Identification and cloning of a cryptococcal deacetylase that produces protective immune responses

Abstract

Cell-mediated immunity plays a crucial role in host defenses against Cryptococcus (Filobasidiella) neoformans. Therefore, the identification of cryptococcal antigens capable of producing T-cell-mediated responses, such as delayed-type hypersensitivity (DTH) reactions, may be useful in the development of immune-based strategies to control cryptococcosis. In order to characterize DTH-producing antigens, culture supernatants from the unencapsulated Cap-67 strain were separated by anion-exchange chromatography. After further fractionation by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis, a purified protein with an apparent molecular mass of 25 kDa was found to produce DTH, as evidenced by increased footpad swelling in mice immunized with culture supernatants, relative to unimmunized mice. The 20-amino-acid N-terminal sequence of the 25-kDa protein was used to search data of the C. neoformans Genome Project. Based on the genomic DNA sequence, a DNA probe was used to screen a lambda cDNA library prepared from strain B3501. Clones were isolated containing the full-length gene (d25), which showed homology with a number of polysaccharide deacetylases from fungi and bacteria. The recombinant d25 protein expressed in Escherichia coli was similar to the natural one in DTH-producing activity. Moreover, immunization with either the natural or the recombinant protein prolonged survival and decreased fungal burden in mice challenged with the highly virulent C. neoformans strain H99. In conclusion, we have described the first cryptococcal gene whose product, a 25-kDa extracellular polysaccharide deacetylase, has been shown to induce protective immunity responses.

FIG. 1.

Induction of DTH by cryptococcal supernatants (sups). Mice were immunized with 280 μg of lyophilized supernatants from the A9759 C. neoformans strain (A9759 sups) (A) or with B. dermatitidis (10⁷ cells) (B). Control nonimmunized mice received PBS only. Seven days later, mice were challenged in the right hind pad by the inoculation of A9759 or Cap-67 supernatants (35 μg) or B. dermatitidis (5 × 10⁴ cells). Results indicate differences in weight between the right hind pad (challenged with the test preparation) and the left one (inoculated with vehicle only). Data represent means ± standard deviations of six observations conducted during three separate experiments. ∗, P < 0.05, relative to nonimmunized mice, by one-way analysis of variance and the Student-Keuls-Newman test.

FIG. 2.

Induction of DTH by different anion-exchange chromatography fractions. Mice were immunized with 280 μg of concentrated supernatants from the A9759 strain (A9759). Control nonimmunized mice received PBS only. Seven days later, mice were challenged in the right hind pad by the inoculation of 10 μg (protein content) of peak 1, 2, or 3 preparations. Twenty-four hours after challenge, mice were sacrificed and hind pad weights were determined. Results indicate differences in weight between the right hind pad (challenged with the test preparation) and the left one (inoculated with vehicle only). Data represent means ± standard deviations of six observations conducted during three separate experiments. ∗, P < 0.05, relative to nonimmunized mice, by one-way analysis of variance and the Student-Keuls-Newman test.

FIG. 3.

DTH induction by preparative SDS-PAGE fractions. Mice were immunized or injected with PBS as indicated in the legend to Fig. 2. Footpad testing was done with 10 μg of the designated antigen fractions. Data represent means ± standard deviations of six observations conducted during three separate experiments. ∗, P < 0.05, relative to nonimmunized mice, by one-way analysis of variance and the Student-Keuls-Newman test.

FIG. 4.

SDS-PAGE and Northern blot analysis of the 25-kDa deacetylase. (A) SDS-PAGE analysis of the purified 25-kDa natural protein. (B) d25 Northern blot analysis. The arrow indicates the size of the d25 mRNA band. (C) SDS-PAGE analysis of the purified d25-GST fusion protein. (D) SDS-PAGE analysis of the recombinant d25 protein after cleavage of the GST moiety.

FIG. 5.

Induction of DTH by the 25-kDa protein. Mice were immunized or injected with PBS as indicated in the legend to Fig. 2. Results indicate differences in weight between the right hind pad (challenged with the test preparation) and the left one (inoculated with vehicle only). Data represent means ± standard deviations of six observations conducted in three separate experiments. ∗, P < 0.05, relative to nonimmunized mice, by one-way analysis of variance and the Student-Keuls-Newman test.

FIG. 6.

Complete DNA and deduced amino acid sequence of the d25. The boxed sequence indicates a putative signal peptide, and the bars indicate introns of 55-, 53-, 56-, and 52-bp from the 5′ to 3′ end. White lettering on a black background indicates the N-terminal sequence obtained from the natural protein.

FIG. 7.

Alignment of similar amino acid sequences from d25 and polysaccharide deacetylase domain consensus sequence (PDDCS). White lettering on a black background indicates identity; + indicates conserved amino acid substitutions.

FIG. 8.

Induction of DTH by the d25 recombinant protein. Mice were immunized or injected with PBS as indicated in the legend to Fig. 2. Results indicate differences in weight between the right hind pad (challenged with 10 μg of each test preparation) and the left one (inoculated with vehicle only). Data represent means ± standard deviations of six observations conducted in three separate experiments. ∗, P < 0.05, relative to nonimmunized mice, by one-way analysis of variance and the Student-Keuls-Newman test. sups, supernatants.

FIG. 9.

Mean numbers of cryptococcal CFU in livers, spleens, and lungs from immune and control mice at 3, 5, and 7 days after intravenous challenge with 7.5 × 10³ viable C. neoformans cells. Three mice per group were used for CFU determinations. The data are from one representative experiment of three producing similar results. SD, standard deviations.

FIG. 10.

Percent survival of GST-, 25-kDa-, d25-GST-, and Cap-67-immunized mice and control mice after challenge to the mice with 7.5 × 10³ viable C. neoformans cells. Mice were treated 7 days before challenge with the designated preparations. Ten mice per group were observed over a 30-day period.