SUT1p interaction with Cyc8p(Ssn6p) relieves hypoxic genes from Cyc8p-Tup1p repression in Saccharomyces cerevisiae.

SUT1 is a hypoxic gene encoding a nuclear protein that belongs to the Zn[II]2Cys-6 family. It has been shown that constitutive expression of SUT1 induces exogenous sterol uptake in aerobically growing Saccharomyces cerevisiae cells. A differential display approach was used to identify genes whose transcription is modified upon SUT1 induction. Within the promoter sequence of one of these genes, DAN1, we identified the region responsive to SUT1 and showed that it has a strong repressive activity when cloned in the vicinity of distinct promoters. Upon SUT1 constitutive expression in aerobiosis, the repression is released, allowing enhanced transcription of the reporter gene. We provide evidence that the repression is promoted by the Cyc8p(Ssn6p)-Tup1p co-repressor and that release of repression is the result of a physical interaction between Sut1p and Cyc8p. Moreover, genetic data suggest that complete derepression of the reporter gene requires a functional Cyc8p. In addition, we show that Sut1p is involved in the induction of hypoxic gene transcription when the cells are shifted from aerobiosis to anaerobiosis.

Toxoplasma gondii: purification and characterization of an immunogenic metallopeptidase.

A Toxoplasma gondii aminopeptidase specific for the fluorogenic substrate L-arginine 7-amino-4-methylcoumarin was identified in cell-free extract. This enzyme was purified by high-performance liquid chromatography using first size exclusion, then anion exchange, followed by a second size exclusion. The purified enzyme exhibited a pl of 4.7 by chromatofocusing and had an apparent molecular weight of 110 kDa, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. The purification factor was 80.9 and the yield was 14%. The optimal activity was at pH 7.4 and was strongly inhibited by EDTA and o-phenanthroline. Antibodies against this T. gondii metallopeptidase were detected by immunoprecipitation and immunoblotting in human sera obtained from patients undergoing toxoplasmosis.

A Candida albicans metallopeptidase degrades constitutive proteins of extracellular matrix.

Among potential virulence factors of Candida albicans, enzymes seem to play an important role. Many studies concern the secreted aspartic proteinases (saps), and the degradation of some components of the subendothelial extracellular matrix by the isoenzyme sap2 has been proved. Nevertheless, other proteolytic enzymes could be involved in the pathogenicity of the yeast. We studied the degradation of four constitutive proteins of the extracellular matrix: type I and IV collagens, laminin and fibronectin, by a 95-kDa metallopeptidase, localised in the cell wall of C. albicans. Each of these constituents was incubated with the purified enzyme and its degradation products analysed by an electrophoretic method. We observed that type I collagen and fibronectin were totally degraded by the enzyme whereas type IV collagen and laminin were only partially degraded. The C. albicans metallopeptidase may play a role in the degradation of the subendothelial extracellular matrix components. This enzyme could facilitate the migration of the yeast in the tissues after crossing the endothelial layer, allowing the fungal invasion of target organs.

Association of a myosin immunoanalogue with cell envelopes of Aspergillus fumigatus conidia and its participation in swelling and germination.

A myosin immunoanalogue was identified in conidia of Aspergillus fumigatus by Western blotting, indirect immunofluorescence assay, and gold immunoelectron microscopy with two different antimyosin antibodies. The distribution pattern of this protein was followed during the early stages of germination. A single 180-kDa polypeptide, detected predominantly in a cell envelope extract, was found to cross-react with monoclonal and polyclonal antibodies raised against vertebrate muscle myosin. Immunoelectron microscopy permitted precise localization of this polypeptide, indicating that myosin analogue was mainly distributed along the plasma membrane of resting and swollen conidia. In germinating conidia, indirect immunofluorescence microscopy revealed myosin analogue at the periphery of germ tubes, whereas actin appeared as dispersed punctate structures in the cytoplasm that were more concentrated at the site of germ tube emergence. A myosin ATPase inhibitor, butanedione monoxime, greatly reduced swelling and blocked germination. In contrast, when conidia were treated with cytochalasin B, an inhibitor of actin polymerization, swelling was not affected and germination was only partially reduced. Butanedione monoxime-treated conidia showed accumulation of cytoplasmic vesicles and did not achieve cell wall reorganization, unlike swollen conidia. Collectively, these results suggest an essential role for this myosin analogue in the deposition of cell wall components during germination of A. fumigatus conidia and therefore in host tissue colonization.

Improved immunodiagnosis of human candidiasis by an enzyme-linked immunosorbent assay using a Candida albicans 52-kilodalton metallopeptidase.

An immunogenic aminopeptidase of Candida albicans was purified by high-performance liquid chromatography. It was then used for the development of an enzyme-linked immunosorbent assay to detect antibodies directed against this antigen in sera from patients with candidiasis. This enzyme specifically cleaves the L-Arg-7-amino-4-methyl-coumarin substrate at pH 7.4 and was detected in the crude extract of different C. albicans isolates. Sera used for this study were obtained from healthy blood donors or from patients with one of the following: systemic candidiasis, aspergillosis, cryptococcosis, toxoplasmosis, or malaria. The statistical analysis demonstrates significant differences between absorbency values obtained with sera from patients with candidiasis and with sera from the other groups (P = 0.000001). Diagnostic parameters show high diagnostic specificity of 97% and a sensitivity of 83% at a cutoff value of 0.425 and suggest the usefulness of this aminopeptidase for the diagnosis of systemic candidiasis.

