Ornithine decarboxylase in Paracoccidioides brasiliensis

Ornithine decarboxylase in Paracoccidioides brasiliensis, a dimorphic human pathogenic fungus, was more active at 37° C in the yeast phase and at 30° C in the mycelial phase. In contrast to other fungal systems, yeast growth and mycelium-to-yeast transition in P. brasiliensis were accompanied by a high activity of ornithine decarboxylase at the onset of the budding process, the activity of which was inhibited by 1,4-diamino-2-butanone. The activity of ornithine decarboxylase remained at a basal level during vegetative growth of both the mycelial phase and the late stage of yeast phase, and also through the yeast-to-mycelium transition. Received: 18 December 1995 / Accepted: 8 March 1996     

Enzyme-linked immunosorbent assay (ELISA) in the paracoccidioidomycosis

An enzyme-linked immunosorbent assay (ELISA) for detection and quantification of antibodies antiParacoccidioides brasiliensis is described. Polystyrene plates have been used as solid phase to absorb P. brasiliensis metabolic yeast phase antigen. Twenty sera of proven paracoccidioidomycosis, 11 of histoplasmosis due Histoplasma capsulatum, 20 of aspergillosis and 20 human normal sera were tested. Ninety-five percent of the paracoccidioidomycosis sera had O.D. superior to 0.150 (from 0.163 to 2.650) at 1/400 serum dilution. ELISA assay was compared with counterimmunoelectrophoresis and erythro-immunoassay tests; a correlation was observed only with erythro-immunoassay. ELISA test should give new perspectives for the serodiagnosis of paracoccidioidomycosis.     

Adenosine triphosphatase in yeast phaseParacoccidioides brasiliensis

We have previously detected various enzymatic activities inP. brasiliensis. In the present study we have examined Adenosine Triphosphatase (ATPase) activity in yeast phase cultures ofP. brasiliensis of increasing age. We employed Wachstein and Meisel's method and electron microscopy and found specific electron-dense deposits indicating ATPase activity to be present in the cytoplasm around vacuoles. Their distribution varied according to age. Deposits decreased or became absent in old cultures. We assume that inP. brasiliensis, ATPase is involved (as in other systems) with transport of Na⁺ and K⁺.     

Identification of genes preferentially expressed in the pathogenic yeast phase of Paracoccidioides brasiliensis, using suppression subtraction hybridization and differential macroarray analysis

Paracoccidioides brasiliensis, a thermodimorphic fungus, is the causative agent of paracoccidioidomycosis (PCM), the most prevalent systemic mycosis in Latin America. Pathogenicity appears to be intimately related to the dimorphic transition from the hyphal to the yeast form, which is induced by a shift from environmental temperature to the temperature of the mammalian host. Little information is available on the P. brasiliensis genes that are necessary during the pathogenic phase. We have therefore undertaken Suppression Subtraction Hybridization (SSH) and macroarray analyses with the aim of identifying genes that are preferentially expressed in the yeast phase. Genes identified by both procedures as being more highly expressed in the yeast phase are involved in basic metabolism, signal transduction, growth and morphogenesis, and sulfur metabolism. In order to test whether the observed changes in gene expression reflect the differences between the growth conditions used to obtain the two morphological forms rather than differences intrinsic to the cell types, we performed real-time RT-PCR experiments using RNAs derived from both yeast cells and mycelia that had been cultured at 37°C and 26°C in either complete medium (YPD or Sabouraud) or minimal medium. Twenty genes, including AGS1 (-1,3-glucan synthase) and TSA1 (thiol-specific antioxidant), were shown to be more highly expressed in the yeast cells than in the hyphae. Although their levels of expression could be different in rich and minimal media, there was a general tendency for these genes to be more highly expressed in the yeast cells.Communicated by C P. Hollenberg     

Epithelial cells treated with genistein inhibit adhesion and endocytosis of<Emphasis Type="Italic"> Paracoccidioides brasiliensis</Emphasis>

