Lower filamentation rates of Candida dubliniensis contribute to its lower virulence in comparison with Candida albicans.

Candida albicans and C. dubliniensis are very closely related yeast species. In this study, we have conducted a thorough comparison of the ability of the two species to produce hyphae and their virulence in two infection models. Under all induction conditions tested C. albicans consistently produced hyphae more efficiently than C. dubliniensis. In the oral reconstituted human epithelial model, C. dubliniensis isolates grew exclusively in the yeast form, while the C. albicans strains produced abundant hyphae that invaded and caused significant damage to the epithelial tissue. In the oral-intragastric infant mouse infection model, C. dubliniensis strains were more rapidly cleared from the gastrointestinal tract than C. albicans. Immunosuppression of Candida-infected mice caused dissemination to internal organs by both species, but C. albicans was found to be far more effective at dissemination than C. dubliniensis. These data suggest that a major reason for the comparatively low virulence of C. dubliniensis is its lower capacity to produce hyphae.

Phase-specific gene expression underlying morphological adaptations of the dimorphic human pathogenic fungus, Coccidioides posadasii.

Coccidioides posadasii is a dimorphic fungal pathogen that grows as a filamentous saprobe in the soil and as endosporulating spherules within the host. To identify genes specific to the pathogenic phase of Co. posadasii, we carried out a large-scale study of gene expression in two isolates of the species. From the sequenced Co. posadasii genome, we chose 1,000 open reading frames to construct a 70-mer microarray. RNA was recovered from both isolates at three life-cycle phases: hyphae, presegmented spherules, and spherules releasing endospores. Comparative hybridizations were conducted in a circuit design, permitting comparison between both isolates at all three life-cycle phases, and among all life-cycle phases for each isolate. By using this approach, we identified 92 genes that were differentially expressed between pathogenic and saprobic phases in both fungal isolates, and 43 genes with consistent differential expression between the two parasitic developmental phases. Genes with elevated expression in the pathogenic phases of both isolates included a number of genes that were involved in the response to environmental stress as well as in the metabolism of lipids. The latter observation is in agreement with previous studies demonstrating that spherules contain a higher proportion of lipids than saprobic phase tissue. Intriguingly, we discovered statistically significant and divergent levels of gene expression between the two isolates profiled for 64 genes. The results suggest that incorporating more than one isolate in the experimental design offers a means of categorizing the large collection of candidate genes that transcriptional profiling typically identifies into those that are strain-specific and those that characterize the entire species.

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

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.

A vaccine against coccidioidomycosis is justified and attainable.

Coccidioides is a fungal pathogen of humans which can cause a life-threatening respiratory disease in immunocompetent individuals. Recurrent epidemics of coccidioidal infections in Southwestern United States has raised the specter of awareness of this soil-borne microbe, particularly among residents of Arizona and Southern California, and has galvanized research efforts to develop a human vaccine against coccidioidomycosis. In this review, we discuss the rationale for such a vaccine, examine the features of host innate and acquired immune response to Coccidioides infection, describe strategies used to identify and evaluate vaccine candidates, and provide an update on progress toward development of a vaccine against this endemic pathogen.

Profiling gene expression in Coccidioides posadasii.

Coccidioides posadasii is a dimorphic fungal pathogen which grows as a filamentous saprobe in the soil and multicellular parasitic form in host lung tissue. Studies of gene expression profiles during saprobic and parasitic phase development can provide clues about morphogenetic regulation and may lead to the discovery of molecular targets for novel antifungal drugs. Suppression-subtractive hybridization (SSH) and quantitative real-time polymerase chain reaction (QRT-PCR) were used to identify and quantify differential gene expression during in vitro growth of Coccidioides. DNA fragments obtained from the subtraction of cDNA pools derived from the saprobic and parasitic phase RNA preparations were each cloned into an appropriate vector and subjected to sequence analysis. Semi-quantitative, reverse transcription polymerase chain reaction (RT-PCR) experiments were first conducted to assess whether these inserts represented differentially expressed genes. Nucleotide sequences of the partial and full-length genes selected by RT-PCR were obtained by genome walking and rapid amplification of cDNA ends (RACE) methods. QRT-PCR analysis of the expression of these genes during saprobic and parasitic cell growth was then conducted using DNA standard curves normalized to a constitutively expressed control gene. Four C. posadasii genes whose expression is essentially restricted to the parasitic cycle were discovered using this approach. These genes include homologues of OPS1 (encodes opsin-related protein), MDR1 (multidrug resistance protein), ALDR1 (aldehyde reductase), and PSP1 (hypothetical lipid transporter/flippase protein). The combined applications of SSH and QRT-PCR permit global analysis of gene expression patterns in C. posadasii.

