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PubMed2004

Coccidioides posadasii infection alters the expression of pulmonary surfactant proteins (SP)-A and SP-D.

BACKGROUND: Coccidioidomycosis or Valley Fever is caused by Coccidioides in Southwest US and Central America. Primary pulmonary infection is initiated by inhalation of air-borne arthroconidia. Since, lung is the first organ that encounters arthroconidia, different components of the pulmonary innate immune system may be involved in the regulation of host defense. Pulmonary surfactant proteins (SP)-A and SP-D have been recognized to play an important role in binding and phagocytosis of various microorganisms, but their roles in Coccidioides infection are not known. METHODS: In this study, we studied the changes in amounts of pulmonary SP-A, SP-D and phospholipid in murine model of Coccidioides posadasii infection, and binding of SP-A and SP-D to Coccidioidal antigens. Mice were challenged intranasally with a lethal dose of C. posadasii (n = 30 arthroconidia) and bronchoalveolar lavage fluid (BALF) samples were collected on day 10, post infection. In another group of animals, mice were immunized with protective formalin killed spherule (FKS) vaccine prior to infection. The concentrations of BALF SP-A, SP-D, total phospholipid were measured using enzyme linked immunosorbent assay and biochemical assays. RESULTS: We found that in lavage fluid samples of C. posadasii infected mice, the concentrations of total phospholipid, SP-A and SP-D were 17 % (SEM 3.5, p < 0.001), 38 % (SEM 5.8, p < 0.001) and 4 % (SEM 1.3, p < 0.001) of those in lavage fluid samples of non-infected control mice, respectively. However, the concentrations of SP-A and SP-D remained unchanged in BALF samples of C. posadasii protected mice after immunization with FKS vaccine. Also, we found that both SP-A and SP-D bind to Coccidiodal antigens. CONCLUSION: Our results suggest that the C. posadasii infection perturbs the pulmonary SP-A, SP-D, and phospholipids, potentially enabling the disease progression and promoting fungal dissemination.

Adaptation, Physiological↗
PubMed2006

Reexamination of Coccidioides spp. reserved in the Research Center for Pathogenic Fungi and Microbial Toxicoses, Chiba University, based on a multiple gene analysis.

The Research Center for Pathogenic Fungi and Microbial Toxicoses, Chiba University is the only organization in Japan to possess a series isolates of Coccidioides spp., which are the most virulent pathogenic fungi and which are treated as biosafety level 3 microorganisms. Recently, the genus Coccidioides has been classified into two species, C. immitis and C. posadasii, based on their endemic areas and genotyping; the former species is endemic to the state of California, and the latter is endemic to other parts of North and South America. We reevaluated 19 isolates of Coccidioides immitis stored in our center using a multiple gene analysis. Five isolates were identified as C. immitis and 14 as C. posadasii. Their sequence information in GenBank will help to identify the two genospecies of Coccidioides spp.

Coccidioides↗
PubMed1964

THE TREATMENT OF COCCIDIOIDAL MENINGITIS. THE USE OF AMPHOTERICIN B IN A GROUP OF 25 PATIENTS.

In a study of 25 patients the usefulness of amphotericin B in the control of meningeal infection produced by Coccidioides immitis was established. Initial treatment must be intensive, consisting of intravenous and intraspinally administered amphotericin B. Serologic evaluation of coccidioidal disease provides the most important single criterion for determining the course of the meningeal infection and for estimating the response of the patient to amphotericin B therapy. Final control of coccidioidal meningitis rests upon the prevention of relapse after completion of initial intensive therapy. This requires continued suppressive fungistasis by regular intracisternal injections of amphotericin B at intervals of three to seven days after the patient returns home. Such suppressive cisternal therapy does not replace the initial intensive use of both intravenously and intraspinally administered amphotericin B. This "local" type of inhibition of C. immitis is without toxic effect upon the kidney, the red blood cells or the serum potassium values which may be associated with the intravenous administration of amphotericin B. Such intraspinal therapy, by lowering the total intravenous dosage required in the initial phase of treatment, results in a proportionate decrease in the degree of nephrotoxicity produced by amphotericin B. The total intravenous dosage given ordinarily should not exceed 5 grams. The long-term therapeutic plan as outlined permits the development of an adequate immune mechanism that appears essential to complete recovery from coccidioidal meningitis. The importance of such immunity in the recovery process has been previously indicated and confirmed by detailed study of a patient who required immunosuppression for successful homotransplantation of a kidney.

Administration, Intravenous↗
PubMed1988

Enzyme-linked immunosorbent assay for antigens of Coccidioides immitis: human sera interference corrected by acidification-heat extraction.

