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Role of bats in the ecology of Paracoccidioides brasiliensis: the survival of Paracoccidioides brasiliensis in the intestinal tract of frugivorous bat, Artibeus lituratus.

The fruit-eating bat, Artibeus lituratus, was fed known quantities of viable yeast cells and mycelial particles of Paracoccidioides brasiliensis in an attempt to assess the role of this animal in the distribution of this agent in nature. Results of mycosal cultures of the stomach, upper intestine, lower intestine and rectum clearly showed that the fungal cells were unable to survive more than 8 hours in the digestive tract of the bat. The mycelial particles were more susceptible than the yeast and were killed before passing to the rectum. The fungus died rapidly in the voided fecal material. These findings indicate the improbability of isolating P. brasiliensis from the digestive tract of wild captured bats and show that A. lituratus probably plays no role in the distribution of this fungus in nature.

Animals

Induction of antigen-specific T suppressor cells by soluble Paracoccidioides brasiliensis antigen.

In naturally acquired paracoccidioidomycosis, patients have depressed in vivo and in vitro cell-mediated immune (CMI) responses to Paracoccidioides brasiliensis antigen. In addition, it has been reported that these patients have significant levels of circulating paracoccidioidal antigen in their sera. The primary purpose of this investigation was to assess the effects of P. brasiliensis antigen on the CMI responses in a mouse model. On the basis of findings with other fungal agents, we predicted that circulating paracoccidioidal antigen may be inducing suppressor cells which modulate the CMI response. In this study, we show (i) that a soluble P. brasiliensis culture filtrate antigen (Pb.Ag) emulsified in complete Freund adjuvant and injected subcutaneously into mice induces reasonably high levels of delayed-type hypersensitivity (DTH) in CBA/J mice; (ii) that Pb.Ag elicits DTH reactions specific for P. brasiliensis when injected into footpads of immunized mice; and (iii) that an intravenous injection of Pb.Ag induces a population of lymph node and spleen cells which, upon adoptive transfer, suppress the afferent limb of the DTH response to paracoccidioidal antigen. The afferent suppressor cells can be detected in spleens as early as 5 days after Pb.Ag treatment, are present in significant numbers by 7 days in both spleens and lymph nodes, and are virtually absent by 14 days. In contrast, at 14 days after antigen injection, efferent suppressor cells were detected in spleens and lymph nodes. The Pb.Ag-induced afferent suppressor cells specifically inhibit the antiparacoccidioidal DTH response. They are nylon wool-nonadherent cells, and their activity is abrogated by anti-Thy-1 and complement treatment, indicating that they are T lymphocytes. The phenotype of these afferent suppressor T cells is L3T4+ Lyt-1+2- I-J+. The Pb.Ag-specific suppressor cells described in this paper are similar to the Ts1 cells in the azobenzenearsonate, 4-hydroxy-3-nitrophenyl acetyl, and cryptococcal models of suppression of the DTH response and to the afferent suppressor cells in the dinitrofluorobenzene contact sensitivity system.

Animals

Characterization of efferent T suppressor cells induced by Paracoccidioides brasiliensis-specific afferent T suppressor cells.

Previously, we reported that Paracoccidioides brasiliensis culture filtrate antigen (Pb.Ag) when injected i.v. into mice induces antigen-specific suppressor cells which down-regulate the anti-P. brasiliensis delayed-type hypersensitivity (DTH) response. The suppressor cells are present in both spleens and lymph nodes of Pb.Ag-treated animals and suppress the afferent limb but not the efferent limb of the DTH response to P. brasiliensis. The suppressor cells induced by Pb.Ag are L3T4+ Lyt-1+2- I-J+ T cells and are considered to be equivalent to the Ts1 cells described for other antigen-specific suppressor cell pathways. This report provides data which show that Ts1 cells induced by Pb.Ag or a soluble factor derived from Ts1 cells (TsF1) stimulates the production of second-order or efferent suppressor cells. The second-order suppressor cells are detectable in spleens and lymph nodes of mice 7 days after injection of Ts1 cells or TsF1 and are specific in suppressing the paracoccidioidal DTH response. In addition, the second-order suppressor cells are T cells with an L3T4- Lyt-2+ I-J+ phenotype and are effective in suppressing only the efferent limb of the P. brasiliensis DTH response. On the basis of the characteristics defined in this study, the paracoccidioidal second-order suppressor cells are equivalent to the Ts2 cells described for other antigen-specific suppressor-cell pathways. Thus, the suppressive circuit induced by Pb.Ag is similar to the suppressor-cell pathways that regulate the DTH responses to azobenzenearsonate, 4-hydroxy-3-nitrophenyl acetyl, lysozyme, and Cryptococcus neoformans antigen. We propose that such a suppressor-cell circuit as defined here with the murine model could be responsible for the depressed cell-mediated immune responses observed in paracoccidioidomycosis patients who have antigen circulating in their sera.