Purification and characterization of urease isolated from the pathogenic fungus Coccidioides immitis.

Coccidioides immitis, the causative agent of San Joaquin Valley fever (coccidioidomycosis), produces a urease which has been suggested to contribute to the virulence of this fungal pathogen. Urease catalyzes the hydrolysis of urea and has been proposed to at least partly account for alkalinity of the microenvironment in which C. immitis grows due to the release of ammonia and ammonium ions. The C. immitis urease was purified to homogeneity (1048-fold) from the mycelial cytosol by chromatographic fractionation. The sequence of 12 N-terminal amino-acid residues of the purified, native polypeptide was identical to that predicted by the translated urease gene sequence which has been reported. The isolated enzyme exhibited a specific activity in the presence of urea of 1750 micromol min(-1) mg(-1) protein, has a native molecular mass of 450 kDa, revealed a Km for urea of 4.1 mM, had a pH optimum of 8.0 and is heat stable. Hydroxyurea, acetohydroxamic acid (AHA) and boric acid each inhibited activity of the purified enzyme. Urease activity was enhanced by the presence of 5-10 mM concentrations of Mg2+ or Mn2+, but inhibited by Li+, Ni2+, Cu2+ or Zn2+. The reversible urease inhibitor, AHA, blocked enzyme activity in the crude mycelial cytosolic fraction when added at a concentration of 10 mM. On the other hand, 10 mM AHA added to 4-day-old mycelial cultures only partially decreased the amount of ammonium detected in the culture medium. It is evident, therefore, that C. immitis urease activity does not account for the total amount of ammonia secreted during in vitro growth of the pathogen. Other metabolic sources of ammonia, which may also contribute to the virulence of C. immitis, are under investigation.

Recombinant urease and urease DNA of Coccidioides immitis elicit an immunoprotective response against coccidioidomycosis in mice.

Coccidioides immitis antigens which stimulate a T helper cell 1 (Th1) pathway of host immune response are considered to be essential components of a vaccine against coccidioidomycosis. Recombinant urease (rURE) and recombinant heat shock protein 60 (rHSP60) of C. immitis were expressed in Escherichia coli and tested as vaccine candidates in BALB/c mice. A synthetic oligodeoxynucleotide which contained unmethylated CpG dinucleotides and was previously shown to enhance a murine Th1 response was used as an immunoadjuvant. T cells isolated from the spleens and lymph nodes of the rURE- and rHSP60-immune mice showed in vitro proliferative responses to the respective recombinant protein, but only those T lymphocytes from rURE-immunized mice revealed markedly elevated levels of expression of selected Th1-type cytokine genes. BALB/c mice immunized subcutaneously with rURE and subsequently challenged by the intraperitoneal (i.p.) route with a lethal inoculum of C. immitis arthroconidia demonstrated a significant reduction in the level of C. immitis infection compared to control animals. rHSP60 was much less effective as a protective antigen. Evaluation of cytokine gene expression in lung tissue and levels of recombinant urease-specific immunoglobulins (immunoglobulin G1 [IgG1] versus IgG2a) in murine sera at 12 days after challenge provided additional evidence that immunization with rURE stimulated a Th1 response to the pathogen. Urease was further evaluated by expression of the URE gene in a mammalian plasmid vector (pSecTag2A.URE) which was used to immunize mice by the intradermal route. In this case, 82% of the vector construct-immunized animals survived more than 40 days after i.p. infection, compared to only 10% of the mice immunized with the vector alone. In addition, 87% of the pSecTag2A.URE-immunized survivors had sterile lungs and spleens. These data support the need for further evaluation of the C. immitis urease as a candidate vaccine against coccidioidomycosis.

Cloning and expression of the gene which encodes a tube precipitin antigen and wall-associated beta-glucosidase of Coccidioides immitis.