We describe the distortion caused by sera from patients with coccidioidomycosis in an enzyme-linked immunosorbent assay (ELISA) for coccidioidal antigen. In experimental mixtures without exogenous antigen, immune sera produced an antigen-like effect. In contrast, in mixtures with a relatively large concentration of exogenous antigen, immune sera obscured antigen activity. In both circumstances, preparing specimens with an acidification-heat extraction procedure before assay rectified serum interference, and only in specimens with no exogenous antigen was the correction incomplete. Other evidence, including direct measurement of anticoccidioidal antibody after extraction, suggested that the residual antigen-like activity was not from persistent antibody. Incorporating the extraction procedure into the ELISA of clinical specimens, we found antigen activity in nine of 15 specimens. We conclude that acidification-heat extraction of sera improves detection of coccidioidal antigens and that coccidioidal antigens are present in some patients with coccidioidal infection.

Acids↗
PubMedDate not supplied

Amphotericin B and imidazole therapy for coccidioidal meningitis in children.

Coccidioidal meningitis is a fatal form of Coccidioides immitis infection. Amphotericin B (AMB) therapy has reduced mortality but is itself toxic, and experience with it in very young children is meager. We are treating six children for coccidioidal meningitis diagnosed at 19 to 74 months of age. All had acute hydrocephalus and ventriculitis. The first four patients were initially treated with AMB but were changed to imidazole therapy (miconazole and ketoconazole). In the last two patients therapy was begun with the imidazoles. Ommaya reservoirs for cisternal therapy have been of limited usefulness. AMB therapy has been limited by local and systemic toxicity and by failure in one case. All children have improved with 15 to 22 mg oral ketoconazole per kg per day and 3 to 5 mg intraventricular miconazole for instillation therapy, including those with noncommunicating hydrocephalus in whom the intraventricular drug does not reach the basilar cisterns. Peak concentrations of ketoconazole in ventricular fluid were 0.08 to 5.6 micrograms/ml. Shunt obstruction and bacterial superinfections have been the major causes of morbidity. Imidazole therapy of coccidioidal meningitis may be more effective than is AMB in young children, and it offers the advantages of fewer side effects and not requiring intrathecal or cisternal administration.

Amphotericin B↗
PubMed2000

Comparison of fluconazole and itraconazole in a rabbit model of coccidioidal meningitis.

Coccidioidal meningitis is a devastating disease that requires long-term therapy with little hope of cure. A rabbit model of coccidioidal meningitis was used to compare the therapeutic efficacies of fluconazole (FCZ) and itraconazole (ITZ). Hydrocortisone-treated male New Zealand white rabbits were infected intracisternally with 5.0x10(4) to 5.4x10(4) arthroconidia of Coccidioides immitis. Oral treatment with polyethylene glycol 200 (PEG) (n = 9), FCZ (n = 8; 80 mg/kg of body weight/day), or ITZ (n = 8; 80 mg/kg/day) began 5 days after infection and continued for 28 consecutive days. Both FCZ and ITZ reduced the number of CFU of C. immitis organisms in the spinal cord and brain compared with the number in PEG-treated animals (P< or =0.003), but the results for FCZ and ITZ were not different from each other. Histopathologic severity (semiquantitative scoring system by an observer blinded to treatment) was equally reduced in both FCZ and ITZ treatment groups compared with that in controls (P< or =0.0004). Both treatments resulted in lower cerebrospinal fluid (CSF) protein concentrations and leukocyte counts and faster clearing of C. immitis from CSF compared with the results for PEG-treated controls. Neither drug affected CSF glucose levels. Both compounds were effective at reducing neurological and systemic signs and extending survival (P< or =0.014). FCZ was more effective at reducing head and body shakes, posture changes, and incontinence; ITZ was more effective at reducing continuous fever. Mean levels of FCZ and ITZ in the serum and CSF were determined by bioassay; at 17 to 26 h postdosing, levels were 28.1 to 40.0 and 22.4 to 29.9 microg/ml, respectively, for FCZ and 0.77 to 2.51 and 0 microg/ml, respectively, for ITZ. The sera of most animals developed antibody to C. immitis, but azole treatment attenuated antibody development in CSF and its titer. In conclusion, both FCZ and ITZ were efficacious, but neither was curative in a rabbit model of coccidioidal meningitis.

Animals↗
PubMed2002

Efficacy of intravenous liposomal amphotericin B (AmBisome) against coccidioidal meningitis in rabbits.

