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Published on: June 15, 2018
Characterization of a serodiagnostic complement fixation antigen of Coccidioides posadasii expressed in the
J-J Yu1, T N Kirkland, L K Hall
1Department of Medical Microbiology and Immunology, Medical College of Ohio, Toledo, Ohio, USA.
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.
Insights
A novel fungal expression system was developed to produce coccidioidal antigens for diagnosing coccidioidomycosis. This system successfully expressed a key antigen, showing high diagnostic efficacy in patient samples.
Area of Science:
- Medical Mycology
- Molecular Biology
- Protein Expression
Background:
- Coccidioides species cause human coccidioidomycosis.
- Developing effective diagnostic antigens is crucial for disease management.
Purpose of the Study:
- To establish a novel expression system for coccidioidal proteins in a related, nonpathogenic fungus.
- To produce and characterize a recombinant coccidioidal antigen for diagnostic applications.
Main Methods:
- Utilized a Coccidioides posadasii heat shock protein 60 (HSP60) promoter for gene expression in Uncinocarpus reesii.
- Constructed a coccidioidal protein expression vector (pCE) for expressing the C. posadasii chitinase gene (CTS1).
- Induced recombinant Cts1 protein (rCts1(Ur)) expression by temperature elevation in transformed U. reesii.
Main Results:
- The expressed rCts1(Ur) exhibited chitinolytic activity comparable to the native protein.
- rCts1(Ur) demonstrated serodiagnostic efficacy similar to commercial antigens in immunodiffusion-complement fixation tests.
- Enzyme-linked immunosorbent assay (ELISA) using rCts1(Ur) correctly identified 96.1% of coccidioidomycosis patients.
Conclusions:
- The novel fungal expression system is effective for producing coccidioidal antigens.
- Recombinant Cts1 produced in U. reesii shows high diagnostic potential for coccidioidomycosis.
- This system offers a valuable tool for developing improved diagnostic antigens.