We report the structure and expression of the Coccidioides immitis BGL2 gene which encodes a previously characterized 120-kDa glycoprotein of this fungal respiratory pathogen. The glycoprotein is recognized by immunoglobulin M tube precipitin (TP) antibody present in sera of patients with coccidioidomycosis, a reaction which has been used for serodiagnosis of early coccidioidal infection. The deduced amino acid sequence of BGL2 shows 12 potential N glycosylation sites and numerous serine-threonine-rich regions which could function as sites for O glycosylation. In addition, the protein sequence includes a domain which is characteristic of family 3 glycosyl hydrolases. Earlier biochemical studies of the purified 120-kDa TP antigen revealed that it functions as a beta-glucosidase (EC 3.2.1.21). Its amino acid sequence shows high homology to several other reported fungal beta-glucosidases which are members of the family 3 glycosyl hydrolases. Results of previous studies have also suggested that the 120-kDa beta-glucosidase participates in wall modification during differentiation of the parasitic cells (spherules) of C. immitis. In this study we showed that expression of the BGL2 gene is elevated during isotropic growth of spherules and the peak of wall-associated BGL2 enzyme activity correlates with this same phase of parasitic cell differentiation. These data support our hypothesis that the 120-kDa beta-glucosidase plays a morphogenetic role in the parasitic cycle of C. immitis.

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.

Molecular characterization and influence on fungal development of ALP2, a novel serine proteinase from Aspergillus fumigatus.

A novel subtilisin-related serine proteinase (ALP2) [EC 3.4.21.48] with a broad range of activity between pH 4.5 and 11.0 was released from a cell wall fraction of Aspergillus fumigatus by an alkaline pH shift. The enzyme which was not detected in the culture supernatant was partially purified by phenylbutylamine agarose chromatography. The N-terminal sequence revealed that ALP2 is the same protein identified as the major allergen of A. fumigatus in patients suffering from extrinsic bronchial asthma (Shen et al. 1999, Int. Arch. Allergy Immunol. 119, 259-264). Based on this N-terminal sequence and on a conserved region of fungal subtilisins, a specific PCR probe was generated and the ALP2 genomic and cDNA were isolated from corresponding phage libraries. ALP2 shares a 49% identity with the vacuolar proteinase B (PrB) of Saccharomyces cerevisiae. In addition there is a 78% identity with PEPC, a serine proteinase which has been described in Aspergillus niger. Targeted disruption of the ALP2-encoding gene resulted in a slightly decreased speed of vegetative growth and in a more than 80% reduction of sporulation in the alp2-negative mutants, correlated with an approximately 50% reduction of the median diameter of conidiophore vesicles. The requirement of ALP2 for regular sporulation, in addition to its role in allergic asthma, raises further interest in cellular proteinases in respect to morphogenesis and pathogenesis in A. fumigatus.

Molecular cloning and targeted deletion of PEP2 which encodes a novel aspartic proteinase from Aspergillus fumigatus.

An aspartic proteinase PEP2 [EC 3.4.23.25] was purified from a cell wall fraction of Aspergillus fumigatus. The enzyme, which showed a broad range of activity from pH 2.0 to 7.0 and migrated as a single band of 39 kDa in SDS-PAGE, was not detected in the culture supernatant. A specific gene probe was designed on the basis of the N-terminal sequence of the native protein, and the PEP2 genomic and cDNA were isolated from corresponding libraries. The deduced amino acid sequence of PEP2 consists of 398 amino acids. A signal sequence of 18 amino acids and a proregion of another 52 amino acids were identified. The mature protein consists of 328 amino acids which include the two DTG-motifs of the active site common to almost all pepsin-like enzymes. PEP2 showed a 64% identity with the vacuolar proteinase A (PrA), of Saccharomyces cerevisiae, and an 88% identity with PEPE, an aspartic proteinase of Aspergillus niger. Recombinant PEP2 was overexpressed in Pichia pastoris and the active enzyme was secreted into the culture supernatant. Targeted deletion of PEP2 did not affect vegetative growth or cell and colony morphology. Identification of proteinases, such as PEP2, which are apparently associated with the Aspergillus cell wall raises new interest in these molecules with respect to their possible function in the pathogenesis of invasive aspergillosis.

Disruption of the gene which encodes a serodiagnostic antigen and chitinase of the human fungal pathogen Coccidioides immitis.

