| Autor: |
Mandel, M. Alejandra, Grace, Greg G., Orsborn, Kris I., Schafer, Fredda, Murphy, Juneann W., Orbach, Marc J., Galgiani, John N. |
| Zdroj: |
Infection and Immunity; November 2000, Vol. 68 Issue: 11 p6196-6201, 6p |
| Abstrakt: |
ABSTRACTWhen mice are vaccinated with a culture filtrate fromCryptococcus 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. neoformansstrain B3501, we prepared a culture filtrate (CneF-Cap67) similar to that used for preparing the commonly used skin test antigen made with C. neoformans184A (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-lengthDHA1gene 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 DHA1revealed 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 byDHA1. Recombinant DHA1 protein expressed inEscherichia coliwas shown to elicit DTH reactions similar to those elicited by CneF-Cap67 in mice immunized against C. neoformans. Thus, DHA1is the first gene to be cloned from C. neoformanswhose product has been shown to possess immunologic activity. |
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