Characterization of an Uncinocarpus reesii-expressed recombinant tube precipitin antigen of Coccidioides posadasii for serodiagnosis.

Early and accurate diagnosis of coccidioidomycosis, also known as Valley fever, is critical for appropriate disease treatment and management. Current serodiagnosis is based on the detection of patient serum antibodies that react with tube precipitin (TP) and complement fixation (CF) antigens of Coccidioides. IgM is the first class of antibodies produced by hosts in response to coccidioidal insults. The highly glycosylated ß-glucosidase 2 (BGL2) is a major active component of the TP antigen that stimulates IgM antibody responses during early Coccidioides infection. The predominant IgM epitope on BGL2 is a unique 3-O-methyl-mannose moiety that is not produced by commonly used protein expression systems. We genetically engineered and expressed a recombinant BGL2 (rBGL2ur), derived from Coccidioides, in non-pathogenic Uncinocarpus reesii, a fungus phylogenetically related to the Coccidioides pathogen. The rBGL2ur protein was purified from the culture medium of transformed U. reesii by nickel affinity chromatography, and the presence of 3-O-methyl mannose was demonstrated by gas chromatography. Seroreactivity of the purified rBGL2ur protein was tested by enzyme-linked immunosorbent assays using sera from 90 patients with coccidioidomycosis and 134 control individuals. The sensitivity and specificity of the assay with rBGL2ur were 78.8% and 87.3%, respectively. These results were comparable to those obtained using a proprietary MiraVista Diagnostic (MVD) IgM (63.3% sensitivity; 96.3% specificity), but significantly better than the ID-TP assay using non-concentrated patient sera (33.3% sensitivity; 100% specificity). Expression of rBGL2ur in U. reesii retains its antigenicity for coccidioidomycosis serodiagnosis and greatly reduces biosafety concerns for antigen production, as Coccidioides spp. are biological safety level 3 agents.

Total and Lectin-Binding Proteome of Spherulin from Coccidioides posadasii.

Coccidioides is a virulent dimorphic fungus that causes coccidioidomycosis (valley fever) in mammals, including humans. Although the genome has been sequenced, a proteomic analysis does not exist. To address this gap in proteomic knowledge, we generated the proteome of spherulin (a well-studied lysate of fungal spherules) and identified 1390 proteins. Some of the proteins included glycosylation enzymes, which led us to hypothesize that fungal glycosylation patterns may be different from those of mammals and could be exploited to detect Coccidioides in tissues. We performed lectin-based immunohistochemistry on formalin-fixed paraffin-embedded human patients' lung tissues. GSL-II (Griffonia simplificonia lectin II) and sWGA (succinylated wheat germ agglutinin) lectins bound specifically to endospores and spherules in infected lungs. To identify lectin-binding glycoproteins in spherulin, we performed lectin-affinity chromatography, followed by LC-MS/MS. A total of 195 glycoproteins from spherulin bound to GSL-II, 224 glycoproteins bound to sWGA, and 145 glycoproteins bound to both lectins. This is the first report of the specific reactivity of GSL-II and sWGA lectins to Coccidioides endospores and spherules in infected human tissues and the first listing of the Coccidioidal proteome from spherulin using sequences present in three Coccidioides databases: RefSeq, SwissProt, and The Broad Institute's Coccidioides Genome project.

Role of Coccidioides Antigen Testing in the Cerebrospinal Fluid for the Diagnosis of Coccidioidal Meningitis.