Disruption of genes in medically important fungi has proved to be a powerful tool for evaluation of putative virulence factors and identification of potential protein targets for novel antifungal drugs. Chitinase has been suggested to play a pivotal role in autolysis of the parasitic cell wall of Coccidioides immitis during the asexual reproductive cycle (endosporulation) of this systemic pathogen. Two chitinase genes (CTS1 and CTS2) of C. immitis have been cloned. Preliminary evidence has suggested that expression of CTS1 is markedly increased during endospore formation. The secreted CTS1 chitinase has also been shown to react with patient anti-Coccidioides complement-fixing (CF) antibody and is a valuable aid in the serodiagnosis of coccidioidomycosis. To examine the role of CTS1 in the morphogenesis of parasitic cells, the CTS1 gene was disrupted by a single, locus-specific crossover event. This resulted in homologous integration of a pAN7.1 plasmid construct that contained a 1.1-kb fragment of the chitinase gene into the chromosomal DNA of C. immitis. Results of Southern hybridizations, immunoblot analyses of culture filtrates using both CTS1-specific murine antiserum and serum from a patient with confirmed coccidioidal infection, an immunodiffusion test for CF antigenicity, and substrate gel electrophoresis assays of chitinase activity confirmed that the CTS1 gene was disrupted and nonfunctional. This is the first report of a successful targeted gene disruption in C. immitis. However, loss of CTS1 function had no effect on virulence or endosporulation. Comparative assays of chitinase activity in the parental and Deltacts1 strains suggested that the absence of a functional CTS1 gene can be compensated for by elevated expression of the CTS2 gene. Current investigations are focused on disruption of CTS2 in the Deltacts1 host to further evaluate the significance of chitinase activity in the parasitic cycle of C. immitis.

Vital staining of fungal elements in deep-seated mycotic lesions during experimental murine mycoses using the parenterally applied optical brightener Blankophor.

Optical brighteners of the diaminostilbene type are fluorescent dyes which are popular diagnostic tools in the mycology laboratory. While these dyes are conventionally used for the in vitro diagnosis of mycoses, their low toxicity and chemical reactivity have led us to investigate their potential use for in vivo staining of fungal elements in mycotic tissue. In mice we have established deep-seated candidiasis, cryptococcosis, aspergillosis and zygomycosis, as well as coccidioidomycosis, histoplasmosis and blastomycosis. After establishment of infection, which mostly required immunosuppression, a single dose of 100 microl of an aqueous solution (2.2 x 10(-4) M) of the optical brightener Blankophor P fluessig (4,4'-Bis [(4-anilino-6-substituted-1,3,5-triazine-2-yl) amino] stilbene-2,2'-disulfonic acid) was injected by the tail vein and the animals were sacrificed 1 h later. Sections of freshly prepared target organs were directly subjected to epifluorescence microscopy using an appropriate filter kit. In most cases, fluorescent fungal elements could be detected in the murine tissue. There was little evidence for uptake of the dye by non-infected tissues. It is suggested that radioactive labeling may render parenteral Blankophor suitable for radiographic localization of deep-seated mycotic foci in the host.

Sequence, expression and functional analysis of the Coccidioides immitis ODC (ornithine decarboxylase) gene.

The ornithine decarboxylase (ODC) gene of the human respiratory fungal pathogen, Coccidioides immitis (Ci) was cloned, sequenced, chromosome-mapped, and expressed in Escherichia coli (Ec). The genomic, cDNA and translated sequences are presented. Transformation of an ODC null mutant strain of Ec (EWH 319) with the Ci ODC gene was conducted to confirm function of the protein encoded by the fungal gene. Activity of the enzyme by the bacterial transformant was inhibited by 1, 4-diamino-2-butanone (DAB), a known inhibitor of eukaryotic ODC. Temporal expression of the Ci ODC gene during the parasitic cell cycle is constitutive, based on results of RT PCR. However, results of enzyme activity assays of cell homogenates obtained at different stages of parasitic cell development in vitro showed that the functional protein is present only during periods of isotropic growth and segmentation, and these morphogenetic events can be arrested by the addition of DAB. The observed absence of a difference in steady-state mRNA transcript amounts, and the developmentally correlated variation in levels of enzyme activity, suggest a translational or post-translational mechanism of ODC regulation. Since no PEST sequence was detected in the Ci ODC, enzyme regulation by programmed protein degradation as reported for many other eukaryotic ODCs may not occur in this case. ODC activity appears to play a key role in the morphogenesis of Ci, and the enzyme could be a rational target for therapy of disseminated coccidioidomycosis.