Purification and characterization of N-acetylglucosaminidase from Trypanosoma cruzi.

N-Acetyl-beta-D-glucosaminidase activity was recovered in cell-free extracts of Trypanosoma cruzi epimastigotes. This enzyme was identified on the basis of its ability to hydrolyze the fluorogenic substrate 4-methylumbelliferyl-N-acetyl-beta-D-glucosaminide. This activity was purified to apparent homogeneity by anion exchange and molecular sieve high-performance liquid chromatography. It eluted at a native molecular weight of approximately 48,000 Da and migrated as a single band upon reducing or nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The optimum pH of the activity was around pH 5.4 and the enzyme gave a single peak of activity on a chromatofocusing column with an isoelectric point of 4.2. The enzyme hydrolyzed 4-methylumbelliferyl-GlcNAc, suggesting that it should be characterized as a N-acetyl-beta-D glucosaminidase, with a K(m) value of 1.5 mM from Lineweaver-Burk plots. Many inhibitors as potential enzyme effectors were investigated.

Purification and characterisation of a metallopeptidase of Candida albicans.

A novel aminopeptidase was purified by high performance liquid chromatography from a cytosoluble 100,000 g extract of Candida albicans on the basis of its ability to cleave L-arginine 7-amino-4-methylcoumarin. The purification factor was 36 and the yield was 20%. The native enzyme had a mol. wt of 52 kDa as demonstrated by SDS-PAGE in the presence or absence of reducing conditions and exhibited an iso-electric point of 4.3. The aminopeptidase showed optimum activity at pH 7.2, a Michaelis constant of c. 50 microM and a Vmax at 19 mM AMC released/min/mg of protein for L-Arg-AMC. This enzyme was shown to cleave at low affinity L-leucine-7-amino-4-methylcoumarin as demonstrated by the spectrofluorimetric method. The enzyme was strongly inhibited by specific metallo-enzyme inhibitors-EDTA and o-phenanthroline. Furthermore, there is evidence that a similar or identical enzyme occurs in other C. albicans clinical isolates and other Candida spp.

Detection and immunolocalization of human erythrocyte spectrin immunoanalogues in Toxoplasma gondii (Protozoan, Parasite).

We demonstrated here the presence of proteins antigenically related to human erythroid spectrin in the parasitic protozoan Toxoplasma gondii. A high molecular weight doublet (M(r) 245-240,000), present in equimolar ratio, and low molecular weight poly-peptides (M(r) 75,000) were reacted with monoclonal and polyclonal anti-human erythroid spectrin antibodies on electroblotted nitro-cellulose sheets. Indirect immunofluorescence assay clearly showed that these proteins were localized in the anterior pole of the organism. Immunogold staining further revealed specific labeling of conoid, rhoptries, micronemes, and dense granules of the apical complex. The presence of the M(r) 245-240,000 doublet and the M(r) 75,000 spectrin-like proteins in the anterior pole of T. gondii may probably be consistent with a structural stabilizer function in its organelles which are suspected to be involved in the process of host cell invasion.

Detection of myosin immunoanalogue in the yeast Candida albicans.

Detection and localization of myosin immunoanalogue protein in the yeast Candida albicans were achieved by immunoblotting, indirect immunofluorescence assay, and immunoelectron microscopy. A polypeptide with an M(r) about 110,000, from cytosolic extract and insoluble fraction in the corresponding membrane pellet, was reacted with polyclonal and monoclonal antibodies raised against vertebrate muscle myosin. This protein was located by immunofluorescence and immunoelectron microscopy in the cell cortex along the plasmalemma, in the cytoplasm, and in the septum corresponding to bud scar region situated between the yeast-mother cell and the bud.

N-acetylglucosamine-binding proteins on Plasmodium falciparum merozoite surface.

Plasmodium falciparum merozoite surface is specifically labelled with a neoglycoprotein bearing N-acetylglucosamine (GlcNAc) residues in a sugar-dependent manner, as shown by affinity cytochemistry in fluorescence and electron microscopy. To ascertain the nature of the sugar receptor, merozoite proteins were blotted and tested by a two-step method using biotinylated GlcNAc-bovine serum albumin (BSA) and streptavidin-peroxidase conjugate. Three parasite proteins were specifically revealed and designated as Pf 120, Pf 83 and Pf 45 GlcNAc-binding proteins. These proteins bind to a gel substituted with GlcNAc and are specifically eluted with 300 mM GlcNAc. Using a rabbit antiserum raised against Pf 83, the Pf 120 GlcNAc-binding protein, in addition to Pf 83, was labelled by Western blotting. Comparative analyses with an antibody against the Pf 83 MSP derived from the P. falciparum merozoite surface protein (Pf MSP) indicated that the Pf 83 GlcNAc-binding protein is not related to the fragment of the Pf MSP antigen. Similarly, the Pf 83 GlcNAc-binding protein is not related to the apical membrane antigen 1 (AMA 1) which also has the same molecular mass. Therefore the Pf 120, Pf 83 and Pf 45 GlcNAc-binding proteins which are located on the merozoite surface and recognize GlcNAc residues could be involved in the binding of merozoites to the glycoconjugates of the surface of the red blood cells.