Animals

A case of paracoccidioidal granuloma in norther Nigeria.

A case of Paracoccidioidal granuloma in Northern Nigeria is presented. The fungus could be cultured and identified as Paracoccidioides brasiliensis. Histology revealed a fungal lesion due to a yeast like organism. The patient responded to long acting Sulphormethoxine (Fanasil). A variation from the classic histopathological description and association of pustular psoriasiform lesions on the soles with a possibility of 'Id' reaction to the fungus are discussed.

Adult

Acidic Stress Induces Proteomic Reprogramming and Virulence-Associated Adaptation in Paracoccidioides brasiliensis.

Paracoccidioidomycosis (PCM) is a neglected systemic mycosis whose etiologic agents must adapt to acidic host niches such as phagolysosomes. Here, we used quantitative liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based proteomics to define the response of Paracoccidioides brasiliensis to acidic stress (pH 4.5) versus control pH (6.5) after 5 and 24 h. We identified and quantified 4374 proteins, including 327 and 722 differentially abundant proteins at 5 and 24 h, respectively, revealing time-dependent proteomic reprogramming. Enrichment analyses highlighted proteolysis, protein metabolism, organonitrogen metabolism, MAPK- and SNF1-like signaling, central carbon metabolism, tyrosine metabolism, and ergosterol biosynthesis as major acid-responsive processes. Complementary assays showed pH-dependent extracellular proteolytic activity, increased adhesion to A549 pulmonary epithelial cells, and dynamic ergosterol remodeling. The proteomic data further indicated increased abundance of moonlighting proteins linked to adhesion and metabolic enzymes associated with ATP generation and melanin precursor production. Together, these findings indicate that P. brasiliensis adapts to acidic environments through coordinated regulation of proteostasis, metabolism, signaling, host-cell interaction, and membrane homeostasis, supporting survival and virulence potential in acidic host microenvironments.

Paracoccidioides

Studies on the relationship between the pathogenicity of Paracoccidioides brasiliensis in mice and its growth rate under different oxygen atmospheres.

We performed comparative studies of the pathogenicity of six strains of Paracoccidioides brasiliensis (Bt-9, Bt-4, Pb-9, Pb-18, Bt-7 and B-1183) for young adult male ddY mice and the growth rate of each strain under different oxygen atmospheres (aerobic, micro-aerobic and anaerobic atmospheres) at 37 degrees C. 10(6) units of yeast cells were intravenously injected into each mouse. The pathogenicity of each isolate was determined by a scoring system based on organ culture and histopathological findings. The growth rates under different oxygen atmospheres were determined by a scoring system in which 300 fungal units per strain were counted. The strain Bt-9 showed the greatest pathogenicity, followed by Bt-4, Pb-9 and Pb-18 had on intermediate rank of pathogenicity. Bt-7 and B-1183 were the least pathogenic of the strains tested. Except for strain Bt-7 all strains showed an excellent growth under an aerobic atmosphere. Bt-4 and Bt-9 also showed excellent growth under a micro-aerobic atmosphere, followed by Pb-9, whereas the growth of Pb-18, Bt-7 and B-1183 was limited. There was a correlation between the growth rate under a micro-aerobic atmosphere and the pathogenicity of a strain. The growth rate of P. brasiliensis under a micro-aerobic atmosphere strongly correlated to its pathogenicity.

Aerobiosis

Scanning electron microscopy of the conidia produced by the mycelial form of Paracoccidioides brasiliensis.

The conidia produced by the mycelial form of Paracoccidioides brasiliensis were examined by scanning electron microscopy for the first time. Several different conidial types were characterized. These included intercalary arthroconidia, several types of septate conidia that are formed from other conidia, pedunculate conidia, and terminal hyphal conidia. In addition, the ultrastructure of the supporting pedestal of the pedunculate conidium was found to be separated from the mother conidium by a septum in some instances, and at other times it was not.

Microscopy, Electron, Scanning

Digestion of killed Paracoccidioides brasiliensis by neutrophils.

We previously described an in vitro assay showing that neutrophils (PMNs) from patients with paracoccidioidomycosis (PARA) have a specific digestive deficiency against suspensions of live Paracoccidioides brasiliensis. We now report that this defect is equally detectable against autoclaved, but not Amphotericin B-killed P. brasiliensis. The use of autoclaved suspensions facilitates the use of our in vitro assay. It might allow the development of an in vitro intradermal test for digestion of fungi. Differential digestive ability of phagocytes against live (or autoclaved) and Amphotericin-B killed fungi is of conceptual interest. It may be relevant in understanding therapeutic effect of Amphotericin B.

Amphotericin B

Electron microscopic study of conidia produced by the mycelium of Paracoccidioides brasiliensis.

