Antigenic similarities to Paracoccidioides brasiliensis in thermo-dependent dimorphic fungi isolated from soil in Botucatu,SP, Brazil

We compared the antigenic characteristics of two thermo-dependent dimorphic fungi isolated from soil in Botucatu, an endemic area of paracoccidioidomycosis (PCM) and Paracoccidioides brasiliensis. The soil isolates grew as cerebriform colonies at 37 °C (yeast form) and as cottonous colonies at 25 °C (mycelial form). No pathogenicity for ddY mice or hamsters were observed. In immunodiffusion test, there were precipitation bands between the 2 soil isolates and pooled PCM patient sera. There were also common precipitation bands at 21, 50 and 58 kDa between the soil isolates antigens and PCM patient sera by Western-blotting, but no gp43 kDa band. No gene for gp 43 kDa protein was detected in the soil isolates by PCR. The fact that these isolates were obtained from an endemic area of PCM and there were some antigenic similarities between the soil isolates and P. brasiliensis in immunodiffusion test and Western-blotting may have some importance in epidemiological surveys done with paracoccidioidin as well interfering with the immune response of the exposed population. This revised version was published online in August 2006 with corrections to the Cover Date.     

Differential expression of cytoplasmic proteins during yeast bud and germ tube formation in Candida albicans

Changes in the identity and quantity of proteins synthesized during morphogenesis may result from alterations in gene expression in the dimorphic yeast Candida albicans. Stationary phase yeast cells, upon resuming growth at 25 degrees C, form budding yeast and at 37 degrees C form germ tubes. In order to identify proteins associated with morphogenesis, we compared cytoplasmic proteins synthesized during germ tube and bud formation. Proteins synthesized during this period were labeled at four intervals with either [3H]leucine or [35S]methionine and separated by two-dimensional polyacrylamide gel electrophoresis. This study shows that, of the 230 proteins resolved on each gel, 5 were specific to the yeast morphology and 2 proteins showed reduction in net synthesis in the mycelial phase. There were, however, no mycelium-specific proteins at any labeling period. The majority of proteins were common to both morphologies and showed no major shift in number during resumption of growth. The observations reported here suggest that differential gene expression occurs during morphogenesis of C. albicans.     

Neonatal exposure to oestrogens alters the protein profiles and gene expression in the genital tract of adult male mice.

After neonatal administration of supraphysiological doses of oestradiol, the concentration of tissue proteins, in adult mice, was significantly reduced by 39, 45 and 56% in epididymis, vas deferens and seminal vesicle respectively. The protein profiles showed persistent alterations. In epididymis, 4 protein bands were differentially increased (14.4, 43 and 67 kDa) or reduced (24 kDa) in oestrogenized males. In vas deferens, 4 proteins were increased (14.4, 49,67 and 76 kDa) and one (34 kDa) virtually absent. In seminal vesicle, about 20 proteins of varying molecular weights (12-140 kDa) were differentially increased or decreased. Testosterone substitution, at adulthood, was unable to reverse these effects. Treatments with oestradiol during adult life induced persistent alterations in the protein profiles of the 3 organs but, in contrast to neonatal treatment, these alterations could be reversed by androgen therapy. A cDNA library has been constructed with RNA prepared from adult seminal vesicle and screened by differential hybridization. Neonatal oestrogenization strongly reduced the abundance of some mRNA species. Eleven recombinants containing putative oestrogen-sensitive sequences were isolated. Two of them, having an insert of about 500 base pairs, were used for dot-blot hybridization. Results showed that the two clones contained sequences which were differently regulated by androgens.     

The antigenic and catalytically active formamidase of Paracoccidioides brasiliensis: protein characterization, cDNA and gene cloning, heterologous expression and functional analysis of the recombinant protein

Paracoccidioides brasiliensis is a well-characterized pathogen of humans. To identify proteins involved in the fungus-host interaction, P. brasiliensis yeast proteins were separated by liquid isoelectric focusing, and fractions were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis. Immunoreactive bands were detected with pooled sera of patients with P. brasiliensis infection. A protein species with a molecular mass of 45 kDa was subsequently purified to homogeneity by preparative gel electrophoresis. The amino acid sequence of four endoproteinase Lys-C-digested peptides indicated that the protein was a formamidase (FMD) (E.C. 3.5.1.49) of P. brasiliensis. The complete cDNA and a genomic clone (Pbfmd) encoding the isolated FMD were isolated. An open reading frame predicted a 415-amino acid protein. The sequence contained each of the peptide sequences obtained from amino acid sequencing. The Pbfmd gene contained five exons interrupted by four introns. Northern and Southern blot analysis suggested that there is one copy of the gene in P. brasiliensis and that it is preferentially expressed in mycelium. The complete coding cDNA was expressed in Escherichia coli to produce a recombinant fusion protein with glutathione S-transferase (GST). The purified recombinant protein was recognized by sera of patients with proven paracoccidioidomycosis and not by sera of healthy individuals. The recombinant 45-kDa protein was shown to be catalytically active; FMD activity was detected in P. brasiliensis yeast and mycelium.     

