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PubMed1998

Evidence of idiotypic modulation in the immune response to gp43, the major antigenic component of Paracoccidioides brasiliensis in both mice and humans.

Paracoccidioidomycosis (PCM) is a systemic mycosis endemic in Latin America, with a high prevalence in Brazil, Argentina, Colombia and Venezuela. The aetiologic agent of disease is a thermal dimorphic fungus, Paracoccidioides brasiliensis. A glycoprotein of 43,000 D (gp43) is the major antigen of P. brasiliensis. Antibodies directed to this antigen are detected in the sera of all patients with PCM. Gp43 binds to laminin, thus participating in adhesion, invasion and pathogenesis of the fungus. As the role of antibodies in PCM is not fully understood, we decided to investigate the outcome of mice immunization with three distinct anti-gp43 MoAbs (17c, 8a and 24a) coupled with keyhole limpet haemocyanin (KLH). Results show not only the expected presence of anti-Id (AB2) antibodies in the sera of these animals but also a spontaneous and increasing amount of anti-anti-Id (AB3) antibodies after the third course of immunization. Hybridomas producing both AB2 and AB3 MoAbs were obtained using spleen cells from mice immunized with MoAb 17c. AB3 MoAbs were also obtained with spleen cells of mice immunized with MoAbs 8a and 24a. It was also shown that human PCM patients' sera with high titres of anti-gp43 antibodies generate anti-Id antibodies. These data suggest that the immune response to P. brasiliensis can be spontaneously modulated by the idiotypic network.

Animals↗
PubMed2001

Modulatory effect of prostaglandins on human monocyte activation for killing of high- and low-virulence strains of Paracoccidioides brasiliensis.

The effect of indomethacin (Indo), a cyclo-oxygenase inhibitor, on the monocyte-mediated killing of a low- (Pb265) and a high- (Pb18) virulence strain of Paracoccidioides brasiliensis was examined. The Pb18 strain was not killed by either non-activated or interferon-gamma (IFN-gamma) -activated human monocytes but these cells did show fungicidal activity if pretreated with Indo. In contrast with IFN-gamma, tumour necrosis factor-alpha (TNF-alpha) was very effective at stimulating the fungicidal activity of monocytes. While the low-virulence strain, Pb265, could not be killed by monocytes, cells preincubated with IFN-gamma demonstrated fungicidal activity. The killing of this strain was also induced by pretreatment of monocytes with Indo. The results suggest a negative role for prostaglandins, which are synthesized via the cyclo-oxygenase pathway, in the regulation of monocyte-mediated killing of virulent and avirulent strains of P. brasiliensis and that TNF-alpha generation during the fungus-monocyte interaction is more important in the killing of Pb265 than Pb18.

Adult↗
PubMed1999

Differential expression of an hsp70 gene during transition from the mycelial to the infective yeast form of the human pathogenic fungus Paracoccidioides brasiliensis.

We have isolated and characterized cDNA and genomic clones that encode a 70 kDa heat shock protein (Hsp70) from the dimorphic human pathogenic fungus Paracoccidioides brasiliensis. The gene encodes a 649-amino-acid protein showing high identity with other members of the hsp70 gene family. The hsp70 gene is induced during both heat shock of yeast cells at 42 degrees C and the mycelial to yeast transition. A differential expression of this gene can be observed between mycelial and yeast forms, with a much higher level of expression in the yeast. We found two introns of 178 and 72 nucleotides in the P. brasiliensis hsp70 gene. Splicing of these introns is regulated during the heat shock process and possibly during infection. In order to analyse the differential accumulation of unspliced mRNA following cellular differentiation and/or heat shock, reverse transcriptase-polymerase chain reaction (RT-PCR) experiments were carried out. The temperature-induced mycelial to yeast transition results in the transient accumulation of unspliced hsp70 mRNA transcripts. Yeast cells, after adaptation at 36 degrees C, seem to be more proficient at splicing, at least with respect to hsp70 mRNA because, during a severe heat shock (42 degrees C), the unspliced form of this mRNA does not accumulate. The mycelial to yeast differentiation will have the adaptational effect of increasing the resistance of the organism to environmental stress, which may be necessary for parasite survival in the mammalian host.