The ultrastructure of asexual spores (conidia) produced by the mycelial form of Paracoccidioides brasiliensis was studied for the first time with transmission electron microscopy, using thin sections of aldehyde-osmium-fixed and epoxy-resin-embedded samples. The various types of conidia observed in the sections correlated well with previous light-microscopic descriptions. These types were intercalary or apical conidia, depending on their location along the originating hyphae. As in previous studies they were characterized as arthroconidia, aleuriospores and sessile or pedunculate pyriform conidia. The sporogenous cells were clearly distinguished from hyphal cells by the thickness and appearance of their cell walls. Copious fibrillar material (glycocalyx) detected at the cell surface was stained with ruthenium red during the fixation process. Typical subcellular organelles (nucleus, nucleolus, mitochondria, ribosomes, etc) were found in most of the sections. It was concluded that the spores produced by the mycelial phase of P. brasiliensis possess all attributes of viable and physiologically competent eukaryotic cells.

Humans

Histological and ultrastructural study of the inflammation evoked by Paracoccidioides brasiliensis antigen in previously immunized mice.

Bentonite particles uncoated and coated with soluble antigen of Paracoccidioides brasiliensis (Pb) were intravenously injected into mice with and without previous immunization with Pb antigen. The inflammatory reaction around the bentonite emboli in small lung vessels was quantitated and morphologically studied by light and electron (EM) microscopy, 2 to 8 days after challenge. In control nonimmunized animals, coated and uncoated bentonite particles caused mild and nonspecific inflammation made up by macrophages. By EM, they formed loosely aggregated clusters with cytoplasm containing few organelles and borders without interdigitation. In immunized mice injected with coated bentonite particles, the inflammatory area was significantly greater than that in nonimmunized animals in all periods of study with maximum difference at day 2. The inflammatory process at days 2 and 4 was characterized as mature granulomata, composed of macrophages with great number of organelles in the cytoplasm, large euchromatic nuclei and prominent nucleoli. Altogether these findings indicated a lesion with high metabolic activity, compatible with a granulomatous hypersensitivity reaction. At days 6 and 8, there was a change from mature to epithelioid granulomata, well demonstrated by EM which showed macrophages with characteristically interdigitated cytoplasmic borders. The results strengthen the importance of cellular immunity in the genesis of epithelioid granuloma in paracoccidioidomycosis and reinforce the usefulness of the present model in studies of the inflammatory cellular sequency and events in this mycosis.

Animals

Paracoccidioides brasiliensis: cell wall structure and virulence. A review.

Paracoccidioides brasiliensis is the causative agent of paracoccidioidomycosis or South American blastomycosis. Many aspects of the disease and its agent are unknown. One of the most important factors regarding the infection and the host-parasite relationships seems to be the fungal cell wall whose biochemical aspects are reviewed here. Biochemical studies, done mainly by Kanetsuna et al., (21,22), have demonstrated that the yeastlike (Y) and the mycelial (M) forms have chitin as a common polysaccharide, with alpha-1, 3-glucan in the Y form and beta-1, 3-glucan in the M form. These polysaccharides are fibrillar and determine to some degree the fungal shape. Moreover, an amorphous galactomannan is found in the cell wall of the M form. This compound is responsible for the antigenic properties of the cell wall (1). Recent studies (30-33) suggest that the cell wall does not possess a stable chemical structure but a rather changing one, as a function of the environment in which the fungus is grown. At the same time, the cell wall composition seems to correlate with the degree of virulence of the particular strain. From these observations it may be deduced that the constituent polysaccharides of P. brasiliensis cell wall, play an important role in the active protection of the fungus against the defensive mechanisms of the host.

Antigens, Fungal

Effects of cyanein and ramihyphin A on the dimorphism of Paracoccidioides brasiliensis.

As a preliminary step in the study of dimorphism in Paracoccidioides brasiliensis, the effects of cyanein and ramihyphin A were studied. These antibiotics have been reported to induce morphological changes in fungi. The results obtained suggest that ramihyphin A induce swelling of hyphae while partially inhibiting Y leads to M transformation with the production of an incipient and swollen mycelium. Cyanein did not affect the mycelial morphology as did ramihyphin A. However, the Y leads to M transformation was inhibited and, also, the M leads to Y transformation was blocked with the production of a few yeast cells which were not released from the mycelium.

Antifungal Agents

The 43-kDa glycoprotein from the human pathogen Paracoccidioides brasiliensis and its deglycosylated form: excretion and susceptibility to proteolysis.

