Identification and cloning of an aspartyl proteinase from Coccidioides immitis.

A 45 kDa protein was isolated from a soluble vaccine prepared from formaldehyde-killed spherules of Coccidioides immitis. From the N-terminal amino acid sequence, the protein yielded a 17-amino-acid peptide that was homologous to sequences of other fungal aspartyl proteinases. The coccidioidal cDNA encoding the proteinase was amplified using oligonucleotide primers designed from the 45 kDa N-terminal amino acid sequence and a fungal aspartyl proteinase consensus amino acid sequence. The PCR product was cloned and sequenced, and the remaining 5' upstream and 3' downstream cDNA was amplified, cloned, and sequenced. The cDNA encoding the coccidioidal aspartyl proteinase open reading frame was cloned and the fusion protein containing a C-terminal His-tag expressed in E. coli. The recombinant aspartyl proteinase was purified by immobilized metal affinity chromatography. This recombinant protein will be used for further studies to evaluate its antigenicity, including protective immunogenicity.

Evaluation of the susceptibility of Coccidioides immitis to lufenuron, a chitin synthase inhibitor.

The activity of the chitin synthase inhibitor lufenuron was evaluated in vitro using the spherule-endospore (SE) phase of Coccidioides immitis. The lufenuron was also used to treat mice infected with C. immitis by the respiratory route. In vitro, lufenuron had no effect upon fungal cell growth. Two formulations of lufenuron were evaluated in vivo. Neither the oral nor the injectable lufenuron extended the survival of mice infected with C. immitis when compared with placebo-treated mice.

Protection against lethal murine coccidioidomycosis by a soluble vaccine from spherules.

The formaldehyde-killed, whole-spherule vaccine, which is protective against lethal challenge of laboratory animals with Coccidioides immitis, was fractionated. It yielded a soluble, multicomponent, subcellular fraction termed the 27K vaccine. This vaccine, when it was accompanied by adjuvant, protected mice against lethal intranasal and intravenous challenge with C. immitis.

Use of a recombinant Coccidioides immitis complement fixation antigen-chitinase in conventional serological assays.

The coccidioidal complement fixation (CF) antigen has been cloned previously, and the fusion protein has been expressed in Escherichia coli. The recombinant CF (rCF) antigen was affinity purified by adsorption-desorption to chitin, and its reactivity was studied by using sera containing coccidioidal antibodies. The affinity-purified rCF antigen formed a line of identity with an immunodiffusion (ID) CF reference antigen (coccidioidin) derived from mycelial-phase Coccidioides immitis and was reactive with human, canine, and equine sera containing coccidioidal antibody. The affinity-purified rCF antigen yielded no detectable reaction with Blastomyces of Histoplasma antiserum by ID. The affinity-purified rCF antigen fixed complement with positive human sera and, even when used at lower concentrations, yielded titers comparable to those obtained with the coccidioidin. The reactivity of the affinity-purified rCF antigen was further evaluated by enzyme immunoassay, in which it manifested good sensitivity (96.9%) and specificity (100%) when evaluated with 43 human patients' sera. Thus, the affinity-purified rCF antigen has yielded reactions comparable to those of crude coccidioidal antigens in conventional CF, IDCF, and enzyme immunoassay.

Cloning and expression of the complement fixation antigen-chitinase of Coccidioides immitis.