Involvement of an alternative oxidase in oxidative stress and mycelium-to-yeast differentiation in Paracoccidioides brasiliensis

Paracoccidioides brasiliensis is a thermodimorphic human pathogenic fungus that causes paracoccidioidomycosis (PCM), which is the most prevalent systemic mycosis in Latin America. Differentiation from the mycelial to the yeast form (M-to-Y) is an essential step for the establishment of PCM. We evaluated the involvement of mitochondria and intracellular oxidative stress in M-to-Y differentiation. M-to-Y transition was delayed by the inhibition of mitochondrial complexes III and IV or alternative oxidase (AOX) and was blocked by the association of AOX with complex III or IV inhibitors. The expression of P. brasiliensis aox (Pbaox) was developmentally regulated through M-to-Y differentiation, wherein the highest levels were achieved in the first 24 h and during the yeast exponential growth phase; Pbaox was upregulated by oxidative stress. Pbaox was cloned, and its heterologous expression conferred cyanide-resistant respiration in Saccharomyces cerevisiae and Escherichia coli and reduced oxidative stress in S. cerevisiae cells. These results reinforce the role of PbAOX in intracellular redox balancing and demonstrate its involvement, as well as that of other components of the mitochondrial respiratory chain complexes, in the early stages of the M-to-Y differentiation of P. brasiliensis.     

Topology of ER processing alpha-mannosidase of Saccharomyces cerevisiae.

The yeast specific alpha-mannosidase which converts Man9GlcNAc to a single isomer of Man8GlcNAc is involved in N-linked oligosaccharide processing in the endoplasmic reticulum (ER). Sequence analysis of the structural gene for this enzyme suggested that it is a type II transmembrane protein (Camirand et al., 1991). To firmly establish its membrane topology, the gene was transcribed in vitro and translation was performed in a reticulocyte lysate with and without dog pancreas microsomal membranes. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of [35S]methionine-labelled products showed that the largest band formed corresponded in size to the 63 kDa peptide expected from the alpha-mannosidase gene product. It was transformed into a 4 kDa larger endoglycosidase H-sensitive band in the presence of microsomal membranes. This glycosylated translation product was completely protected from proteinase K digestion in the absence of detergent. These results demonstrate that the yeast ER alpha-mannosidase is a type II membrane protein, like Golgi enzymes involved in N-linked glycosylation.     

Cell wall proteins of Candida albicans

Proteins were solubilized from cell wall fractions of Candida albicans and separated by polyacrylamide gel electrophoresis. Cell walls were isolated from 25 and 37 degrees C growing and stationary phase yeast cultures and from germ tubes. The 42 protein bands detected by dye binding were observed in all wall extracts, regardless of the temperature, growth state, or morphology of the culture. The carbohydrate content of most bands was below the detectable limit of the periodic acid Schiff reagent. The protein complement revealed by autoradiography of radiolabeled proteins was half that detected by staining. Two bands showed greater intensity from cultures grown at 37 degrees C. The radio-labeled pattern was similar with both [35S]methionine-and [14C]leucine-labeled proteins and either pulse- or continuous-labeled proteins.     

Transcriptome analysis and molecular studies on sulfur metabolism in the human pathogenic fungus Paracoccidioides brasiliensis

The dimorphic pathogenic fungus Paracoccidioides brasiliensis can grow as a prototroph for organic sulfur as a mycelial (non-pathogenic) form, but it is unable to assimilate inorganic sulfur as a yeast (pathogenic) form. Temperature and the inability to assimilate inorganic sulfur are the single conditions known to affect P. brasiliensis mycelium-to-yeast (M-Y) dimorphic transition. For a comprehensive evaluation of genes that have their expression modulated during the M-Y transition in different culture media, we performed a large-scale analysis of gene expression using a microarray hybridization approach. The results of the present work demonstrate the use of microarray hybridization analysis to examine gene expression during the M-Y transition in minimal medium and compare these results with the M-Y transition in complete medium. Our results showed that about 95% of the genes in our microarray are mainly responding to the temperature trigger, independently of the media where the M-Y transition took place. As a preliminary step to understand the inorganic sulfur inability in P. brasiliensis yeast form, we decided to characterize the mRNA accumulation of several genes involved in different aspects of both organic and inorganic sulfur assimilation. Our results suggest that although P. brasiliensis cannot use inorganic sulfur as a single sulfur source to initiate both M-Y transition and Y growth, the fungus can somehow use both organic and inorganic pathways during these growth processes.     

