The Cryptococcus neoformans gene DHA1 encodes an antigen that elicits a delayed-type hypersensitivity reaction in immune mice

• Published in: Infection and immunity Vol. 68; no. 11; pp. 6196 - 6201
• Main Authors: MANDEL, M. Alejandra, GRACE, Greg G, ORSBORN, Kris I, SCHAFER, Fredda, MURPHY, Juneann W, ORBACH, Marc J, GALGIANI, John N
• Format: Journal Article
• Language: English
• Published: Washington, DC American Society for Microbiology 01-11-2000
• Subjects: Amino Acid Sequence; Animals; Antigens, Fungal - chemistry; Antigens, Fungal - genetics; Antigens, Fungal - immunology; Base Sequence; Biological and medical sciences; Cloning, Molecular; Cryptococcus neoformans; Cryptococcus neoformans - genetics; Cryptococcus neoformans - immunology; DHA1 gene; DHA1 protein; Fundamental and applied biological sciences. Psychology; Fungal and Parasitic Infections; Genes, Fungal; Growth, nutrition, metabolism, transports, enzymes. Molecular biology; Hypersensitivity, Delayed - etiology; Immunization; Lentinus edodes; Mice; Mice, Inbred CBA; Microbiology; Molecular Sequence Data; Molecular Weight; Mycology; Immunization; Yeast; Immune response; Cryptococcus neoformans; Rodentia; Delayed hypersensitivity; Cellular immunity; Fungi; Antigen; Vertebrata; Mammalia; Immunogenicity; Mouse; Fungi Imperfecti; Recombinant protein; Molecular cloning; Thallophyta; Structural analysis
• ISSN: 0019-9567; 1098-5522
• DOI: 10.1128/IAI.68.11.6196-6201.2000

When mice are vaccinated with a culture filtrate from Cryptococcus neoformans (CneF), they mount a protective cell-mediated immune response as detected by dermal delayed-type hypersensitivity (DTH) to CneF. We have identified a gene (DHA1) whose product accounts at least in part for the DTH reactivity. Using an acapsular mutant (Cap-67) of C. neoformans strain B3501, we prepared a culture filtrate (CneF-Cap67) similar to that used for preparing the commonly used skin test antigen made with C. neoformans 184A (CneF-184A). CneF-Cap67 elicited DTH in mice immunized with CneF-184A. Deglycosylation of CneF-Cap67 did not diminish its DTH activity. Furthermore, size separation by either chromatography or differential centrifugation identified the major DTH activity of CneF-Cap67 to be present in fractions that contained proteins of approximately 19 to 20 kDa. Using N-terminal and internal amino acid sequences derived from the 20-kDa band, oligonucleotide primers were designed, two of which produced a 776-bp amplimer by reverse transcription-PCR (RT-PCR) using RNA from Cap-67 to prepare cDNA for the template. The amplimer was used as a probe to isolate clones containing the full-length DHA1 gene from a phage genomic library prepared from strain B3501. The full-length cDNA was obtained by 5' rapid amplification of cDNA ends and RT-PCR. Analysis of DHA1 revealed a similarity between the deduced open reading frame and that of a developmentally regulated gene from Lentinus edodes (shiitake mushroom) associated with fruiting-body formation. Also, the gene product contained several amino acid sequences identical to those determined biochemically from the purified 20-kDa peptide encoded by DHA1. Recombinant DHA1 protein expressed in Escherichia coli was shown to elicit DTH reactions similar to those elicited by CneF-Cap67 in mice immunized against C. neoformans. Thus, DHA1 is the first gene to be cloned from C. neoformans whose product has been shown to possess immunologic activity.