Urease as a virulence factor in experimental cryptococcosis.

Urease catalyzes the hydrolysis of urea to ammonia and carbamate and has been found to be an important pathogenic factor for certain bacteria. Cryptococcus neoformans is a significant human pathogenic fungus that produces large amounts of urease; thus we wanted to investigate the importance of urease in the pathogenesis of cryptococcosis. We cloned and sequenced the genomic locus containing the single-copy C. neoformans urease gene (URE1) and used this to disrupt the native URE1 in the serotype A strain H99. The ure1 mutant strains were found to have in vitro growth characteristics, phenoloxidase activity, and capsule size similar to those of the wild type. Comparison of a ure1 mutant with H99 after intracisternal inoculation into corticosteroid-treated rabbits revealed no significant differences in colony counts recovered from the cerebrospinal fluid. However, when these two strains were compared in both the murine intravenous and inhalational infection models, there were significant differences in survival. Mice infected with a ure1 strain lived longer than mice infected with H99 in both models. The ure1 strain was restored to urease positivity by complementation with URE1, and two resulting transformants were significantly more pathogenic than the ure1 strain. Our results suggest that urease activity is involved in the pathogenesis of cryptococcosis but that the importance may be species and/or infection site specific.

A major cell surface antigen of Coccidioides immitis which elicits both humoral and cellular immune responses.

Multinucleate parasitic cells (spherules) of Coccidioides immitis isolates produce a membranous outer wall component (SOW) in vitro which has been reported to be reactive with antibody from patients with coccidioidal infection, elicits a potent proliferative response of murine immune T cells, and has immunoprotective capacity in a murine model of coccidioidomycosis. To identify the antigenic components of SOW, the crude wall material was first subjected to Triton X-114 extraction, and a water-soluble fraction derived from this treatment was examined for protein composition and reactivity in humoral and cellular immunoassays. Protein electrophoresis revealed that the aqueous fraction of three different isolates of C. immitis each contained one or two major glycoproteins (SOWgps), distinguished by their molecular sizes, which ranged from 58 to 82 kDa. The SOWgps, however, showed identical N-terminal amino acid sequences, and each was recognized by sera from patients with C. immitis infection. Antibody raised against the purified 58-kDa glycoprotein (SOWgp58) of the Silveira isolate was used for Western blot and immunolocalization analyses. Expression of SOWgp was shown to be parasitic phase specific, and the antigen was localized to the membranous SOW. The water-soluble fraction of SOW and the purified SOWgp58 were tested for the ability to stimulate proliferation of human peripheral monocytic cells (PBMC). The latter were obtained from healthy volunteers with positive skin test reaction to spherulin, a parasitic-phase antigen of C. immitis, and from volunteers who showed no skin test reaction to the same antigen. The SOW preparations stimulated proliferation of PBMC from skin test-positive but not skin test-negative donors, and the activated cells secreted gamma interferon, which is indicative of a T helper 1 pathway of immune response. Results of this study suggest that SOWgp is a major parasitic cell surface-expressed antigen that elicits both humoral and cellular immune responses in patients with coccidioidal infection.

Isolation and confirmation of function of the Coccidioides immitis URA5 (orotate phosphoribosyl transferase) gene.

The OPRTase (URA5) gene of the human pathogenic fungus, Coccidioides immitis (Ci), was cloned, sequenced, chromosome-mapped and expressed both by transformation of Escherichia coli and by complementation of wdura5Delta, an auxotrophic strain of Wangiella dermatitidis (Wd) with a disrupted URA5 gene. A functional assay of the recombinant URA5 expressed by E. coli was conducted to ensure that the isolated Ci gene encodes the appropriate enzyme. In the absence of a transformation system for Ci, we also used a reported method of introduction of heterologous DNA into cells of the phylogenetically related fungus, Wangiella dermatitidis, to confirm the function of the Ci URA5 gene. Both the genomic and cDNA sequences of the Ci URA5 gene are presented. The transcription start point and two poly(A) addition sites were confirmed. The gene contains a 714-bp ORF that translates a 238-amino-acid (aa) protein of 25.5kDa and pI of 6.5. No introns are present. The translated protein contains a single, putative N-glycosylation site. The deduced Ci protein showed 55-63% aa sequence similarity to reported fungal OPRTases. The URA5 gene was mapped to chromosome IV of Ci, and was shown to be a single copy gene by Southern and Northern hybridizations. Transformation of the wdura5Delta mutant to prototrophy was accomplished by electroporation of Wd yeast cells with the Ci URA5 gene. Cellular uptake of the heterologous DNA was confirmed by Southern hybridization. The stable transformants were unable to grow on a medium containing 5-FOA. Expression of the Ci URA5 gene can be used as a selectable marker for a transformation system, and the latter is essential for molecular studies of this pathogenic fungus.