Freeze-Induced Alterations of Translatable mRNA Populations in Wood Frog Organs

To investigate the roles that gene expression and new protein synthesis play in freezing survival by the wood frog, Rana sylvatica, we compared the in vitro translation products made from mRNA isolated from six tissues (liver, brain, heart, muscle, kidney, gut) of control (5 degrees C), frozen (24 h at -2.5 degrees C), and thawed (24 h at 5 degrees C after 24 h frozen) frogs. [(35)S]Methionine-labeled proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and located by fluorography. Results indicated specific changes in the translatable populations of mRNA in tissues of freezing-exposed frogs that were largely reversed upon thawing. Differential protein expression was greatest in the comparison of liver from control versus frozen frogs with proteins ranging from 45 to 14.8 kDa identified as enhanced or unique to the frozen state. One unique protein appeared in skeletal muscle (116 kDa) of freeze-exposed frogs while another (52.5 kDa) was enhanced. Analysis of brain and heart each revealed the presence of one protein unique to the frozen state in each (58.9 and 5.9 kDa, respectively) whereas no change in the pattern of in vitro translation products was seen in gut (stomach + intestine combined) or kidney between the three experimental states. These freeze-induced alterations in the populations of translatable mRNA suggest that changes in the complement of specific proteins underlie various adaptive responses that contribute to the freezing survival of this amphibian.     

Selective affinity labeling of a 27-kDa integral membrane protein in rat liver and kidney with N-bromoacetyl derivatives of L-thyroxine and 3,5,3'-triiodo-L-thyronine

125I-Labeled N-bromoacetyl derivatives of L-thyroxine and L-triiodothyronine were used as alkylating affinity labels to identify rat liver and kidney microsomal membrane proteins which specifically bind thyroid hormones. Affinity label incorporation was analyzed by ethanol precipitation and individual affinity labeled proteins were identified by autoradiography after separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. Six to eight membrane proteins ranging in size from 17 to 84 kDa were affinity labeled by both bromoacetyl-L-thyroxine (BrAcT4) and bromoacetyl-L-triiodothyronine (BrAcT3). Affinity labeling was time- and temperature-dependent, and both reduced dithiols and detergents increased affinity labeling, predominantly in a 27-kDa protein(s). Up to 80% of the affinity label was associated with a 27-kDa protein (p27) under optimal conditions. Affinity labeling of p27 by 0.4 nM BrAc[125I]L-T4 was blocked by 0.1 microM of the alkylating ligands BrAcT4, BrAcT3, or 100 microM iodoacetate, by 10 microM concentrations of the non-alkylating, reversible ligands N-acetyl-L-thyroxine, 3,3',5'-triiodothyronine, 3,5-diiodosalicylate, and EMD 21388, a T4-antagonistic flavonoid. Neither 10 microM L-T4, nor 10 microM N-acetyltriiodothyronine or 10 microM L-triiodothyronine blocked affinity labeling of p27 or other affinity labeled bands. Affinity labeling of a 17-kDa band was partially inhibited by excess of the alkylating ligands BrAcT4, BrAcT3, and iodoacetate, but labeling of other minor bands was not blocked by excess of the competitors. BrAc[125I]T4 yielded higher affinity label incorporation than BrAc[125I]T3, although similar banding patterns were observed, except that BrAcT3 affinity labeled more intensely a 58,000-Da band in liver and a 53,000-55,000-Da band in kidney. The pattern of other affinity labeled proteins with p27 as the predominant band was similar in liver and kidney. Peptide mapping of affinity labeled p27 and p55 bands by chemical cleavage and protease fragmentation revealed no common bands excluding that p27 is a degradation product of p55. These data indicate that N-bromoacetyl derivatives of T4 and T3 affinity label a limited but similar constellation of membrane proteins with BrAcT4 incorporation greater than that of BrAcT3. One membrane protein (p27) of low abundance (2-5 pmol/mg microsomal protein) with a reactive sulfhydryl group is selectively labeled under conditions identical to those used to measure thyroid hormone 5'-deiodination. Only p27 showed differential affinity labeling in the presence of noncovalently bound inhibitors or substrates on 5'-deiodinase suggesting that p27 is likely to be a component of type I 5'-deiodinase in rat liver and kidney.     

Wall mannoproteins of the yeast and mycelial cells of Candida albicans: nature of the glycosidic bonds and polydispersity of their mannan moieties

Zymolyase released between 20 and 25% of the total protein from purified walls of yeast (Y) and mycelial (M) cells of Candida albicans. The material released contained 92% carbohydrate (86% mannose and 6% glucose) and 7% protein. Over 85% of the carbohydrate was N-glycosidically linked to the protein and the rest (less than 15%) was linked O-glycosidically. Highly polydisperse, high molecular mass mannoproteins, resolved by electrophoresis as four defined bands in Y cells and two bands in M cells, had both types of sugar chains. A 34 kDa species found in both types of cells had a single 2.5 kDa N-glycosidically linked sugar chain and a 31.5 kDa protein moiety. Polydispersity in the high molecular mass mannoproteins was due to the N-linked sugar chains (mannan) with a molecular mass between 500 kDa and 20 kDa (average 100 kDa) in Y cells and between 400 kDa and 20 kDa (average 50 kDa) in M cells. Three mannoproteins of 34, 30 and 29 kDa secreted by protoplasts were associated with the high molecular mass mannoproteins, suggesting that this type of interaction might be related to the regeneration of the cell wall.     

