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 (rCts1Ur) was induced in pCE-CTS1-transformed U. reesii by elevating the cultivation temperature. The isolated rCts1Ur 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 rCts1Ur, 74 out of the 77 coccidioidomycosis patients examined (96.1%) were positively identified by enzyme-linked immunosorbent assay. The rCts1Ur 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.

[1]  M. Montero,et al.  Expression regulation of the endochitinase chit36 from Trichoderma asperellum (T. harzianum T-203) , 2002, Current Genetics.

[2]  U. Reichard,et al.  A Parasitic Phase-Specific Adhesin of Coccidioides immitis Contributes to the Virulence of This Respiratory Fungal Pathogen , 2002, Infection and Immunity.

[3]  P. Skuce,et al.  The contribution of molecular biology to the development of vaccines against nematode and trematode parasites of domestic ruminants. , 2001, Veterinary parasitology.

[4]  A. Burt,et al.  Gene genealogies, cryptic species, and molecular evolution in the human pathogen Coccidioides immitis and relatives (Ascomycota, Onygenales). , 2001, Molecular biology and evolution.

[5]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[6]  U. Reichard,et al.  Disruption of the Gene Which Encodes a Serodiagnostic Antigen and Chitinase of the Human Fungal PathogenCoccidioides immitis , 2000, Infection and Immunity.

[7]  T. Kirkland,et al.  The hsp60 gene of the human pathogenic fungus Coccidioides immitis encodes a T-cell reactive protein. , 1997, Gene.

[8]  D. Magee,et al.  Mapping of a Coccidioides immitis-specific epitope that reacts with complement-fixing antibody , 1997, Infection and immunity.

[9]  D. Magee,et al.  Recombinant Coccidioides immitis complement-fixing antigen: detection of an epitope shared by C. immitis, Histoplasma capsulatum, and Blastomyces dermatitidis , 1997, Clinical and diagnostic laboratory immunology.

[10]  D. Pappagianis,et al.  Use of a recombinant Coccidioides immitis complement fixation antigen-chitinase in conventional serological assays , 1996, Journal of clinical microbiology.

[11]  T. White,et al.  Human pathogeneic fungi and their close nonpathogenic relatives. , 1996, Molecular phylogenetics and evolution.

[12]  D. Magee,et al.  Molecular cloning and characterization of the Coccidioides immitis complement fixation/chitinase antigen , 1996, Infection and immunity.

[13]  T. Kirkland,et al.  Isolation and characterization of two chitinase-encoding genes (cts1, cts2) from the fungus Coccidioides immitis. , 1995, Gene.

[14]  G. Cole,et al.  Evidence for a phylogenetic connection between Coccidioides immitis and Uncinocarpus reesii (Onygenaceae). , 1994, Microbiology.

[15]  J. Mccoy,et al.  A Thioredoxin Gene Fusion Expression System That Circumvents Inclusion Body Formation in the E. coli Cytoplasm , 1993, Bio/Technology.

[16]  G. Cole,et al.  A seroreactive 120-kilodalton beta-1,3-glucanase of Coccidioides immitis which may participate in spherule morphogenesis , 1992, Infection and immunity.

[17]  D. Pappagianis,et al.  The coccidioidal complement fixation and immunodiffusion-complement fixation antigen is a chitinase , 1992, Infection and immunity.

[18]  B. Zimmer,et al.  Purification and Amino-Terminal Sequence Analysis of the Complement-Fixing and Precipitin Antigens from Coccidioides immitis , 1990, Journal of clinical microbiology.

[19]  C. Smith,et al.  Pattern of 39,500 serologic tests in coccidioidomycosis. , 1956, Journal of the American Medical Association.

[20]  M. Huppert.  Serology of coccidioidomycosis , 2005, Mycopathologia et mycologia applicata.

[21]  K. Blennow,et al.  Identification of proteins in a human pleural exudate using two‐dimensional preparative liquid‐phase electrophoresis and matrix‐assisted laser desorption/ionization mass spectrometry , 1999, Electrophoresis.

[22]  G. Gooday,et al.  A rapid and sensitive microassay for determination of chitinolytic activity , 1992 .

[23]  P. Punt,et al.  Transformation of filamentous fungi based on hygromycin B and phleomycin resistance markers. , 1992, Methods in enzymology.

[24]  D. Pappagianis.  Epidemiology of coccidioidomycosis. , 1988, Current topics in medical mycology.

[25]  L. Sigler,et al.  Taxonomy of Malbranchea and some other hyphomycetes with arthroconidia , 1976 .