Amino Acid Sequence↗
PubMed2003

Regulation of T helper cell differentiation in vivo by GP43 from Paracoccidioides brasiliensis provided by different antigen-presenting cells.

Paracoccidioidomycosis, endemic in Latin America, is a progressive systemic mycosis caused by Paracoccidioides brasiliensis. The infection can evolve into different clinical forms that are associated with various degrees of suppressed cell-mediated immunity. Assuming that the effector immune response is a consequence of the preferential activation of either Th1 or Th2 subsets, in the present work we evaluated whether the nature of antigen-presenting cells (APCs) can influence the Th1/Th2 balance in vivo. It was observed that the injection of mature dendritic cells (DCs), macrophages and B cells primed the mice and induced a proliferation of T cells in vitro. It was seen that DCs from resistant mice stimulated predominantly interleukin-2 (IL-2) and interferon-gamma (IFN-gamma), whereas macrophages activated IL-10, IL-4 and IFN-gamma-secreting T cells and B cells IL-4 and IL-10 only. Results presented here clearly demonstrate that DC drives the development of cells secreting Th1-derived cytokines, whereas B cells induce the differentiation of a Th2 phenotype in vivo.

Animals↗
PubMed1999

Ultrastructural differences observed in Paracoccidioides brasiliensis yeast phase cells grown on solid and in liquid medium.

An ultrastructural study was conducted on, yeast-like Paracoccidioides brasiliensis cells grown on liquid and solid peptone--yeast extract--glucose medium. A large proportion of cells grown in liquid medium presented cytoplasmic damage compared with the cells grown on solid medium, which remained intact, suggesting that agar plays an important role in the development of this fungus.

Agar↗
PubMed2001

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 degrees C 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.

Animals↗
PubMed2002

Paracoccidioides brasiliensis: virulence and an attempt to induce the dimorphic process with fetal calf serum.

The virulence of Paracoccidioides brasiliensis strains and the induction of the dimorphic process of the fungus were investigated. The strains were kept under mineral oil for different periods of time, except for one that was successively subcultured on agar slants. Four strains with transitional morphology at room temperature were avirulent to experimental animals. Two strains with typical morphology at 36 degrees C, one of them preserved under mineral oil for 16 years and the other maintained by successive subcultures for 10 years, were virulent to mice. These strains caused macro- and microscopic lesions in various organs, with enlargement of the spleen and heart. An attempt to induce the dimorphic process with fetal calf serum (FCS) in P.brasiliensis strains with transitional morphology failed. FCS was probably unable to stimulate the synthesis of Y cell wall alpha-1,3-glucan. The results demonstrate that keeping strains under mineral oil for a long period of time may have altered the enzymatic activities of the proteinases and other virulence factors that participate in the transition process and in pathogenesis.

Animals↗
PubMed2002

Histopathologic and histochemical characterization of calcified structures in hamsters inoculated with Paracoccidioides brasiliensis.

In pathogenicity studies of 31 Paracoccidioides brasiliensis isolates preserved using Castellani's method we intraperitoneally inoculated 104 young adult hamsters and found laminated concentric structures and calcified appearance that resembled Schaumann bodies, in 43 of them, especially in animals with apparently good condition. We characterized these structures histologically and histochemically using different stains (PAS, Grocott, haematoxylin-eosin, Von Kossa). The Von Kossa staining revealed calcium in these structures. Similar structures have been described in patients with sarcoidosis and also in hamsters inoculated with P. brasiliensis. We found no correlation between the presence of these calcifications and serum calcium levels.

Animals↗
PubMed1983

Estradiol binds to a receptor-like cytosol binding protein and initiates a biological response in Paracoccidioides brasiliensis.

