Phenotypical properties associated with virulence from clinical isolates belonging to the Candida parapsilosis complex.

The production of virulence attributes in three reference strains and 11 clinical isolates primarily identified as Candida parapsilosis was evaluated. Morphological and phenotypical tests were not able to discriminate among the three species of the C. parapsilosis complex; consequently, molecular methods were applied to solve this task. After employing polymerase chain reaction-based methods, nine clinical strains were identified as C. parapsilosis sensu stricto and two as C. orthopsilosis. Protease, catalase, and hemolysin were produced by all 14 strains, while 92.9% and 78.6% of strains secreted, respectively, esterase and phytase. No phospholipase producers were detected. Mannose/glucose, N-acetylglucosamine, and sialic acid residues were detected at the surface of all strains, respectively, in high, medium, and low levels. All strains presented elevated surface hydrophobicity and similar ability to form biofilm. However, the adhesion to inert substrates and mammalian cells was extremely diverse, showing typical intrastrain variations. Overall, the strains showed (1) predilection to adhere to plastic over glass and the number of pseudohyphae was more prominent than yeasts and (2) the interaction process was slightly enhanced in macrophages than fibroblasts, with the majority of fungal cells detected inside them. Positive/negative correlations were demonstrated among the production of these virulence traits in C. parapsilosis complex.

Surface phosphatase in Rhinocladiella aquaspersa: biochemical properties and its involvement with adhesion.

Rhinocladiella aquaspersa is an etiologic agent of chromoblastomycosis, a subcutaneous chronic infectious disease. In the present work, we found that the three morphological forms of this fungus (conidia, mycelia and sclerotic bodies) expressed different levels of ecto-phosphatase activity. Our results demonstrated that surface conidial enzyme is an acid phosphatase, inhibited by sodium salts of molybdate, orthovanadate and fluoride and that the inhibition caused by orthovanadate and molybdate was irreversible. The conidial ecto-phosphatase efficiently released phosphate groups from different phosphorylated substrates, causing a higher rate of phosphate removal when p-nitrophenylphosphate was used as substrate. This ecto-enzyme of R. aquaspersa is modulated by Co(2 +) ions and inorganic phosphate (Pi). Accordingly, removal of Pi from the culture medium resulted in a marked (121-fold) increase of ecto-phosphatase activity. Surface phosphatase activity is apparently involved in fungal adhesive properties, since the attachment of R. aquaspersa to epithelial cells was reversed by the pre-treatment of the conidia with orthovanadate, molybdate and anti-phosphatase antibody. Corroborating this finding, conidia with greater ecto-phosphatase activity (grown in Pi-depleted medium) showed higher adherence to epithelial cells than fungi cultivated in the presence of Pi.

Multiple effects of pepstatin A on Trypanosoma cruzi epimastigote forms.

Herein, we have aimed to explore the effects of pepstatin A, a powerful aspartic protease inhibitor, on Trypanosoma cruzi, the etiologic agent of Chagas' disease. Pepstatin A arrested the proliferation of epimastigotes of T. cruzi (clone Dm28c, TcI lineage), in both dose- and time-dependent manner. The IC(50) value was calculated to be 36.2 µM after 96 h of parasite-drug contact. The parasite treatment with pepstatin A resulted in significant morphological alterations, including parasites becoming round in shape, reduction (≈25%) of the parasite size, and parasites presenting parts or the whole flagellum detached from the cell body. Cell lysis was not observed, resulting in a trypanostatic effect. The treatment of different T. cruzi strains, belonging to distinct phylogenetic lineages, with pepstatin A at 36.2 µM resulted in growth inhibition as follows: 28% to Y (TcII), 45% to CL Brener (TcII), 45.4% to 4167 (Z3), and 26.4% to 3663 (Z3) strains. The hydrolysis of a cathepsin D fluorogenic substrate (7-methoxycoumarin-4-acetyl-Gly-Lys-Pro-Ile-Leu-Phe-Phe-Arg-Leu-Lys(DNP)-D: -Arg-amide) by T. cruzi epimastigote extract was inhibited (≈65%) by pepstatin A at 10 µM, suggesting that an aspartic protease may be the intracellular target of this inhibitor. Curiously, pepstatin A induced an increase of 54% and 98%, respectively, in the surface expression of gp63- and calpain-related molecules in epimastigotes, but not in the cruzipain level, as well as stimulated the epimastigote-to-trypomastigote differentiation in a dose-dependent manner. However, approximately 45% of the trypomastigotes had their flagellum detached from the cell body. These results contribute to understand the possible role of aspartic proteases in the physiology of T. cruzi cells, adding new in vitro insights into the possibility of exploiting aspartic protease as promising targets to treat Chagas' disease.

Paracoccidioides brasiliensis enolase is a surface protein that binds plasminogen and mediates interaction of yeast forms with host cells.

Paracoccidioidomycosis (PCM), caused by the dimorphic fungus Paracoccidioides brasiliensis, is a disseminated, systemic disorder that involves the lungs and other organs. The ability of the pathogen to interact with host components, including extracellular matrix (ECM) proteins, is essential to further colonization, invasion, and growth. Previously, enolase (EC 4.2.1.11) was characterized as a fibronectin binding protein in P. brasiliensis. Interaction of surface-bound enolase with plasminogen has been incriminated in tissue invasion for pathogenesis in several pathogens. In this paper, enolase was expressed in Escherichia coli as a recombinant glutathione S-transferase (GST) fusion protein (recombinant P. brasiliensis enolase [rPbEno]). The P. brasiliensis native enolase (PbEno) was detected at the fungus surface and cytoplasm by immunofluorescence with an anti-rPbEno antibody. Immobilized purified rPbEno bound plasminogen in a specific, concentration-dependent fashion. Both native enolase and rPbEno activated conversion of plasminogen to plasmin through tissue plasminogen activator. The association between PbEno and plasminogen was lysine dependent. In competition experiments, purified rPbEno, in its soluble form, inhibited plasminogen binding to fixed P. brasiliensis, suggesting that this interaction required surface-localized PbEno. Plasminogen-coated P. brasiliensis yeast cells were capable of degrading purified fibronectin, providing in vitro evidence for the generation of active plasmin on the fungus surface. Exposure of epithelial cells and phagocytes to enolase was associated with an increased expression of surface sites of adhesion. In fact, the association of P. brasiliensis with epithelial cells and phagocytes was increased in the presence of rPbEno. The expression of PbEno was upregulated in yeast cells derived from mouse-infected tissues. These data indicate that surface-associated PbEno may contribute to the pathogenesis of P. brasiliensis.