Induction of T Helper Type 1 Responses by a Polysaccharide Deacetylase fromCryptococcus neoformans

Abstract

A 25-kDa cryptococcal deacetylase (d25) was found here to induce cell proliferation, as well as secretion of interleukin 2 and gamma interferon, but not interleukin 4, in spleen cells from d25-immunized or Cryptococcus neoformans-infected mice. The gamma interferon, but not the interleukin 2, response was required for the protective activities of d25 immunization in a murine cryptococcosis model.The encapsulated fungus Cryptococcus (Filobasidiella) neoformans is an opportunistic pathogen that often infects patients with immune defects, although cryptococcosis has been reported also in individuals with an intact immune system (12,22). Cryptococcal infections are particularly frequent in AIDS patients, often requiring lifelong suppressive therapy to prevent a relapse (19). Nevertheless, conventional antifungal therapy has toxic side effects, and survivors may progress to fatal disseminated disease despite long-term treatment (24). These observations indicate the need to develop alternative strategies to control cryptococcosis, including active, passive, or adoptive immunotherapy. Type 1 cytokines, such as interleukin 12 (IL-12) and gamma interferon (IFN-␥), are necessary for the development of immune-protective responses (4,7,20). In contrast, type 2 cytokines, such as interleukin 4 (IL-4) and interleukin 5, are not protective and may be responsible for destructive lung pathology (11, 13). While many studies have focused on capsular glucuronoxylomannan, the main virulence factor of C. neoformans, little is known of protein antigens capable of inducing cell-mediated immunity (1, 21). At least four cryptococcal proteins with T-cell-stimulating properties have been identified, and the corresponding genes have been cloned (2,10,15,18). Two of them, with respective molecular masses of 98 and 25 kDa, have a polysaccharide deacetylase domain (2, 15). Immunization with the 25-kDa deacetylase (d25) was shown to protect mice from lethal experimental cryptococcosis (2).The present study was undertaken to assess the ability of d25 to produce T1-type responses, as measured by lymphocyte proliferation and cytokine production, and to assess the role of such responses in the protective activities of d25 immunization. Mice used in this study were housed under specific-pathogenfree conditions in enclosed filter top cages of the Department of Pathology and Experimental Microbiology of the University of Messina (Messina, Italy). The mice were fed clean food and water ad libitum. All of the procedures described in this work were in agreement with the guidelines of the National Institute of Health for handling of laboratory animals. The studies performed here have been approved by relevant national and institutional committees.Recombinant and natural d25 (rd25 and nd25, respectively) were produced and purified as previously described (2). Endotoxin was removed from rd25 preparations using repeated treatments with polymyxin-agarose beads (Bio-Rad Laboratories, Milan, Italy), according to the instructions of the manufacturer. Endotoxin c...

Chitosan, the Deacetylated Form of Chitin, Is Necessary for Cell Wall Integrity in Cryptococcus neoformans

“…Based on the data presented in this report, we are naming three of these genes CDA for chitin deacetylase and one FPD for fungal polysaccharide deacetylase. Two of the putative deacetylases, CDA2 and FPD1, encode proteins named MP98 and D25, respectively, which have been previously described as immunoreactive (3,27). All of the chitin/polysaccharide deacetylase proteins are homologs of both the Cda1p and Cda2p proteins of S. cerevisiae with E values of Ͻe Ϫ13 .…”

“…This result suggests that Fpd1/D25 does not deacetylate chitin during vegetative growth. Although Fpd1/ D25 has been identified as a secreted protein that induces a protective immune response in mice (3,4), it seems unlikely that it would function as a chitin deacetylase during an infection. Since it is secreted, it could potentially deacetylate polysaccharides other than chitin found in its environment.…”

“…Interestingly, one of the C. neoformans chitin/polysaccharide deacetylases, MP98 (encoded by CDA2), was isolated in a screen for cryptococcal antigens that stimulated an immune response in C. neoformans-reactive CD4 ϩ mouse T-cell hybridomas (27) and a second polysaccharide deacetylase, D25 (encoded by FPD1), that induces a protective immune response in mice has also been reported (3). This indicates that at least two of the potential chitin/polysaccharide deacetylases are expressed in vivo and are immunogenic.…”

“…Cda1, Cda2/MP98, and Cda3 are predicted to be GPI anchored, suggesting that they transverse the plasma membrane and/or attach to the cell wall to deacetylate chitin as it is being extruded through the plasma membrane. No GPI anchor is predicted for Fpd1/D25; however, it is an immunoreactive secreted protein (3,4).…”

Fusion of a Novel Genetically Engineered Chitosan Affinity Protein and Green Fluorescent Protein for Specific Detection of Chitosan In Vitro and In Situ

“…Chitin deacetylase was shown to be present at the plasma membrane of the early stages of the life cycle in the human-pathogenic fungus Encephalitozoon cuniculi (9). Cryptococcus neoformans, the causal agent of cryptococcosis in humans, was shown to possess four putative chitin deacetylase genes, and two of its chitin deacetylases have been reported to induce immune responses in the host, indicating that they are expressed during host tissue colonization (3,7,24). Deletion mutants of the chitin deacetylases in this opportunistic fungal pathogen showed that chitosan is important during vegetative growth; it appears to help in maintaining cell integrity and to aid in bud separation (3).…”