioides brasiliensis strains, primers were designed and tested on genomic DNA of this and other pathogenic fungi. They were specific and highly sensitive for P. brasiliensis DNA. Positive results were obtained when these were tested in clinical samples.

EFFECT OF MACROPHAGE BLOCKADE ON THE RESISTANCE OF INBRED MICE TO PARACOCCIDIOIDES-BRASILIENSIS INFECTION

The effect of macrophage blockade on the natural resistance and on the adaptative immune response of susceptible (B10.D2/oSn) and resistant (A/Sn) mice to Paracoccidioides brasiliensis infection was investigated. B10.D2/oSn and A/Sn mice previously injected with colloidal carbon were infected ip with yeast cells to determine the 50% lethal dose, and to evaluate the anatomy and histopathology, macrophage activation, antibody production and DTH reactions. Macrophage blockade rendered both resistant and susceptible mice considerably more susceptible to infection, as evidenced by increased mortality and many disseminated lesions. P. brasiliensis infection and/or carbon treatment increased the ability of macrophages from resistant mice to spread up to 25 days after treatment. In susceptible mice the enhanced spreading capacity induced by carbon treatment was impaired at ail assayed periods except at 1 week after infection. Macrophage blockade enhanced DTH reactions in resistant mice, but did not alter these reactions in susceptible mice, which remained anergic. To the contrary, macrophage blockade enhanced specific antibody production by susceptible mice, but did nor affect the low levels produced by resistant mice. The effect of macrophage blockade confirms the natural tendency of resistant animals to mount DTH reactions in the course of the disease and the preferential antibody response developed by susceptible mice after P. brasiliensis infection. on the whole, macrophage functions appear to play a fundamental role in the natural and acquired resistance mechanisms to P. brasiliensis infection.

AN IMPROVED CULTURE-MEDIUM FOR DETECTING LIVE YEAST PHASE CELLS OF PARACOCCIDIOIDES-BRASILIENSIS

The plating efficiency of standard mycological media such as brain heart infusion (BHI) agar is poor for Paracoccidioides brasiliensis. We prepared a water-extract of yeast phase cells of P. brasiliensis and examined it for growth-enhancing activity for the fungus. The water-extract, when added to BHI agar to a concentration of 5%, improved the plating efficiency of the medium for the fungus to some extent, but the degree of improvement was considerably varied among P. brasiliensis isolates. By contrast, when the water-extract was added in combination with horse serum (4%), the plating efficiency was highly improved (to 94-99%) for all the P. brasiliensis isolates employed. The growth-enhancing factor(s) in the water-extract was heat-stable and heating at 120-degrees-C for 15 min had little, if any, effect on growth-enhancing activity.

GRANULOMATOUS REACTION IN THE HAMSTER-CHEEK POUCH INDUCED BY KILLED PARACOCCIDIOIDES-BRASILIENSIS

We have studied the role of Paracoccidioides brasiliensis viability in the morphology of paracoccidioidomycotic granulomas in the hamster cheek pouch, an immunologically privileged site. Naive (N = 75) and previously sensitized (N = 50) two-month old male hamsters (Mesocricetus auratus) were inoculated into the pouch with 5 x 10(5) live or heat- or formalin-killed fungi. Previously sensitized animals presented a positive footpad test and immunodiffusion demonstrable antibodies (titer 1/32), at the time of sacrifice; naive animals were always negative for those immunological tests. The histological results showed that, like viable P. brasiliensis, killed fungi evoke typical epithelioid granulomas in 100% of animals, even in the absence of immunodiffusion or footpad test detectable immune response. The granulomas elicited by killed fungi were devoid of giant cells or a mononuclear cell halo, suggesting that live proliferating fungi or their products may be involved in these events.

