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Characterization of a serodiagnostic complement fixation antigen of Coccidioides posadasii expressed in the nonpathogenic Fungus Uncinocarpus reesii.

ABSTRACT: Coccidioides spp. (immitis and posadasii) are the causative agents of human coccidioidomycosis. In this study, we developed a novel system to overexpress coccidioidal proteins in a nonpathogenic fungus, Uncinocarpus reesii, which is closely related to Coccidioides. A promoter derived from the heat shock protein gene (HSP60) of Coccidioides posadasii was used to control the transcription of the inserted gene in the constructed coccidioidal protein expression vector (pCE). The chitinase gene (CTS1) of C. posadasii, which encodes the complement fixation antigen, was expressed using this system. The recombinant Cts1 protein (rCts1(Ur)) was induced in pCE-CTS1-transformed U. reesii by elevating the cultivation temperature. The isolated rCts1(Ur) showed chitinolytic activity that was identical to that of the native protein and had serodiagnostic efficacy comparable to those of the commercially available antigens in immunodiffusion-complement fixation tests. Using the purified rCts1(Ur), 74 out of the 77 coccidioidomycosis patients examined (96.1%) were positively identified by enzyme-linked immunosorbent assay. The rCts1(Ur) protein showed higher chitinolytic activity and slightly greater seroreactivity than the bacterially expressed recombinant Cts1. These data suggest that this novel expression system is a useful tool to produce coccidioidal antigens for use as diagnostic antigens.

SUBMITTER: Yu JJ

PROVIDER: S-EPMC1287831 | biostudies-literature | 2005 Nov

REPOSITORIES: biostudies-literature

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Characterization of a serodiagnostic complement fixation antigen of Coccidioides posadasii expressed in the nonpathogenic Fungus Uncinocarpus reesii.

Yu J-J JJ Kirkland T N TN Hall L K LK Wopschall J J Smith R C RC Hung C-Y CY Chen X X Tarcha E E Thomas P W PW Cole G T GT

Journal of clinical microbiology 20051101 11

Coccidioides spp. (immitis and posadasii) are the causative agents of human coccidioidomycosis. In this study, we developed a novel system to overexpress coccidioidal proteins in a nonpathogenic fungus, Uncinocarpus reesii, which is closely related to Coccidioides. A promoter derived from the heat shock protein gene (HSP60) of Coccidioides posadasii was used to control the transcription of the inserted gene in the constructed coccidioidal protein expression vector (pCE). The chitinase gene (CTS1 ...[more]

PMID: 16272471

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Project description: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.

Dataset Information

Characterization of a serodiagnostic complement fixation antigen of Coccidioides posadasii expressed in the nonpathogenic Fungus Uncinocarpus reesii.

Publications

Characterization of a serodiagnostic complement fixation antigen of Coccidioides posadasii expressed in the nonpathogenic Fungus Uncinocarpus reesii.

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