title: Simple method for screening Candida species isolates for the presence of secreted proteinases: a tool for the prediction of successful inhibitory treatment.
abstract: 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.
authors.affiliation: Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic.
authors.forename: Jií
authors.initials: J
authors.lastname: Dostál
authors.forename: Petr
authors.initials: P
authors.lastname: Hamal
authors.forename: Libuse
authors.initials: L
authors.lastname: Pavlícková
authors.forename: Milan
authors.initials: M
authors.lastname: Soucek
authors.forename: Tomás
authors.initials: T
authors.lastname: Ruml
authors.forename: Iva
authors.initials: I
authors.lastname: Pichová
authors.forename: Olga
authors.initials: O
authors.lastname: Hrusková-Heidingsfeldová
mesh_terms: D002175:Candida / Q000201:enzymology / Q000302:isolation & purification*; D003470:Culture Media; D010450:Endopeptidases / Q000032:analysis* / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008828:Microbiological Techniques / Q000379:methods*; D010447:Peptide Hydrolases / Q000378:metabolism
chemical_list: D003470:Culture Media; D010450:Endopeptidases; D010447:Peptide Hydrolases