Paracoccidioidomycosis, a disease caused by Paracoccidioides brasiliensis, which is endemic to Latin America, is much more common in men than women, suggesting a role for hormonal factors. We recently showed that two other yeasts possess steroid binding proteins and postulated that these receptor-like molecules represented a mechanism by which the hormonal milieu of the host might influence an infecting pathogen. Therefore, we examined P. brasiliensis for a sex steroid binding protein. Because tritiated steroids rapidly dissociated from the other fungal binding proteins, we developed a fast binding method with Sephadex G-50 microcolumns speeded by centrifugation. This method detected specific binding of [3H]estradiol in P. brasiliensis cytosol. Other tritiated steroid hormones, including testosterone and corticosterone, failed to exhibit specific binding. Scatchard analysis of [3H]estradiol binding showed an apparent dissociation constant (Kd) of 1.7 X 10(-8) M and a maximal binding capacity (Nmax) of 235 fmol/mg of protein. Susceptibility to trypsin indicated the binding site was protein in nature. The protein had a Stokes radius of approximately equal to 32 A by HPLC exclusion column and a sedimentation coefficient of 4.4 S by sucrose gradient, consistent with an apparent Mr of approximately equal to 60,000. Competition experiments revealed that estrone, estriol, and progesterone had 25% of the affinity of estradiol, whereas diethylstilbestrol, androgens, and corticosteroids had low affinity. Investigation of steroid hormone actions in P. brasiliensis indicated that estradiol inhibited the fungal transformation from mycelial form to yeast form, the initial step of infection. This suppressive effect was dose-dependent and not found with testosterone. We hypothesize that endogenous estrogens in the host, acting through the cytosol binding protein in the fungus, inhibit mycelial-to-yeast transformation, thus explaining the resistance of women to paracoccidioidomycosis.

Binding, Competitive↗
PubMed1996

Cloning, characterization, and epitope expression of the major diagnostic antigen of Paracoccidioides brasiliensis.

The 43,000-Da glycoprotein (gp43) of Paracoccidioides brasiliensis is an immunodominant antigen for antibody-dependent and immune cellular responses in patients with paracoccidioidomycosis. In order to identify the peptide epitopes involved in the immunological reactivities of the gp43 and to obtain highly specific recombinant molecules for diagnosis of the infection, genomic and cDNA clones representing the entire coding region of the antigen were sequenced. The gp43 open reading frame was found in a 1,329-base pair fragment with 2 exons interrupted by an intron of 78 nucleotides. The gene is present in very few copies per genome, as indicated by Southern blotting and chromosomal megarestriction analysis. A single transcript of 1.5 kilobase pairs was verified in the yeast phase. The gene encodes a polypeptide of 416 amino acids (Mr 45,947) with a leader peptide of 35 residues; the mature protein has a single N-glycosylation site. The deduced amino acid sequence showed similarities of 56-58% with exo-1,3- beta-D-glucanases from Saccharomyces cerevisiae and Candida albicans. However, the gp43 is devoid of hydrolase activity and does not cross-react immunologically with the fungal glucanases. Internal and COOH-terminal gene fragments of the gp43 were expressed as recombinant fusion proteins, which reacted with antibodies elicited against the native antigen.

Amino Acid Sequence↗
PubMed2005

Transcriptional profiles of the human pathogenic fungus Paracoccidioides brasiliensis in mycelium and yeast cells.

Paracoccidioides brasiliensis is the causative agent of paracoccidioidomycosis, a disease that affects 10 million individuals in Latin America. This report depicts the results of the analysis of 6,022 assembled groups from mycelium and yeast phase expressed sequence tags, covering about 80% of the estimated genome of this dimorphic, thermo-regulated fungus. The data provide a comprehensive view of the fungal metabolism, including overexpressed transcripts, stage-specific genes, and also those that are up- or down-regulated as assessed by in silico electronic subtraction and cDNA microarrays. Also, a significant differential expression pattern in mycelium and yeast cells was detected, which was confirmed by Northern blot analysis, providing insights into differential metabolic adaptations. The overall transcriptome analysis provided information about sequences related to the cell cycle, stress response, drug resistance, and signal transduction pathways of the pathogen. Novel P. brasiliensis genes have been identified, probably corresponding to proteins that should be addressed as virulence factor candidates and potential new drug targets.

