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Infection and Immunity, November 2000, p. 6196-6201, Vol. 68, No. 11
0019-9567/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

The Cryptococcus neoformans Gene DHA1 Encodes an Antigen That Elicits a Delayed-Type Hypersensitivity Reaction in Immune Mice

M. Alejandra Mandel,1 Greg G. Grace,2,3 Kris I. Orsborn,2,3 Fredda Schafer,4 Juneann W. Murphy,4 Marc J. Orbach,1 and John N. Galgiani2,3,*

Medical and Research Services, Veterans Affairs Medical Center,2 and Department of Medicine, College of Medicine,3 and Department of Plant Pathology, College of Agriculture,1 University of Arizona, Tucson Arizona, and Department of Microbiology, University of Oklahoma, Oklahoma City, Oklahoma4

Received 28 June 2000/Accepted 25 July 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.

* Corresponding author. Mailing address: Valley Fever Center for Excellence (1-111), 3601 South Sixth Ave., Tucson, AZ 85723. Phone: (520) 792-1450 ext. 6793. Fax: (520) 629-4738. E-mail: spherule{at}u.arizona.edu.

Infection and Immunity, November 2000, p. 6196-6201, Vol. 68, No. 11
0019-9567/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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