The predicted secretome of the opportunistic fungal pathogen Candida albicans contains more than 200 ORFs of diverse and often unknown function. Majority of the secreted proteins that have been experimentally evaluated to date are hydrolytic enzymes (proteinases, phospholipases and lipases). Acting on the interface between the pathogen and the host, the secreted hydrolases may enable invasion of host tissues, help the pathogen to avoid host defense mechanisms, or allow the microorganism to utilize host cell macromolecules as a source of nutrients. Aspartic proteinases constitute the best-characterized family of the C. albicans secreted proteins. Number of studies addressed also secreted phospholipases. Lipases and N-acetylhexosaminidase have received less attention thus far. Many ORFs that are predicted to encode secreted proteins await characterization.

The yeasts of the genus Candida are opportunistic pathogens associated with the rising incidence of life-threatening infections in immunocompromised individuals. Secretion of aspartic proteinases has been determined to be one of the virulence factors of the pathogenic Candida species. To analyze the extracellular proteolytic activities of a large number of Candida clinical isolates, we developed a screening system based on a solid medium containing hemoglobin as the sole nitrogen source. The cleavage of hemoglobin by the secreted proteinases results in formation of clearance zones. The visibility of such zones was enhanced by addition of an acid-base indicator. Using this system, we assessed 245 clinical isolates of Candida from patients in the hospital of the Faculty of Medicine, Palacky University, Olomouc, Czech Republic, for the presence of secreted aspartic proteases (Saps). We also used the test plates for rapid semiquantitative testing of Sap inhibitors. Most of the pepstatin analogs affected the formation of the zones of clearance as well as the growth of Candida albicans , C. tropicalis , and C. parapsilosis colonies. By contrast, the human immunodeficiency virus proteinase inhibitors saquinavir, ritonavir, nelfinavir, and indinavir had no effect on the Candida strains tested. These results are in agreement with the inhibition constants obtained for the individual inhibitors with purified Saps. Thus, the plates containing hemoglobin proved to be an appropriate tool for the rapid and reliable assessment of Sap production and inhibition.

The human fungal pathogen Candida parapsilosis possesses at least three genes encoding secreted aspartic proteinases. Whereas the Sapp1p isoenzyme ha

Numerous Δ12-, Δ15- and multifunctional membrane fatty acid desaturases (FADs) have been identified in fungi, revealing great variability in the enzymatic specificities of FADs involved in biosynthesis of polyunsaturated fatty acids (PUFAs). Here, we report gene isolation and characterization of novel Δ12/Δ15- and Δ15-FADs named CpFad2 and CpFad3, respectively, from the opportunistic pathogenic yeast Candida parapsilosis. Overexpression of CpFad3 in Saccharomyces cerevisiae strains supplemented with linoleic acid (Δ9,Δ12-18:2) and hexadecadienoic acid (Δ9,Δ12-16:2) leads to accumulation of Δ15-PUFAs, i.e., α-linolenic acid (Δ9,Δ12,Δ15-18:3) and hexadecatrienoic acid with an unusual terminal double bond (Δ9,Δ12,Δ15-16:3). CpFad2 produces a range of Δ12- and Δ15-PUFAs. The major products of CpFad2 are linoleic and hexadecadienoic acid (Δ9,Δ12-16:2), accompanied by α-linolenic acid and hexadecatrienoic acid (Δ9,Δ12,Δ15-16:3). Using GC/MS analysis of trimethylsilyl derivatives, we identified ricinoleic acid (12-hydroxy-9-octadecenoic acid) as an additional product of CpFad2. These results demonstrate that CpFAD2 is a multifunctional FAD and indicate that detailed analysis of fatty acid derivatives might uncover a range of enzymatic selectivities in other Δ12-FADs from budding yeasts (Ascomycota: Saccharomycotina).

Secreted aspartic proteinases (Sap) play a role in the virulence of pathogenic Candida spp. Candida parapsilosis possesses three genes encoding these enzymes: SAPP1, SAPP2, and SAPP3. We analyzed the expression of the SAPP1 and SAPP2 genes and the production of Sapp1p and Sapp2p proteinases in the presence of different nitrogen sources. While the SAPP2 transcript was present under all of the conditions tested, expression of SAPP1 was induced only by the presence of exogenous protein as the sole nitrogen source. The concentration of Sapp1p in the medium upon induction was at least one order of magnitude higher than the concentration of Sapp2p in all media tested in this study. Enzymological characterization of purified Sapp1p and Sapp2p demonstrated that Sapp2p has a more restricted substrate specificity and significantly lower catalytic activity than Sapp1p. Homology models of Sapp1p and Sapp2p revealed structural motifs that may be responsible for the differences between these two enzymes. Our results indicate that C. parapsilosis secretes a low level of Sapp2p proteinase with narrow substrate specificity and low proteolytic activity under most conditions, while expression and secretion of a higher amount of catalytically efficient Sapp1p enzymes is triggered in the presence of exogenous protein serving as a nitrogen source.

Opportunistic pathogens of the genus Candida produce secreted aspartic proteinases (Saps) that play an important role in virulence. Saps are synthesized as zymogens, but cell-free culture supernatants of Candida spp. contain only mature Saps. To study the zymogen conversion, the gene encoding a precursor of C. parapsilosis proteinase Sapp1p was cloned, expressed in E. coli and the product was purified. When placed in acidic conditions, the precursor was autocatalytically processed, yielding an active proteinase. The self-activation proceeded through an intermediate product and the resulting enzyme was one amino acid shorter than the authentic enzyme. This truncation did not cause changes in proteinase activity or secondary structure compared to the authentic Sapp1p. Accurate cleavage of the pro-mature junction, however, required a processing proteinase. A crude membrane fraction prepared from C. parapsilosis cells contained an enzyme with Kex2-like activity, which processed the Sapp1p precursor at the expected site. The pro-segment appeared to be indispensable for Sapp1p to attain an appropriate structure. When expressed without the pro-segment, the Sapp1p mature domain was not active and had a lower content of alpha-helical conformation, as measured by circular dichroism. A similar effect was observed when a His(6)-tag was linked to the C-terminus of Sapp1p or its precursor.