BACKGROUND: Coccidioidal meningitis (CM), a common cause of chronic meningitis in endemic area, is usually diagnosed by detection of anti-Coccidioides antibodies in cerebrospinal fluid (CSF), and findings may be negative in up to one-third of cases. CSF cultures and cytology are infrequently positive. Antigen detection has been used for the diagnosis of other forms of coccidioidomycosis and meningitis caused by other mycoses. The purpose of this study was to assess the diagnostic utility of CSF Coccidioides antigen (CAg) detection for the diagnosis of CM. METHODS: The medical records of patients with clinically suspected meningitis, in whom CSF was tested for Coccidioides antibodies and CAg, were retrospectively reviewed, and CSF CAg testing was prospectively conducted in patients with CM. All specimens were submitted for CAg testing. RESULTS: Thirty-six patients with 42 episode of CM were studied. The sensitivity and specificity of CAg were 93% and 100%, respectively. Cultures of CSF were positive in 7%, antibodies were demonstrated by immunodiffusion in 67% and complement fixation in 70%, and immunoglobulin M and G antibodies were demonstrated by enzyme immunoassay in 8% and 85%, respectively. CONCLUSIONS: Testing CSF for CAg is a useful addition to diagnostic methods in suspected CM and complements testing with CSF antibodies and culture.

Anti-inflammatory and immunomodulatory effect of an extract of Coccidioides posadasii in experimental arthritis.

Trying to surpass host defenses, fungal infections alter the immune response. Components from nonpathogenic fungi present therapeutic anti-inflammatory and immunomodulating activities. This study reveals that proteins present in a Coccidioides posadasii extract provide anti-inflammatory benefit in experimental arthritis. Zymosan was given intra-articularly to rats and mice, and groups were pretreated with C. posadasii extract either per os or intraperitoneally. Controls received the vehicle. Acute hypernociception was evaluated using articular incapacitation and von Frey methods. Cell influx and cytokine levels were assessed in joint exudates. Joint damage was evaluated by histopathology and determination of glycosaminoglycan content of the cartilage. Synovia was evaluated for cell death and inducible nitric oxide synthase (iNOS) expression using TUNEL and immunohistochemistry, respectively. Pretreatment with C. posadasii extract significantly inhibited acute and chronic cell influx, hypernociception, and provoked reduction of glycosaminoglycan loss while reducing chronic synovitis, cell death, and iNOS expression. Reduction/alkylation of C. posadasii extract abrogated these effects. C. posadasii administration did not alter TNF-α, IL-1ß, IL-17, and γ-interferon levels, whereas IL-10 levels were significantly reduced. Data reveal that a C. posadasii extract reduces iNOS expression that is associated with inhibition of synovial apoptosis and decrease in IL-10 levels released into zymosan-inflamed joints. Characterization of active components excluded charged carbohydrates while pointing to a protein as responsible for these effects. In summary, systemic administration of components from a pathogenic fungus provides anti-inflammatory effects, being species-independent and orally active. Besides adding to understand host response against fungi, the results may lead to therapeutic implications.

The Protein Maker: an automated system for high-throughput parallel purification.

The Protein Maker is an automated purification system developed by Emerald BioSystems for high-throughput parallel purification of proteins and antibodies. This instrument allows multiple load, wash and elution buffers to be used in parallel along independent lines for up to 24 individual samples. To demonstrate its utility, its use in the purification of five recombinant PB2 C-terminal domains from various subtypes of the influenza A virus is described. Three of these constructs crystallized and one diffracted X-rays to sufficient resolution for structure determination and deposition in the Protein Data Bank. Methods for screening lysis buffers for a cytochrome P450 from a pathogenic fungus prior to upscaling expression and purification are also described. The Protein Maker has become a valuable asset within the Seattle Structural Genomics Center for Infectious Disease (SSGCID) and hence is a potentially valuable tool for a variety of high-throughput protein-purification applications.

Multivalent recombinant protein vaccine against coccidioidomycosis.

Coccidioidomycosis is a human respiratory disease that is endemic to the southwestern United States and is caused by inhalation of the spores of a desert soilborne fungus. Efforts to develop a vaccine against this disease have focused on identification of T-cell-reactive antigens derived from the parasitic cell wall which can stimulate protective immunity against Coccidioides posadasii infection in mice. We previously described a productive immunoproteomic/bioinformatic approach to the discovery of vaccine candidates which makes use of the translated genome of C. posadasii and a computer-based method of scanning deduced sequences of seroreactive proteins for epitopes that are predicted to bind to human major histocompatibility (MHC) class II-restricted molecules. In this study we identified a set of putative cell wall proteins predicted to contain multiple, promiscuous MHC II binding epitopes. Three of these were expressed by Escherichia coli, combined in a vaccine, and tested for protective efficacy in C57BL/6 mice. Approximately 90% of the mice survived beyond 90 days after intranasal challenge, and the majority cleared the pathogen. We suggest that the multicomponent vaccine stimulates a broader range of T-cell clones than the single recombinant protein vaccines and thereby may be capable of inducing protection in an immunologically heterogeneous human population.