A chitinase had been isolated from the culture filtrates of Coccidioides immitis endosporulating spherules and from hyphae and shown to be the coccidioidal complement fixation (CF) and immunodiffusion-CF antigen. In the present study, we made use of our previously determined amino-terminal (N-terminal) sequence of the CF-chitinase to design degenerate oligonucleotide primers and to amplify and sequence a PCR product that coded for the N-terminal portion of the CF-chitinase. The PCR product was used as a hybridization probe to screen a developing spherule-(lambda)ZAP cDNA library, and three hybridizing clones were selected. These clones were converted into their pBluescript expression plasmid form in Escherichia coli and induced to express their recombinant proteins. Lysate from only one clone, pCTS 4-2A, yielded an enzymatically functional CF-chitinase and a line of identity with control immunodiffusion-CF-positive antigen. The pCTS 4-2A insert was sequenced and found to contain a deduced open reading frame coding for a 427-amino-acid polypeptide with an approximate molecular weight of 47 kDa. When purified by a chitin adsorption-desorption method, the recombinant protein exhibited virtually identical characteristics to those of the original C. immitis CF-chitinase. Nondenaturing gels of the pCTS 4-2A E. coli lysates and the purified C. immitis and recombinant CF-chitinase revealed proteins that had chitinase activity and similar relative electrophoretic mobilities. The appearance and relative levels of hybridizing RNA from the developing spherules-endospores (SEs) and hyphae correlated with the appearance or presence and level of CF-chitinase enzyme activity found in SEs culture filtrate and in cellular extracts of developing SE and hyphae. Thus, a functional recombinant CF-chitinase antigen was produced in E. coli and was used in serological diagnostic applications. These results also suggest a functional role for this chitinase in SE development and maturation.

Cloning and sequence analysis of the cDNA for a protein from Coccidioides immitis with immunogenic potential.

We have cloned and sequenced the cDNA encoding an immunoreactive protein from the pathogenic fungus Coccidioides immitis which stimulates human T cells and has been associated with protective vaccines in mice. The transcript contained an open reading frame encoding 194 amino acids with a calculated molecular weight of 19.5 kDa, a 151 base 5' untranslated region (UTR), and a 468 base 3'UTR. A four member repeat motif, usually thr-ala-glu-pro, exists for amino acids 98 through 141. Deduced amino acid sequence derived from the cDNA was identical with previously determined internal amino acid sequence from the native protein, and goat antiserum raised against the purified fungal protein reacted with an inducible fusion protein translated from this cDNA. Using this cDNA to produce recombinant protein will allow direct testing of its role in human immunity to coccidioidomycosis and may lead to new diagnostic tests.

Characterization of Coccidioides immitis isolates by restriction fragment length polymorphisms.

The marked increase in the number of cases of coccidioidomycosis in California in 1992 led to a study of isolates from various patients and environmental sources by restriction fragment length polymorphism (RFLP) analysis. Of 15 different isolates, most of the isolates (13 of 15) from California and 1 from Venezuela yielded one main RFLP pattern with evidence of two subgroups. The other two isolates (both from patients in the San Joaquin Valley of California) yielded a different RFLP pattern.

Amino-terminal sequence analysis of the Coccidioides immitis chitinase/immunodiffusion-complement fixation protein.

A chitinase isolated from Coccidioides immitis was subjected to amino-terminal protein sequence analysis. The resulting 18-amino-acid sequence was compared with the previously reported amino acid sequence of coccidioidal immunodiffusion-complement fixation (IDCF) antigen. From the homology of the two sequences, the results support the identification of the IDCF antigen with a chitinase.

Molecular cloning and selection of genes regulated in Aspergillus development.

Over 350 clones homologous to poly(A)+ RNAs that are significantly more prevalent in conidiating cultures of Aspergillus nidulans than in somatic cells have been selected from a recombinant DNA library formed between nuclear DNA and lambda Charon 4A. The procedure used for this selection involved in situ hybridization to a cDNA probe which had been selectively depleted of sequences represented in somatic cells by complement hybridization. Five of these clones have been characterized further. All but one encoded poly(A)+ RNAs that were at least ten times more prevalent in conidiating cultures than in somatic cells. One clone hybridized to a single, developmentally regulated RNA. The three others were complementary to several RNAs having different molecular weights, each of which was more prevalent in condiating cultures than in vegetative cells. These results and quantitative aspects of the selection procedure suggest that developmentally controlled poly(A)+ RNA coding regions may not be distributed randomly in the Aspergillus genome.