A second gene for a secreted aspartate proteinase in Candida albicans

Abstract

The gene (PRAII) encoding a secreted aspartate proteinase of Candida albicans has been cloned and sequenced. The nucleotide and deduced amino acid sequences ofPRAII are 77 and 73% identical, respectively, with the reported sequences of PRAIO also cloned from C. albicans. Southern analyses indicated that the genome of each strain examined (ATCC 10231 and ATCC 10261) contains PRAIO and PRAII. Northern (RNA) analyses showed that PRAII was expressed at a much higher level than was PRA10 when secretion of the proteinase by strain ATCC 10261 was induced with albumin.The opportunistic pathogen Candida albicans secretes an aspartate proteinase (EC 3.4.23.6) Enzyme purification and protein analysis. Ten-liter cultures of strain ATCC 10261 were grown in a medium containing 0.2% yeast extract, 0.2% bovine serum albumin, and 2% (wt/vol) glucose (12 h at 30°C with aeration at 10 liters min-1 and a speed of 400 rpm). After cell harvesting, the medium was adjusted from pH 3.0 to pH 7.0 with 6 M NaOH and concentrated to 400 to 500 ml by ultrafiltration (10,000 molecular weight cutoff). After dialysis for 12 h against 10 mM sodium citrate buffer (pH 6.8), the concentrate was applied to a DEAE-Sephacel column (26 by 2.5 cm) equilibrated with the same buffer, and the column was washed

Analysis of secreted aspartic proteinases from Candida albicans: purification and characterization of individual Sap1, Sap2 and Sap3 isoenzymes

“…Sullivan, unpublished). An earlier report by Wright et al (1992) had indicated some expression of Sapl in 10261 but the full-length probe used in that experiment would have allowed cross-hybridization to occur. It is clear then that evidence from both mRNA and secreted protein levels shows that Sap2 is the major isoenzyme expressed and that strain 10231 is a significantly greater producer of Sap3 than is 10261 under the conditions used.…”

“…It is based o n the well-known ability of aspartic proteinases, with their generally low isoelectric points, to bind to anionexchange resins, and was first used by Remold et al (1968) for Candida Sap. Two such steps have been employed in this study: the 'traditional' DEAE chromatography (in this case as per Riichel, 1981 and Wright et al, 1992) followed by a much faster and higher resolution separation with Mono Q (strong anion exchanger). However, it was not necessary to include the DEAE step as can be seen in Fig.…”

“…It was subsequently characterized further by Ruchel (1981) and since then a number of reports on the purification, activity and substrate specificity of this enzyme have appeared (Negi et al, 1984;Kaminishi et al, 1986;Ray & Payne, 1990;Bannerjee et al, 1991;Yamamoto et al, 1992;White et al, 1993;Morrison et al, 1993a, b ;Fusek et al, 1994;Tsushima et al, 1994). The SAP2 gene itelf was cloned and sequenced by Wright et al (1992), being the second gene in the family to be identified. Most recently, the crystal structure of Sap2 from two different strains of C. albicans was determined (Cutfield et al, 1995;Abad-Zapatero et al, 1996), revealing details of the binding site for peptide-based G. SMOLENSKI a n d OTHERS inhibitors.…”

Detection ofCandida albicansmRNA in Archival Histopathology Samples by Reverse Transcription-PCR

“…Six C. albicans genes were selected for the study: the multiple-copy-number 18S rRNA gene, the actin (ACT1) gene (10), the EFB1 gene (11), the secreted aspartic proteinase 2 (SAP2) gene (26), and the alcohol dehydrogenase (Adh) 1 and 2 (ADH1 and ADH2) genes (2,24). PCR primers for detection of cDNA (Table 1) were designed to amplify PCR products smaller than 200 bp because of the reported degradation and chemical modification of RNA recovered from FFPE samples (12).…”

Design of a Novel Antimicrobial Peptide Activated by Virulent Proteases

“…Although S. cerevisiae has several extracellular proteases (35), the proteolytic activity of C. albicans is significantly greater than that of S. cerevisiae because of the Sap family. In vitro, C. albicans can grow using proteins as a sole nitrogen source, whereas in vivo it survives in the blood stream utilizing serum albumin (36). In addition, the specific linker of our peptide was optimized for Sap isozymes.…”