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.

Institute of Organic Chemistry and Biochemistry Academy of Sciences of the Czech Republic

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Bodanszky, M., and A. Bodanszky. 1984. The practice of peptide synthesis. Springer-Verlag, Berlin, Germany.

Brožková, K., I. Křížová, L. Pavlíčková, M. Hradilek, M. Fusek, T. Ruml, M. Souček, and I. Pichová. 1999. Peptidomimetic inhibitors of extracellular aspartic proteinases of Candida albicans and Candida tropicalis. Collect. Czech. Chem. Commun. 64:130-137.

Capobianco, J. O., C. G. Lerner, and R. C. Goldman. 1992. Application of a fluorogenic substrate in the assay of proteolytic activity and in the discovery of a potent inhibitor of Candida albicans aspartic proteinase. Anal. Biochem. 204:96-102. PubMed

Caugant, D. A., and P. Sandven. 1993. Epidemiological analysis of Candida albicans strains by multilocus enzyme electrophoresis. J. Clin. Microbiol. 31:215-220. PubMed PMC

Dan, M., F. Poch, and D. Levin. 2002. High rate of vaginal infection caused by non-C. albicans Candida species among asymptomatic women. Med. Mycol. 40:383-386. PubMed

Dean, D. A., and K. W. Burchard. 1996. Fungal infection in surgical patients. Am. J. Surg. 171:374-382. PubMed

De Viragh, P. A., D. Sanglard, G. Togni, R. Falchetto, and M. Monod. 1993. Cloning and sequencing of two Candida parapsilosis genes encoding acid proteases. J. Gen. Microbiol. 139:335-342. PubMed

Fidel, P. L., Jr., J. A. Vazquez, and J. D. Sobel. 1999. Candida glabrata: review of epidemiology, pathogenesis and clinical disease with comparison to C. albicans. Clin. Microbiol. Rev. 12:80-96. PubMed PMC

Fusek, M., E. A. Smith, M. Monod, B. Dunn, and S. I. Foundling. 1994. Extracellular aspartic proteinases from Candida albicans, Candida tropicalis and Candida parapsilosis yeast differ substantially in their specificities. Biochemistry 33:9791-9799. PubMed

Gilfillan, G. D., D. J. Sullivan, K. Haynes, T. Parkinson, D. C. Coleman, and N. A. R. Gow. 1998. Candida dubliniensis: phylogeny and putative virulence factors. Microbiology 144:829-838. PubMed

Haynes, K. 2001. Virulence in Candida species. Trends Microbiol. 9:591-596. PubMed

Hrušková-Heidingsfeldová, O., J. Dostál, P. Hamal, J. Pazlarová, T. Ruml, and I. Pichová. 2001. Enzymological characterization of secreted proteinases from Candida parapsilosis and Candida lusitaniae. Collect. Czech. Chem. Commun. 66:1707-1719.

Hube, B., and J. Naglik. 2001. Candida albicans proteinases: resolving the mystery of a gene family. Microbiology 147:1997-2005. PubMed

Koelsch, G., J. Tang, J. A. Loy, M. Monod, K. Jackson, S. I. Foundling, and X. Lin. 2000. Enzymic characteristic of secreted aspartic proteases of Candida albicans. Biochim. Biophys. Acta 1480:117-131. PubMed

Lerner, C. G., and R. C. Goldman. 1993. Stimuli that induce production of Candida albicans extracellular aspartyl proteinase. J. Gen. Microbiol. 139:1643-1651. PubMed

Monod, M., G. Togni, B. Hube, and D. Sanglard. 1994. Multiplicity of genes encoding secreted aspartic proteinases in Candida species. Mol. Microbiol. 13:357-368. PubMed

Odds, F. C., and A. B. Abbott. 1980. A simple system for the presumptive identification of Candida albicans and differentiation of strains within the species. Sabouraudia 18:301-317. PubMed

Pfaller, M., R. N. Jones, G. V. Doern, H. S. Sader, R. J. Hollis, and S. A. Messer for the SENTRY Participant Group. 1998. International surveillance of bloodstream infections due to Candida species: frequency of occurrence and antifungal susceptibilities of isolates collected in 1997 in the United States, Canada, and South America for the SENTRY Program. J. Clin. Microbiol. 36:1886-1889. PubMed PMC

Pichová, I., L. Pavlíčková, J. Dostál, E. Dolejší, O. Hrušková-Heidingsfeldová, J. Weber, T. Ruml, and M. Souček. 2001. Secreted aspartic proteinases of Candida albicans, Candida tropicalis, Candida parapsilosis and Candida lusitaniae: inhibition with peptidomimetic inhibitors. Eur. J. Biochem. 268:2669-2677. PubMed

Ray, T. L., and C. D. Payne. 1990. Comparative production and rapid purification of Candida acid proteinase from protein-supplemented cultures. Infect. Immun. 58:508-514. PubMed PMC

Ruchel, R., F. De Bernardis, T. L. Ray, P. A. Sullivan, and T. G. Cole. 1992. Candida acid proteinases. J. Med. Vet. Mycol. 30:123-132. PubMed

Ruchel, R. 1981. Properties of a purified proteinase from the yeast Candida albicans. Biochim. Biophys. Acta 659:99-113. PubMed

Ruchel, R., K. Uhlemann, and B. Boning. 1983. Secretion of aspartic proteinases by different species of the genus Candida. Zentbl. Bacteriol. Microbiol. Hyg. 255:537-548. PubMed

Safdar, A., V. Chaturvedi, E. W. Cross, S. Park, E. M. Bernard, D. Armstrong, and D. S. Perlin. 2001. Prospective study of Candida species in patients at a comprehensive cancer center. Antimicrob. Agents Chemother. 45:2129-2133. PubMed PMC

Saiman, L., E. Ludington, M. Pfaller, S. Rangel-Frausto, R. T. Wiblin, J. Dawson, H. M. Blumberg, J. E. Patterson, M. Rinaldi, J. E. Edwards, R. P. Wenzel, and W. Jarvis. 2000. Risk factors for candidemia in neonatal intensive care unit patients. Pediatr. Infect. Dis. J. 19:319-324. PubMed

Smolenski, G., P. A. Sullivan, S. M. Cutfield, and J. F. Cutfield. 1997. Analysis of secreted aspartic proteinases from Candida albicans: purification and characterization of individual Sap1, Sap2 and Sap3 isoenzymes. Microbiology 143:349-356. PubMed

Stewart, K., and C. Abad-Zapatero. 2001. Candida proteases and their inhibition: prospects for antifungal therapy. Curr. Med. Chem. 8:941-948. PubMed

Verduyn Lunel, F. M., J. F. Meis, and A. Voss. 1999. Nosocomial fungal infections: candidemia. Diagn. Microbiol. Infect. Dis. 34:213-220. PubMed

Wu, T., L. P. Samaranayake, W. K. Leung, and P. A. Sullivan. 1999. Inhibition of growth and secreted aspartyl proteinase production in Candida albicans by lysozyme. J. Med. Microbiol. 48:721-730. PubMed

Zaugg, C., M. Borg-von Zepelin, U. Reichard, D. Sanglard, and M. Monod. 2001. Secreted aspartic proteinase family of Candida tropicalis. Infect. Immun. 69:405-412. PubMed PMC

Distribution of secreted aspartyl proteinases using a polymerase chain reaction assay with SAP specific primers in Candida albicans isolates

Secreted aspartate proteinases, a virulence factor of Candida spp.: occurrence among clinical isolates