Biochemical properties of the concanavalin A-binding 43-kDa glycoprotein (gp43) of Paracoccidioides brasiliensis and its deglycosylated form were compared. Deglycosylation was achieved by treatment with trifluoromethanesulfonic acid, endoglycosidase H, N-glycanase, or metabolically, by growing cells with tunicamycin. The resulting antigen in all cases had Mr 38,000, and probably derived from the gp43 by loss of N-linked high-mannose oligosaccharide chains. The presence of galactopyranose units in the carbohydrate chains was suggested by antigen binding to peanut lectin. Pulse and chase experiments using [35S]methionine metabolic labeling of P. brasiliensis growing in the presence of tunicamycin showed that the N-linked chains of gp43 are not required for antigen secretion. The 38-kDa antigen was more susceptible than the native antigen to the action of papain and pronase, thus indicating a protective role of the carbohydrate moiety against proteolysis. Both forms are equally resistant to endogenous proteases at neutral pH. The gp43, itself, has a proteolytic activity at pH 5-6, but not at neutral pH. Deglycosylation with endoglycosidase H or tunicamycin preserved epitopes in the 38-kDa molecule reactive with (a) antibodies from patients with paracoccidioidomycosis, or rabbit immunized with the gp43 and (b) mouse monoclonal antibodies against the gp43 antigen. The present results provide a basis for the understanding of diagnostic reactions and fungal virulence involving the gp43 exocellular antigen of P. brasiliensis.

Antigens, Fungal

Amino acid synthetic media for fungal pathogens based on aminopeptidase specificities: Histoplasma capsulatum, Blastomyces dermititidis, Paracoccidioides brasiliensis and Cryptococcus neoformans.

The development of simple and chemically defined liquid media for Histoplasma capsulatum, Blastomyces dermatitidis, Paracoccidioides brasiliensis and Crypto-occus neoformans according to their aminopeptidases profiles as amino acid requirement was described. When 1.5% purified agar was added, these media also supported excellent mycelial growth and sporulation of the deep mycoses. H. capsulatum was converted to and maintained in yeast phase when 0.1% L-cystine was added to the solid medium incubated at 37 degrees C.

Amino Acids

Isolation of a specific antigen with alkaline phosphatase activity from soluble extracts of Paracoccidioides brasiliensis.

A specific antigen of Paracoccidioides brasiliensis was isolated from a metabolic extract of the fungus. The extraction was made by specific adsorption to and subsequent elution from a column containing a cross linked polymer to which the antibodies of a monospecific rabbit serum had been covalently attached. The purity of the final product was demonstrated by immunodiffusion analysis of the eluate using immune serum produced in a sensitized rabbit. The purified antigen was shown to have cationic electrophoretic mobility and alkaline phosphatase activity.

Alkaline Phosphatase

Cell wall analysis of an adenine-requiring mutant of the yeast-like form of Paracoccidioides brasiliensis strain IVIC Pb9.

An adenine-requiring mutant of Paracoccidioides brasiliensis strain IVIC Pb9 was isolated after treatment of the yeast-like (Y) form with nitrosoguanidine. Cell wall analysis of this mutant (strain IVIC Pb141) showed an increase in the amount of alpha-1,3-glucan and a virtual disappearance of the antigenic galactomannan. At the same time, a higher degree of virulence was observed for the mutant. These results agree with the hypothesis presented before (16) about a relationship between cell wall polysaccharides and pathogenicity in P. brasiliensis.

Adenine

Isolation and characterization of a Paracoccidioides brasiliensis strain from a dogfood probably contaminated with soil in Uberlândia, Brazil.

We report the isolation of a strain of Paracoccidioides brasiliensis from a dogfood, probably contaminated with soil, in a Brazilian city. The fungus was isolated on appropriate culture media, and when inoculated into a guinea-pig testis produced orchitis with abundant fungal elements. Histopathology of sections of the testicle showed an inflammatory reaction with P. brasiliensis inside monocytes. Immunological identification confirmed the identity of the isolate.

Animal Feed

Antigens of diagnostic value in three isolates of Paracoccidioides brasiliensis.

Yeast cellular extracts of three isolates of Paracoccidioides brasiliensis were tested by the Western-blot technique against 53 sera from patients with paracoccidioidomycosis (PCM). Numerous antigens were recognized by the sera but only five were specific for (58, 57, 48, 44 and 23 kDa). These specific antigens had the same relative molecular mass in the different isolates but their reactivity with sera from PCM patients was variable. An antigen in isolate 688, which was specific for PCM, had an apparent molecular mass of 48 kDa. Although a 48 kDa antigen was also present in isolates B339 and 1789.88 it was not specific for PCM, demonstrating antigenic variability among isolates. The 44 kDa antigen in isolate B339 and the 44 and 48 kDa antigens in isolate 688 reacted with a rabbit antiserum raised against a 43 kDa glycoprotein, a specific antigen used in the diagnosis of PCM. There was no correlation between the specific antigen(s) detected and the clinical form of the disease.

Antibodies, Fungal