We investigated here the potential role of Toll-like receptors (TLR) and the adaptor protein MyD88 in innate immunity responses to Cryptococcus neoformans, a pathogenic encapsulated yeast. Peritoneal macrophages from MyD88 -/-or TLR2 -/-mice released significantly less TNF-a, compared with wild-type controls, after in vitro stimulation with whole yeasts. In contrast, no differences in TNF-a release were noted between macrophages from C3H/HeJ mice, which have a loss of function mutation in TLR4, relative to C3H/HeN controls. When MyD88-or TLR2-deficient mice were infected with low doses of the H99 serotype A strain, all of the control animals, but none of MyD88 -/-and only 38% of the TLR2 -/-animals survived, in association with higher fungal burden in the mutant mice. Both MyD88 -/-and TLR2 -/-animals showed decreased TNF-a, IL12p40 and/or IFN-c expression in various organs during infection. No difference in susceptibility to experimental cryptococcosis was found between C3H/HeJ mice and C3H/HeN controls. In conclusion, our data indicate that TLR2 and MyD88, but not TLR4, critically contribute to anti-cryptococcal defenses through the induction of increased TNF-a, IL-12 and IFN-c expression.

Host defenses against the encapsulated yeast Cryptococcus neoformans involve both humoral and cellmediated immunity. Mannoproteins (MPs) are a heterogeneous class of immunodominant glycoproteins which have been only incompletely characterized. In this study, we report on the molecular features of two novel MPs that are recognized by serum antibodies during cryptococcosis. After fractionation of extracellular cryptococcal products, MPs reacted more strongly than other components with sera from C. neoformans-infected AIDS patients. Further fractionation and Western blot analysis of MPs evidenced the presence of highly reactive bands with molecular masses of 250, 125, 115, and 84 kDa. The 115-and 84-kDa bands contained significant amounts of N-linked oligosaccharides, as shown by decreased molecular mass after peptide-N-glycosidase F treatment. N-terminal amino acid sequences of the two bands were used to search C. neoformans nucleotide databases. Homologous genomic sequences were used to synthesize DNA probes and isolate cDNA clones containing the full-length genes, which were designated MP84 and MP115. Both genes showed the presence of a serine/threonine-rich region, a potential site for heavy glycosylation. MP84 and MP115 showed homology with, respectively, polysaccharide deacetylases and carboxylesterases from other organisms. Recombinant, deglycosylated proteins expressed in Escherichia coli still reacted with sera from patients, albeit more weakly than natural MPs, indicating that at least some of the reactive epitopes were retained in the recombinant forms. In conclusion, we identified two novel MPs that are important targets of antibody responses during cryptococcosis. These data may be useful to devise alternative immunity-based strategies to control the disease.

No 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...

The characterization of proteins secreted by Cryptococcus neoformans is of relevance to the identification of vaccine candidates, because concentrated supernatants from the fungus have been shown to be immunoprotective in previous studies. After fractionation of supernatants by anion exchange chromatography and preparative electrophoresis, we obtained the N-terminal amino acid sequences of 13 major proteins. Using a C. neoformans nucleotide database, we were able to clone and sequence the ORFs coding for 12 of these proteins. Some of the genes are identical to previously described ones, while six encode novel proteins, including four putative mannoproteins. The molecular characterization of these and other secreted products may provide useful information in the development of immune-based strategies to control cryptococcosis.