Monoclonal antibodies against the 43,000 da glycoprotein from Paracoccidioides brasiliensis modulate laminin-mediated fungal adhesion to epithelial cells and pathogenesis

The surface glycoprotein gp43, a highly immunogenic component of Paracoccidioides brasiliensis, is used in the serodiagnosis of paracoccidioidomycosis (PCM) and has recently been shown to specifically bind the extracellular matrix protein laminin, Binding to laminin induces the increased adhesion of the fungus to epithelial cells; a hamster testicle infection model has shown that the gp43-dependent binding of fungal cells to laminin enhances their pathogenicity in vivo. We report on the production and characterization of 12 monoclonal antibodies against the gp43 that recognize peptide sequences in the molecule detecting at least three different epitopes as well as different isoforms of this antigen. MAbs interfered in the fungal pathogenicity in vivo either by inhibiting or enhancing granuloma formation and tissue destruction, Results suggest that P. brasiliensis propagules may start infection in man by strongly adhering to human lung cells, Thus, laminin-mediated fungal adhesion to human lung carcinoma (A549) cells was much more intense than to Madin-Darby canine kidney cells (MDCK), indicating differences in binding affinity, Subsequent growth of fungi bound to the lung cells could induce the granulomatous inflammatory reaction characteristic of PCM. Both steps are greatly stimulated by laminin binding in infective cells expressing gp43.

Crystallization and preliminary X-ray diffraction analysis of the lectin from Canavalia gladiata seeds

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Phylogenetic and evolutionary aspects of Paracoccidioides brasiliensis reveal a long coexistence with animal hosts that explain several biological features of the pathogen

The habitat of the mycelial saprobic form of Paracoccidio ides brasiliensis, which produces the infectious propagula, has not been determined and has proven difficult for mycologists to describe. The fungus has been rarely isolated from the environment, the disease has a prolonged latency period and no outbreaks have been reported. These facts have precluded the adoption of preventive measures to avoid infection. The confirmation of natural infections in nine-banded armadillos (Dasypus novemcinctus) with P. brasiliensis, in high frequency and wide geographic distribution, has opened new avenues for the study and understanding of its ecology. Armadillos belong to the order Xenarthra, which has existed in South America ever since the Paleocene Era (65 million years ago), when the South American subcontinent was still a detached land, before the consolidation of what is now known as the American continent. on the other hand, strong molecular evidence suggests that P. brasiliensis and other dimorphic pathogenic fungi - such as Blastomyces dermatitidis, Coccidioides immitis and Histoplasma capsulatum - belong to the family Onygenaceae sensu Into (order Onygenales, Ascomycota), which appeared around 150 million years ago.P. brasiliensis ecology and relation to its human host are probably linked to the fungal evolutionary past, especially its long coexistence with and adaptation to animal hosts other than Homo sapiens, of earlier origin. Instead of being a blind alley, the meaning of parasitism for dimorphic pathogenic fungi should be considered as an open two-way avenue, in which the fungus may return to the environment, therefore contributing to preserve its teleomorphic (sexual) and anamorphic (asexual) forms in a defined and protected natural habitat. (c) 2006 Elsevier B.V. All rights reserved.

Differential gene expression by Paracoccidioides brasiliensis in host interaction conditions: Representational difference analysis identifies candidate genes associated with fungal pathogenesis

Paracoccidioides brasiliensis causes infection by the host inhalation of airborne propagules of the mycelia phase of the fungus. These particles reach the lungs, and disseminate to virtually all organs. Here we describe the identification of differentially expressed genes in studies of host-fungus interaction. We analyzed two cDNA populations of P. brasiliensis, one obtained from infected animals and the other an admixture of fungus and human blood thus mimicking the hematologic events of the fungal dissemination. Our analysis identified transcripts differentially expressed. Genes related to iron acquisition, melanin synthesis and cell defense were specially upregulated in the mouse model of infection. The upregulated transcripts of yeast cells during incubation with human blood were those predominantly related to cell wall remodeling/synthesis. The expression pattern of genes was independently confirmed in host conditions, revealing their potential role in the infection process. This work can facilitate functional studies of novel regulated genes that may be important for the survival and growth strategies of P. brasiliensis in humans. (c) 2006 Elsevier Masson SAS. All rights reserved.