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.

FIG. 1.

Representations of pCE vector (A) and pCE-CTS1 plasmid (B). The hatched box represents an 18-nucleotide fragment that encodes the His tag. Primer pairs used to amplify CpHSP60 (A-B and C-D), as well as CpCTS1 (E-F), genes are positioned, and their sequences are presented in Table 1. Two XbaI sites used to release the Cts1 expression cassette are indicated.

FIG. 2.

Confirmation of pCE-CTS1 transformants 2, 3, and 8 (lanes 2 to 4, respectively) by PCR amplification of the inserted CpCTS1 gene (A) and detection of the expressed rCts1_Ur protein (B). (A) Ethidium bromide-stained electrophoresis gel of PCR products amplified from a parental strain of U. reesii (lane 1) and the pCE-CTS1 transformants. std., standards. (B) Proteins prepared from cytosol plus culture filtrate of the parental strain (lanes 1) or transformants were separated in a 10% SDS-PAGE and stained with Coomassie blue (left) or detected by anti-His-tag antibodies in a Western blot (right).

FIG. 3.

(A) SDS-PAGE separation of U. reesii-expressed recombinant Cts1 protein (lanes 1 and 2) and partially purified native CpCts1 (lanes 3 and 4). Total proteins were prepared from non-heat-shocked (lane 1) or heat-shocked (lane 2) culture filtrate by ammonia sulfate precipitation. rCts1_Ur was isolated by Ni affinity chromatography (lane 3) and reacted with an anti-His-tag antibody (lane 5; Western blot). (B) Molecular sizes of rCts1_Ur and native CpCts1 (nCts1) were determined by SELDI-TOF mass spectrometry. (C) Deglycosylation of rCts1_Ur using PNGase F. Coomassie blue-stained SDS-PAGE revealed untreated rCts1_Ur (lane 1), enzyme-treated rCts1_Ur (lane 2), and PNGase F alone (lane 3) as a reference. SELDI-TOF mass spectrometry of the enzyme-treated rCts1_Ur is also presented. std., standards.

FIG. 4.

ID-CF assays (A to C) using the isolated rCts1_Ur as an antigen. (A) Samples in wells are reference CF Ag (wells 1 and 4); 1 μg, 0.5 μg, 0.25 μg, or 0.125 μg of rCts1_Ur (wells 2, 3, 5, and 6, respectively); and reference CF antibody (Ab) (well 7). (B) Samples in wells are reference CF Ab (wells 1 and 4), sera from coccidioidomycosis patients (wells 2 and 5), rCts1_Ur (well 6), heat-treated rCts1_Ur (well 3), and reference CF Ag (well 7). (C) Samples in wells are reference CF Ab (wells 1 and 4), sera from patient (wells 2 and 5), sera from healthy individuals (wells 3 and 6), and rCts1_Ur (well 7). (D) Protein samples of reference CF Ag or rCts1_Ur used in the ID-CF assays were separated in an SDS-PAGE (12%) and stained with silver reagents. std., standards.

FIG. 5.

Correlation of CF titer with reactivity at a single dilution in the rCts1_Ur ELISA.

FIG. 6.

Endpoint titers of CF-positive and control sera. The endpoint was defined as an OD of 0.20, which is more than twice the value obtained with human serum binding to blank wells. The lines represent the medians of the two groups.

FIG. 7.

Expression and seroreactivity of rCts1_Ec. (A) SDS-PAGE analysis of expression of rCts1_Ec. Lanes 1 to 4 show electrophoretic separation of cytosolic proteins from bacteria transformed with either pET32b alone (lanes 1 and 2) or pET32b-CTS1 (lanes 3 and 4) without IPTG induction (lanes 1 and 3) and with IPTG induction (lanes 2 and 4). Lanes 5 and 6 show the nickel affinity-purified rCts1_Ec before and after thrombin cleavage, respectively. std., standards. (B) ID-CF assays. Samples in wells are 4 μg, 2 μg, 1 μg, or 0.5 μg of rCts1_Ec (wells 1 to 4, respectively); 0.25 μg (well 5) or 1 μg (well 6) of rCts1_ur; and reference antibody (well 7). (C) Comparison of reactivity of rCts1_Ur (closed circles) and rCts1_Ec (open circles) with sera (1/100 to 1/512,000; twofold dilution) from three coccidioidomycosis patients, using ELISA.