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

Abstract

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.

Identification and Cloning of a Cryptococcal Deacetylase That Produces Protective Immune Responses

“…This is in agreement with a previous study, also using biochemical separation of culture supernatants (20). Of course, our data do not exclude that other cryptococcal products are capable of producing DTH reactions.…”

“…Mandel et al have described a cryptococcal gene, termed DHA1, which encodes a DTH-producing peptide found in culture supernatants (20). Levitz et al have recently characterized a 98-kDa MP that is capable of stimulating a mouse CD4 ϩ -Tcell hybridoma to produce interleukin 2 (18).…”

“…Mandel et al cloned a cryptococcal gene, termed DHA1, which encodes a secreted protein capable of inducing DTH (20). Since this protein only partially accounted for the DTHinducing ability of whole CneF, it is likely that other cryptococcal antigens are also capable of producing cell-mediated responses.…”

Optimal T Cell Responses to Cryptococcus neoformans Mannoprotein Are Dependent on Recognition of Conjugated Carbohydrates by Mannose Receptors

“…One such hybridoma, designated P1D6, has specificity for a serine/threonine-rich MP, MP98, with an apparent molecular mass of 98 kDa. While other laboratories have also identified immunoreactive cryptococcal Ags (24,25), it is likely that a successful vaccine will consist of multiple components able to elicit both T and B cell-mediated immunity.…”

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

“…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).…”