The hsp60 gene of the human pathogenic fungus Coccidioides immitis encodes a T-cell reactive protein.
A heat shock protein-encoding gene (hsp60) from the human respiratory fungal pathogen, Coccidioides immitis (Ci), was cloned, sequenced, chromosome-mapped, expressed and immunolocalized in parasitic cells. Both the genomic and cDNA sequences are presented. The transcription start point and poly (A) addition site were confirmed. The hsp60 gene contains two introns and a 1782-bp ORF which translates a 594-amino acid (aa) protein of 62.4 kDa and pI of 5.6. The translated protein revealed two potential N-glycosylation sites. The deduced HSP60 showed 78-83% aa sequence similarity to reported fungal HSP60 proteins. The hsp60 gene was mapped to chromosome III of Ci and was shown to be a single copy gene by Southern and Northern hybridization. Expression of a 1737-bp cDNA fragment of the hsp60 gene in E. coli resulted in production of a recombinant protein. Amino acid sequence analysis of the recombinant protein confirmed that it was encoded by the Ci hsp60 gene. Antiserum raised in mice against the isolated recombinant protein immunolocalized HSP60 in the cytoplasm and wall of parasitic cells of Ci. The recombinant HSP60 was used to immunize BALB/c mice and was shown to induce proliferation of T cells isolated from lymph nodes of these animals. The hsp60 gene of Ci is the first reported heat-shock protein gene of this human pathogen.
Thomas
P W
PW
Department of Microbiology and Immunology, Medical College of Ohio, Toledo 43614-5806, USA.
Wyckoff
E E
EE
Pishko
E J
EJ
Yu
J J
JJ
Kirkland
T N
TN
Cole
G T
GT
D000595:Amino Acid Sequence; D000818:Animals; D000946:Antigens, Fungal; D001483:Base Sequence; D002473:Cell Wall / Q000737:chemistry; D018834:Chaperonin 60 / Q000032:analysis / Q000235:genetics* / Q000276:immunology*; D002874:Chromosome Mapping; D015825:Chromosomes, Fungal / Q000235:genetics; D003001:Cloning, Molecular; D003045:Coccidioides / Q000235:genetics*; D003593:Cytoplasm / Q000737:chemistry; D004271:DNA, Fungal / Q000032:analysis; D004926:Escherichia coli / Q000235:genetics; D018628:Gene Dosage; D005800:Genes, Fungal / Q000235:genetics*; D006801:Humans; D008213:Lymphocyte Activation / Q000187:drug effects; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013601:T-Lymphocytes / Q000276:immunology*
D000946:Antigens, Fungal; D018834:Chaperonin 60; D004271:DNA, Fungal; D011993:Recombinant Fusion Proteins