Intracellular and oligomeric forms of surfactant-associated apolipoproteins(s) A in the rat

Sulfhydryl-dependent oligomeric forms of the surfactant-associated apolipoprotein(s) A, obtained from particulate preparations of adult rat lung lavage, were characterized by immunoblot analysis and by silver staining of proteins separated by one- and two-dimensional SDS-polyacrylamide gel electrophoresis. Under non-reducing conditions, these proteins migrated as oligomers, Mr approx. 50-70, 115, 160 kDa and greater. The large oligomers were reduced to the apolipoprotein(s) A subunits by treatment with beta-mercaptoethanol; Mr 38 (A3), 32 (A2) and 26 kDa (A1), pI 4.2-4.8. Mr 50 kDa protein was composed of sulfhydryl-dependent homo-dimers of protein(s) A1 (Mr 26 kDa). 55 kDa protein was a hetero-dimer composed primarily of A1 and A2 (Mr 26 and 32 kDa). 62 kDa protein was composed of hetero-dimers of A3 and apolipoprotein A2 (Mr 38 and 32 kDa). 70 kDa protein was a homodimer composed of apolipoprotein A3 A3 (38 kDa). Larger molecular forms were composed primarily of 38 and 32 kDa and lesser amounts of 26 kDa. Treatment with endoglycosidase F reduced A2 and A3 to 26 kDa. Apolipoprotein A1 co-migrated with a protein of Mr 26 kDa immunoprecipitated from [35S]methionine-labelled Type II epithelial cells. Chymotryptic-tryptic peptide maps of apolipoproteins A1, A2 and A3 were identical, suggesting that apolipoproteins A3 and A2 arise through extensive glycosylation of apolipoprotein A1.     

Differential expression of Rho1GTPase and Rho3GTPase during isotropic and polarized growth of Mucor circinelloides

Evidence has been obtained that indicates the presence of small 22 kDa GTP-binding Rho proteins through ADP-ribosylation by Clostridium botulinum C3 exotoxin in Mucor circinelloides. Rho protein was detected at all stages of growth studied. During polarized growth, both under aerobic conditions and during the yeast-mycelia transition, the radiolabeling of the [32P]ADP-ribosylated protein increased when tube formation occurred and decreased as the hyphae branched. However, when Mucor grew isotropically, the Rho protein band was thick and its intensity did not vary significantly even after bud formation and separation of daughter cells. Crude extracts of yeast and mycelial cells exhibited a broad 22 kDa band of the [32P]ADP-ribosylated Rho protein that was resolved into a protein with a pI of 6.0, after two-dimensional electrophoresis, corresponding to the Rho1p homolog. Furthermore, [32P]ADP-ribosylated Rho protein from soluble and particulate extracts of multipolarized mycelial cells obtained from the yeast-mycelia transition was separated into two proteins with pI of 6.0 and 6.4, respectively, after two-dimensional electrophoresis. These correspond to the Rho1p and Rho3p homologs, respectively. Therefore, our results show that an increase in Rho accumulation is associated with polarized growth.     

Chitinase from Paracoccidioides brasiliensis: molecular cloning, structural, phylogenetic, expression and activity analysis

A full-length cDNA encoding a chitinase (Pbcts1) was cloned by screening a cDNA library from the yeast cells of Paracoccidioides brasiliensis. The cDNA consists of 1888 bp and encodes an ORF of 1218 bp corresponding to a protein of 45 kDa with 406 amino acid residues. The deduced PbCTS1 is composed of two signature family 18 catalytic domains and seems to belong to fungal/bacterial class. Phylogenetic analysis of PbCTS1 and other chitinases suggests the existence of paralogs of several chitinases to be grouped based on specialized functions, which may reflect the multiple and diverse roles played by fungi chitinases. Glycosyl hydrolase activity assays demonstrated that P. brasiliensis is able to produce and secrete these enzymes mainly during transition from yeast to mycelium. The fungus should be able to use chitin as a carbon source. The presence of an endocytic signal in the deduced protein suggests that it could be secreted by a vesicular nonclassical export pathway. The Pbcts1 expression in mycelium, yeast, during differentiation from mycelium to yeast and in yeast cells obtained from infected mice suggests the relevance of this molecule in P. brasiliensis electing PbCTS1 as an attractive drug target.