Paracoccidioidomycosis is caused by Paracoccidioides brasiliensis, which although not formally considered an intracellular pathogen, can be internalized by epithelial cells in vitro and in vivo. The mechanisms used by P. brasiliensis to adhere to and invade non-professional phagocytes have not been identified. The signal-transduction networks, involving protein tyrosine kinase (PTK) and protein phosphatase activities, can modulate crucial events during fungal infections. In this study, the involvement of PTK has been investigated in P. brasiliensis adherence and invasion in mammalian epithelial cells. A significant inhibition of the fungal invasion occurred after the pre-treatment of the epithelial cells with genistein, a specific tyrosine kinase inhibitor, indicating that the tyrosine kinase pathway is involved in P. brasiliensis internalization. In contrast, when the fungus was treated, a slight (not significant) inhibition of PTK was observed, suggesting that PTK might not be the fungus’ transduction signal pathway during the invasion process of epithelial cells. An intense PTK immunofluorescence labeling was observed in the periphery of the P. brasiliensis infected cells, little PTK labeling was found in both uninfected cells and yeast cells, at later infection times (8 and 24 h). Moreover, when the epithelial cells were treated with genistein and infected with P. brasiliensis, no labeling was observed, suggesting the importance of the PTK in the infectious process. These results suggest that PTK pathway participates in the transduction signal during the initial events of the adhesion and invasion processes of P. brasiliensis to mammalian epithelial cells.     

Mitochondrial function in the yeast form of the pathogenic fungus <Emphasis Type="Italic">Paracoccidioides brasiliensis</Emphasis>

Differences between the respiratory chain of the fungus Paracoccidioides brasiliensis and its mammalian host are reported. Respiration, membrane potential, and oxidative phosphorylation in mitochondria from P. brasiliensis spheroplasts were evaluated in situ, and the presence of a complete (Complex I–V) functional respiratory chain was demonstrated. In succinate-energized mitochondria, ADP induced a transition from resting to phosphorylating respiration. The presence of an alternative NADH–ubiquinone oxidoreductase was indicated by: (i) the ability to oxidize exogenous NADH and (ii) the lack of sensitivity to rotenone and presence of sensitivity to flavone. Malate/NAD⁺-supported respiration suggested the presence of either a mitochondrial pyridine transporter or a glyoxylate pathway contributing to NADH and/or succinate production. Partial sensitivity of NADH/succinate-supported respiration to antimycin A and cyanide, as well as sensitivity to benzohydroxamic acids, suggested the presence of an alternative oxidase in the yeast form of the fungus. An increase in activity and gene expression of the alternative NADH dehydrogenase throughout the yeast’s exponential growth phase was observed. This increase was coupled with a decrease in Complex I activity and gene expression of its subunit 6. These results support the existence of alternative respiratory chain pathways in addition to Complex I, as well as the utilization of NADH-linked substrates by P. brasiliensis. These specific components of the respiratory chain could be useful for further research and development of pharmacological agents against the fungus.     

Mice Immunization with Radioattenuated Yeast Cells of <Emphasis Type="Italic">Paracoccidiodes brasiliensis</Emphasis>: Influence of the Number of Immunizations

Paracoccidioides brasiliensis is the agent of paracoccidioidomycosis, the most prevalent mycosis in Latin America. Up to the moment no vaccine has been reported. The aim of this study was to evaluate the influence of the number of immunizations on the protection elicited by radioattenuated yeast cells of P. brasiliensis. BALB/c mice were divided into two groups that were immunized once (Group 1) or twice (Group 2), respectively. In each group, mice were divided into sub-groups that were challenged 30, 45, or 60 days after the second immunization. Organ colony-forming units (CFUs) was determined 90 days post-challenge. A significant reduction in CFUs recovery was verified in both groups, but it was higher in Group 2. Histologic alterations were observed only in Group 1. The cytokines IL-4, IL-10, and IFN-γ were produced in mice of Group 1. In Group 2, only IFN-γ was significantly detected. IgG2a predominance relative to IgG1 was also observed in Group 2. Altogether, our results indicated that mice immunized once developed a mixed Th1/Th2 response, which was less efficient in the infection control, while a trend to a Th1 pattern was obtained with two immunizations, promoting optimal elimination of P. brasiliensis yeast cells from mice tissues.     