Immunogenicity of a 48-kilodalton recombinant T-cell-reactive protein of Coccidioides immitis.

The outcome of coccidioidomycosis depends to a large extent on the effectiveness of the T-cell-mediated immune (CMI) response to the fungal pathogen. For this reason, identification of Coccidioides immitis antigens which stimulate T cells is important for understanding the nature of host defense against the organism and essential for the development of an effective vaccine. Here we describe the immunogenicity of a 48-kDa T-cell-reactive protein (TCRP). The antigen is expressed by parasitic cells and localized in the cytoplasm. It stimulates the proliferative response and production of gamma interferon by T cells of mice immunized with C. immitis spherules. Specific antibody reactive with the recombinant TCRP (rTCRP) was detected in sera of patients with confirmed coccidioidal infection, and the highest titers of antibody to the recombinant protein correlated with elevated titers to the serodiagnostic complement fixation antigen of C. immitis. These results suggest that the TCRP is presented to the host during the course of infection. Immunization of BALB/c and C3H/HeN mice with the rTCRP affords a modest but significant level of protection against an intraperitoneal challenge with C. immitis. It is suggested that the rTCRP may be able to contribute to a multicomponent vaccine designed to stimulate CMI response against the parasitic cycle of C. immitis.

Isolation and characterization of the urease gene (URE) from the pathogenic fungus Coccidioides immitis.

The urease (URE)-encoding gene from Coccidioides immitis (Ci), a respiratory fungal pathogen of humans, was cloned, sequenced, chromosome-mapped and expressed. Both the genomic and cDNA sequences are reported. The transcription start point and poly(A)-addition site were confirmed. The URE gene contains eight introns and a 2517-bp ORF that translates a 839-amino-acid (aa) protein of 91.5 kDa and pI of 5.5, as deduced by computer analysis of the nucleotide sequence. The translated protein revealed eight putative N-glycosylation sites. The deduced URE showed comparable levels of homology to reported URE of the jack bean plant (Canavalia ensiformis; 71.8%) and URE of several genera of bacteria (Bp, 71.7%; Hp, 68.3%; Ka, 71.6%; Pm, 71.9%). The URE gene was mapped to chromosome III of Ci and was shown to be a single copy gene by Southern hybridization. Expression of a 1687-bp fragment of the URE gene in E. coli resulted in the production of a 63-kDa recombinant protein that was recognized in an immunoblot by antiserum raised against the Ka URE homolog. This is the first report of a fungal URE gene.

The hsp60 gene of the human pathogenic fungus Coccidioides immitis encodes a T-cell reactive protein.

A heat shock protein-encoding gene (hsp60) from the human respiratory fungal pathogen, Coccidioides immitis (Ci), was cloned, sequenced, chromosome-mapped, expressed and immunolocalized in parasitic cells. Both the genomic and cDNA sequences are presented. The transcription start point and poly (A) addition site were confirmed. The hsp60 gene contains two introns and a 1782-bp ORF which translates a 594-amino acid (aa) protein of 62.4 kDa and pI of 5.6. The translated protein revealed two potential N-glycosylation sites. The deduced HSP60 showed 78-83% aa sequence similarity to reported fungal HSP60 proteins. The hsp60 gene was mapped to chromosome III of Ci and was shown to be a single copy gene by Southern and Northern hybridization. Expression of a 1737-bp cDNA fragment of the hsp60 gene in E. coli resulted in production of a recombinant protein. Amino acid sequence analysis of the recombinant protein confirmed that it was encoded by the Ci hsp60 gene. Antiserum raised in mice against the isolated recombinant protein immunolocalized HSP60 in the cytoplasm and wall of parasitic cells of Ci. The recombinant HSP60 was used to immunize BALB/c mice and was shown to induce proliferation of T cells isolated from lymph nodes of these animals. The hsp60 gene of Ci is the first reported heat-shock protein gene of this human pathogen.