Blotting, Northern↗
PubMed1975

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↗
PubMed1976

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↗
PubMed1977

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↗
PubMed1980

Lipid composition of Paracoccidioides brasiliensis: comparison between the yeast and mycelial forms.

The total lipid extracted from the yeast form of Paracoccidioides brasiliensis was twice as much as from the mycelial form. In both morphological forms neutral lipids were predominant and triglycerides were the greatest single component in the extracts. Relative percentage of phospholipid was higher in Y form than in M form. Phosphatidylcholine was predominant in the former and phosphatidylethanolamine in the latter. Diphosphatidylglycerol was the most unsaturated lipid in both the forms, and in the Y form it was the only individual class of lipid that contained a greater percentage of linoleic than oleic acid. Otherwise, the lipids of Y form, both neutral and polar, were characterized by a greater percentage of oleic acid and those of the M form by the predominance of linoleic acid. Differential distribution of phosphatidylcholine, phosphatidylethanolamine, sterol, oleic and linoleic acid in Y and M forms is interpreted to suggest a possible role of cellular lipid composition in the dimorphic behaviour of P. brasiliensis.

Fatty Acids↗
PubMed1981

Ultrastructural studies on the dimorphism of Paracoccidioides brasiliensis, Blastomyces dermatitidis and Histoplasma capsulatum.

Whole cells or cell walls of the yeastlike and mycelial forms of Paracoccidioides brasiliensis, Blastomyces dermatitidis and Histoplasma capsulatum were treated successively with sodium hydroxide, beta-1,3-glucanase and pronase. The microfibrils in the insoluble residues, probably composed of chitin, were examined in the electron microscope. In the yeastlike form, tightly interwoven, randomly oriented microfibrils were seen. On the other hand, in the mycelial form, a large portion of microfibrils tended to lie in a more or less longitudinal orientation. A role of chitin in the conversion from the yeastlike form to the mycelial form is discussed.

Blastomyces↗
PubMed1982

Variability of cell wall composition in Paracoccidioides brasiliensis: a study of two strains.

A human isolate (IVIC Pb73) of paracoccidioides brasiliensis and a mutant strain (IVIC Pb219) derived from Pb73 were studied with regard to their cell wall composition. Differences were found between these strains and the only other strain (IVIC Pb9) previously studied from this point of view. Besides the expected beta-1,3-linkages, beta-1,2-bonds were also found in the alkali-insoluble glucan of both the parental and the mutant strains in the yeast forms, a rather uncommon linkage in fungal glucans. Also, the alkali-insoluble fraction from the mycelial form was composed of glucose (of which a very small proportion occurred as beta-1,3-glucan) plus a high proportion of galactose (about 50%), and some mannose. The alkali-soluble fraction, usually associated with the immunogenic galactomannan in the mycelial form, also had glucose besides galactose and mannose, but was exclusively composed of mannose in the yeastlike form. These results suggest that, as in other fungi, variations in cell wall composition could be expected between strains of P. brasiliensis.

Amino Sugars↗
PubMed1982

Isolation and wall analysis of dimorphic mutants of Paracoccidioides brasiliensis.

Nitrosoguanidine treatment of Paracoccidioides brasiliensis strain IVIC Pb73 induced a set of mutants affected in their ability to complete dimorphism. Some did not transform to the typical mycelial form, while others did not grow as yeasts. These mutants were preliminarily characterized by microscopic observations and the study of some of their properties: optimal growth temperature, nutritional requirements, virulence and cell wall analyses. Comparison of these properties allowed the classification of the mutants in 4 functional categories with respect to dimorphism.

Amino Acids↗