Polyamines in growth and dimorphism ofParacoccidioides brasiliensis

Putrescine and spermidine were the only polyamines found inParacoccidioides brasiliensis, a dimorphic fungus pathogenic for humans. Free polyamines (putrescine>spermidine) increased during the first 24 h of yeast growth, with a second peak at 42 h, and also during the first 12 h of mycelium-to-yeast transition (spermidine>putrescine). Conjugated and bound polyamines were also quantified. 1,4-Diamino-2-butanone decreased free putrescine and spermidine accumulation by inhibiting the activity of ornithine decarboxylase. The increase in free polyamines corresponds to bud emergence in yeast growth and to the mycelium-to-yeast transition ofP. brasiliensis.Abbreviations DAB 1,4-Diamino-2-butanone - Y Yeasts - M Mycelia - ODC Ornithine decarboxylase     

Growth-promoting factors for yeast cells ofParacoccidioides brasiliensis

Low-density seedings of yeast cells ofParacoccidioides brasiliensis give poor growth (as assessed by plating efficiency test) on conventional mycological agar media, and therefore growth-promoting factors for this fungus were sought. Water-extracts of yeast cells of sixP. brasiliensis isolates were all considerably effective in promoting the growth of low-density seedings ofP. brasiliensis isolates Pb-18 and Hachisuga, but had little effect on isolate Bt-4. Horse serum, at a concentration range of 2–4%, moderately or considerably promoted the growth of theseP. brasiliensis isolates. Combinations of the fungus cell extracts with horse serum were highly effective in promoting the growth of all of the fungal isolates. The fungus cell extracts showed siderophore (microbial iron carrier) activity. An iron-chelator, ethylenediaminetetraacetic acid, at a concentration of 100 μM also highly promoted the growth of the fungal isolates in the presence of horse serum, and ferric ion added to culture medium was considerably effective in the growth promotion. These results suggest that deficient utilization of external iron by the fungus cell is one of the growth-limiting processes for low-density seedings of yeast cells ofP. brasiliensis on conventional mycological agar media.     

Studies on plating efficiency and estimation of viability of suspensions of Paracoccidioides brasiliensis yeast cells

Mild sonication was used to obtain single cell suspensions of Paracoccidioides brasiliensis. These cells were intact by microscopic criteria. Direct cell counts in a given inoculum and colony formation on various media were used to determine plating efficiency. Sonicated and nonsonicated cell suspensions were used to study plating efficiency and to estimate viability by means of vital dyes. Methylene blue, Erythrosin B, and Janus green were unreliable when used with P. brasiliensis, but vital dyes were accurate when tested with Candida albicans.Acridine orange gave more meaningful results of viability. Estimates of viability, however, changed significantly as a result of relatively minor alterations in the composition of the suspending medium.In initial experiments, the plating efficiency of P. brasiliensis was dismally low. It descended abruptly with increasing dilution of inoculum. Efficiency was much improved if horse serum was added to brain heart infusion plates or if glucose glycine yeast extract (GGY) plates were incubated at room temperature and mycelial colonies were counted. With the technique we report, current plating efficiency of sonicated suspensions is of the order of 25 %. Our results and procedures have an important bearing upon those studies concerned with in vitro killing of P. brasiliensis in suspensions or with isolating this fungus from clinical or environmental specimens.     

Expression inEscherichia coli of a gene coding for epitopes of a diagnostic antigen ofParacoccidioides brasiliensis

A methodology to isolate nucleic acids from the human pathogenParacoccidioides brasiliensis was developed that enabled the construction of genomic libraries in bacteriophage λgt11. Among 3000 recombinant phage 20 were selected by hybridization with cDNA synthesized from mRNA isolated fromP. brasiliensis yeast forms, which correspond to the fungus phase found in infected tissues. One of these clones expressed an antigen which was specifically recognized by sera from patients with paracoccidioidomycosis. Antibodies isolated by adsorption to a lysate of this clone bound to the exocellular glycoprotein of 43 kDa (gp43). As shown previously (Pucciaet al., 1986,Infect. Immun.53: 199–206), this is the main diagnostic antigen of paracoccidioidomycosis.     

