The respective control animals received 100 l of PBS, 1 : 1 PBS/alum suspension, or DMSO. Passive immunization was performed by i.p. which found that Sap-deficient mutants are less virulent than parental strains8,9 and that protease inhibitors reduce virulence.10,11 Moreover, isolates obtained from immunocompromised hosts expressed higher levels of Sap activity than those obtained from control patients.12,13 Finally, Saps have been shown to break down several host substrates and to participate in host tissue damage.14,15 Saps are encoded by a multigene family encompassing at least 10 genes16,17 that are differentially regulated at various stages of infection.18 Analyses of secreted Saps composition in cultures of different strains revealed that Sap2 is the most abundant19 and preferentially expressed at the late stages of infection extracts enriched in Sap2 have been successfully used as immunogens to reduce mucosal candidasis in mouse or rat models.22C24 In this work we directly tested whether Sap2 vaccination can protect BALB/c mice from systemic candidiasis. We first provide evidence that Sap2 may account for the B-cell polyclonal activation induced by colonization. Moreover, injection of either native or recombinant Sap2, in the absence of adjuvant, induced a detectable specific AAPK-25 immune response that AAPK-25 was associated with reduced load upon contamination. Materials and methods MiceMale BALB/c and C57BL/6 mice (6C8 weeks old) were purchased from Charles River (Barcelona, Spain). Animals were housed at the animal facilities of the Institute Abel Salazar during the time of the experiments. C. albicansin C57BL/6 mice every 3 months. C. albicans-was grown in Winge medium (03% yeast extract, 02% glucose) for 48 hr at 37 in an orbital incubator. Culture supernatant proteins were concentrated by ultrafiltration with a 10 000-MW cut-off membrane in a VivaFlow system (Vivascience, Hanover, Germany), dialysed against Bistris 20 mm buffer, pH 60, and separated by ion-exchange chromatrography on a DEAE-cellulose (DE52; Whatman, Maidstone, Kent, UK) column that was eluted with a 0C03 m NaCl gradient. Fractions eluted in 005C015 m NaCl (F005C015) were found to be enriched in Sap2 and were concentrated by vacuum dialysis using a 14 000-MW cut-off membrane (Sigma, St Louis, MO). Mannoside constituents were removed from this fraction by affinity chromatography in a concanavalin ACsepharose column (Amersham Pharmacia, Uppsala, Sweden) using a buffer Rat monoclonal to CD4.The 4AM15 monoclonal reacts with the mouse CD4 molecule, a 55 kDa cell surface receptor. It is a member of the lg superfamily,primarily expressed on most thymocytes, a subset of T cells, and weakly on macrophages and dendritic cells. It acts as a coreceptor with the TCR during T cell activation and thymic differentiation by binding MHC classII and associating with the protein tyrosine kinase, lck of 20 mm Tris/05 m NaCl. Sap2 was further purified by a Pepstatin A affinity-chromatography column (Sigma), as described previously.25 As a last purification step, Sap2 preparations were depleted of contaminating endotoxin using a polymixin B column (Pierce, Rockford, IL), and tested by the limulus test (E-toxate; Sigma). All Sap2 preparations used in this study tested endotoxin free. Cloning of recombinant Sap2 (rSap2)The full-length mature Sap2-coding sequence was cloned by nested polymerase chain reaction (PCR) using genomic DNA as a template. Genomic DNA was prepared as described previously. 26 The external and internal primer-pairs were 5-GTTGATTCCTCTTGGTTGTTGA-3, 5-TTTATTCCACCCCTTCATCTTA-3 and 5-GTAAAACTCTCGAGAGACAAGC-3, 5-TTTATTCCACGAATTCATCTTA-3, respectively. The first PCR reaction, made up of 100 ng of template, 15 m MgCl2, 02 mm each dNTP (all from Invitrogen, Life Technologies, CA) and 50 pm each primer, in a final volume of 50 l, was performed as follows: 94 for 2 min 30 seconds, followed by 30 cycles of 30 seconds at 94, 45 seconds at 53 and 2 min at 72, and terminated by a 10-min incubation at 72. The nested PCR reaction was performed using 1 l of the first PCR product in the same PCR conditions. For both amplifications, the DNA polymerase Expand? High Fidelity PCR system (Roche, Basel, Switzerland) was used. The internal primers have been designed to contain an BL21-CodonPLus (DE3) RIL (Stratagene, La Jolla, CA) and expression of recombinant Sap2 was induced by the addition of 1 mm isopropyl–d-thiogalactopyranoside (IPTG). Production of AAPK-25 rSap2made up of pRSET-Sap2 was grown at 37 in 2 16% tryptone, 1% yeast extract, 05% NaCl (YT) medium, supplemented with 2% glucose and ampicillin, to an optical density of 08 at 600 nm. IPTG was then added to a final concentration of 1 1 mm and the culture maintained for an additional 5 hr. Upon analyses, the Sap2 protein was found to be sequestered.