The parasitic cell wall of Coccidioides immitis.

Coccidioides immitis is a human respiratory pathogen characterized by a parasitic cycle that is unique among fungi that cause systemic mycoses. Biochemical, molecular and immunological studies of the cell wall of C. immitis have focused on three distinct events of parasitic cell differentiation: isotropic growth, segmentation and endosporulation. Current investigations of each developmental phase in vitro include the identification, expression analysis, and disruption of synthase and hydrolase genes that are suspected to have key roles in morphogenesis. Temporal expression of families of beta-glucosidase and chitinase genes are of particular interest because their products may participate in wall modification during both isotropic growth and endosporulation and, thereby, represent potential molecular targets for novel antifungal drugs. Furthermore, our immunological studies of these and other isolated parasitic cell-wall components have resulted in the identification of antigens with demonstrated impact on host response to coccidioidal infection. C. immitis has proved to be an excellent model for fungal cell-wall research.

The X-ray structure of a chitinase from the pathogenic fungus Coccidioides immitis.

The X-ray structure of chitinase from the fungal pathogen Coccidioides immitis has been solved to 2.2 A resolution. Like other members of the class 18 hydrolase family, this 427 residue protein is an eight-stranded beta/alpha-barrel. Although lacking an N-terminal chitin anchoring domain, the enzyme closely resembles the chitinase from Serratia marcescens. Among the conserved features are three cis peptide bonds, all involving conserved active site residues. The active site is formed from conserved residues such as tryptophans 47, 131, 315, 378, tyrosines 239 and 293, and arginines 52 and 295. Glu171 is the catalytic acid in the hydrolytic mechanism; it was mutated to a Gln, and activity was abolished. Allosamidin is a substrate analog that strongly inhibits the class 18 enzymes. Its binding to the chitinase hevamine has been observed, and we used conserved structural features of the two enzymes to predict the inhibitors binding to the fungal enzyme.

Coccidioides immitis antigen 2: analysis of gene and protein.

Antigen 2 is a glycosylated protein present in the cell walls of the dimorphic fungus Coccidioides immitis. Using oligodeoxyribonucleotide (oligo) primers based on the sequences of Ag2 cDNA, the gene encoding Ag2 was cloned from genomic DNA derived from the mycelial phase of C. immitis by PCR. Nucleotide (nt) sequence analyses showed a 582 base pair (bp) ORF disrupted by two introns which are 78 bp and 101 bp long. The deduced primary translation product consists of 194 amino acids (aa), contains an N-terminal putative signal sequence to allow transport into the endoplasmic reticulum, and a C-terminal putative signal sequence to enable a GPI anchor addition. Putative GPI anchor/cleavage site and O-glycosylation sites, as well as phosphorylation and myristoylation sites are also present. On the basis of these analyses, we predict that a prepro-Ag2 undergoes a post-translational modification to yield the mature glycosylated Ag2 protein which is anchored on the extracellular plasma membrane of mycelial and spherule-phase cells.

Ubiquinone systems of Coccidioides immitis, the causative agent of coccidioidomycosis.

The ubiquinone (coenzyme Q) systems of eleven strains of Coccidioides immitis were determined by high performance liquid chromatography (HPLC). The ubiquinone profile of the fungi was shown to be homogeneous: in all of the strains, ubiquinone-10 (Q-10) was demonstrated to be the major component, with Q-9 as a minor component. The results imply that the ubiquinone system may serve as an additional phenotypic criterion for identifying the fungus.