Serological Survey of Paracoccidioidomycosis in Sheep

The objective of this study was to evaluate the prevalence of antibodies against Paracoccidioides brasiliensis in sheep from Guarapuava, Paraná State, Brazil. The seroepidemiological study was carried out in 262 sheep. The samples were analyzed by ELISA and immunodiffusion test using P. brasiliensis gp43 and exoantigen as antigens, respectively. Initially, two sheep were immunized with P. brasiliensis to evaluate whether contact with the fungal cells could induce a humoral immune response against gp43 and exoantigen from P. brasiliensis. Both animals produced antibodies against gp43 and exoantigen, the main antigens used for diagnosis and seroepidemiology of paracoccidioidomycosis. A reactivity of 37% was observed to the P. brasiliensis gp43 antigen by ELISA although no reactivity had been observed by the immunodiffusion test. Sheep under extensive grazing system showed higher frequency of positivity to P. brasiliensis (P ≤ 0.05) than those under intensive and semi-intensive systems. These data suggest that sheep may be a useful epidemiological marker of P. brasiliensis presence in the environment and reinforce that contact with soil is an important risk factor for infection.     

Pathogenesis of paracoccidioidomycosis: A histopathological study of the experimental murine infection

The pathogenesis of primary pulmonary P. brasiliensis infection, the systemic dissemination which followed, and the histopathology of the main organs involved was studied in a murine model of chronic paracoccidioidomycosis. Adult male BALB/C mice, were challenged intranasally with 26×10^–6 viable P. brasiliensis yeast cells. We inoculated 86 animals which were sacrificed from 0 h to 20 weeks. As controls, 11 mice were instilled with saline solution, and 48 with 26×10^–6 heat-killed. P. brasiliensis yeast cells. None of the animals receiving saline, exhibited pathologic alterations; 11.6% of those inoculated with the heatkilled cells, revealed mild, transitory acinopulmonary neutrophilic infiltrates. The animals infected with viable cells, developed a systemic process affecting mainly the lungs (46.5%), liver (18.6%), lymphnodes (18.6%), and spleen (3.5%). In this group of animals, lung lesions were detected regularly at all time periods from 3 h to 20 weeks. A multiple bronchoneumonic process was initially observed at 6 h, reached its maximum intensity around the third day, subsided thereafter but did not disappear and reactivated after the fifth week to become stationary until the end of experiments. Dissemination to other organs occurred early, and apparently by the hematogenous route. Initially the inflammatory cell infiltrate was mainly neutrophilic. With time, these cells were gradually replaced by lymphocytes, histiocytes and plasmocytes. Granuloma configuration of the cell infiltrate was distinctly seen around the fifth week, with multinucleated giant cells appearing at the ninth week. Hiliary lymphnode involvement was rare (7%) and primary lung lesions, as seen in tuberculosis and histoplasmosis, were not observed.     

Studies on the relationship between the estrous cycle of BALB/c mice and their resistance to Paracoccidioides brasiliensis infection

A relationship between the estrous cycle and non-specific host resistance to Paracoccidioides brasiliensis yeast cells was examined by using both sexes of adult BALB/c mice. They were divided into 6 groups, including a male group and females at proestrus, estrus, metestrus-I, metestrus-II and diestrus. The mice received yeast cells through three different inoculation routes; intravenous, intraperitoneal and intratracheal. In all of the inoculation routes, the clearance of the yeast cells was influenced by the estrous cycle. The female mice at estrus, which might have high blood estrogen levels, showed a marked clearance of the yeast cells from the blood, peritoneal cavity and lungs. These results suggested that non-specific host resistance to the yeast cells was enhanced by estrogen. All female groups inoculated by the three routes showed higher clearance of the yeast cells than the male group.     

Cytological studies of <Emphasis Type="Italic">Agaricus brasiliensis</Emphasis>

Agaricus brasiliensis is a medicinal mushroom native to Brazil. It was first identified as Agaricus blazei and its scientific name continues to be debated. We examined the cytology of different Brazilian commercial strains of A. brasiliensis and the nuclear behavior of strain CS1 during basidiospore development using fluorescent microscopy. All strains have multinucleate hyphae and no significant differences in nuclei numbers were observed between them. Basidia from A. brasiliensis strain CS1 are typically tetrasporics and produce binucleate basidiospores, demonstrating that a postmeiotic mitosis occurs during basidiospore development. This result suggests that A. brasiliensis is primarily a heterothallic species.