The efficacy of intravenously administered liposomal amphotericin B (AmBisome [AmBi]) for the treatment of experimental coccidioidal meningitis was compared with those of oral fluconazole (FLC) and intravenously administered conventional amphotericin B (AMB). Male New Zealand White rabbits were infected by intracisternal inoculation of arthroconidia of Coccidioides immitis. Starting 5 days postinfection, animals received one of the following: 5% dextrose water diluent; AMB given at 1 mg/kg of body weight; AmBi given at 7.5, 15, or 22.5 mg/kg intravenously three times per week for 3 weeks; or oral FLC given at 80 mg/kg for 19 days. One week after the cessation of therapy, all survivors were euthanatized, the numbers of CFU remaining in the spinal cord and brain were determined, and histological analyses were performed. All AmBi-, FLC-, or AMB-treated animals survived and had prolonged lengths of survival compared with those for the controls (P < 0.0001). Treated groups had significantly lower numbers of white blood cells and significantly lower protein concentrations in the cerebrospinal fluid compared with those for the controls (P < 0.01 to 0.0005) and had fewer clinical signs of infection (e.g., weight loss, elevated temperature, and neurological abnormalities including motor abnormalities). The mean histological scores for AmBi-treated rabbits were lower than those for FLC-treated and control rabbits (P < 0.016 and 0.0005, respectively); the scores for AMB-treated animals were lower than those for the controls (P < 0.0005) but were similar to those for FLC-treated rabbits. All regimens reduced the numbers of CFU in the brain and spinal cord compared with those for the controls (P < or =0.0005). AmBi-treated animals had 3- to 11-fold lower numbers of CFU than FLC-treated rabbits and 6- to 35-fold lower numbers of CFU than AmB-treated rabbits. Three of eight animals given 15 mg of AmBi per kg had no detectable infection in either tissue, whereas other doses of AmBi or FLC cleared either the brain or the spinal cord of infection in fewer rabbits. In addition, clearance of the infection from both tissues was achieved in none of the rabbits, and neither tissue was cleared of infection in AMB-treated animals. Overall, these data indicate that intravenously administered AmBi is superior to oral FLC or intravenous AMB and that FLC is better than AMB against experimental coccidioidal meningitis. These data indicate that AmBi may offer an improvement in the treatment of coccidioidal meningitis. Additional studies are warranted.

Amphotericin B↗
PubMed1989

Pleural effusion in Michigan caused by Coccidioides immitis after travel to an endemic area.

Primary coccidioidal disease is rarely diagnosed in the midwest in the nonimmunocompromised host. Since coast-to-coast travel is common today, many patients may become exposed to Coccidioides immitis while traveling in endemic areas. We present a case of acute coccidioidal pleural effusion in a Michigan woman who had recently visited northeastern Arizona. Her travel history was the single most important factor in the eventual diagnosis of coccidioidal pleural effusion.

Arizona↗
PubMed2000

Identification and cloning of an aspartyl proteinase from Coccidioides immitis.

A 45 kDa protein was isolated from a soluble vaccine prepared from formaldehyde-killed spherules of Coccidioides immitis. From the N-terminal amino acid sequence, the protein yielded a 17-amino-acid peptide that was homologous to sequences of other fungal aspartyl proteinases. The coccidioidal cDNA encoding the proteinase was amplified using oligonucleotide primers designed from the 45 kDa N-terminal amino acid sequence and a fungal aspartyl proteinase consensus amino acid sequence. The PCR product was cloned and sequenced, and the remaining 5' upstream and 3' downstream cDNA was amplified, cloned, and sequenced. The cDNA encoding the coccidioidal aspartyl proteinase open reading frame was cloned and the fusion protein containing a C-terminal His-tag expressed in E. coli. The recombinant aspartyl proteinase was purified by immobilized metal affinity chromatography. This recombinant protein will be used for further studies to evaluate its antigenicity, including protective immunogenicity.

Amino Acid Sequence↗
PubMed2001

Assessment of the human cellular immune response to T27K, a coccidioidal antigen preparation, by flow cytometry of whole blood.

Whole blood flow cytometry was performed among donors with various clinical forms of coccidioidomycosis using T27K, a coccidioidal antigen preparation protective in mice but not previously studied in humans. The median percent of CD3+ lymphocytes (CD3+) producing intracellular interferon-gamma (IFN-gamma) among healthy immune donors was 0.43%, significantly above that for non-immune donors (0.01%) and greater than that for subjects with other forms of coccidioidomycosis, including chronic pulmonary (0.11%), disseminated (0.09%) and concomitant human immunodeficiency virus (HIV) infection (0.07%) (P < or =0.002 for all). No increase in intracellular interleukin (IL)-10 production or apoptosis was noted in samples incubated with T27K. Among 14 HIV-infected patients with concomitant coccidioidomycosis, seven of eight patients whose peripheral blood CD4 concentration was > 200 cells microl(-1) had > 0.06% of CD3+ produce intracellular IFN-gamma, compared to none of six whose peripheral blood CD4+ lymphocyte concentration was < or =200 cells microl(-1) (P = 0.005). These data indicate that there is a specific human cellular immune response to T27K as a coccidioidal antigen and that this response can be categorized based on the clinical status of the coccidioidally infected patient.

AIDS-Related Opportunistic Infections↗
PubMed1995

Persistent coccidioidal seropositivity without clinical evidence of active coccidioidomycosis in patients infected with human immunodeficiency virus.

We retrospectively identified 13 patients infected with human immunodeficiency virus (HIV) who had persistently positive coccidioidal serological tests without evidence of active coccidioidomycosis--to our knowledge, a heretofore undescribed phenomenon. The median duration of follow-up was 19 months. Five patients developed active coccidioidomycosis during this follow-up period; the median interval from the initial positive serological test to the development of active disease was 23 months. There were no significant differences between patients who developed active coccidioidomycosis and those who did not with regard to CD4 lymphocyte count or previous antifungal therapy. Moreover, when all 13 patients were compared with 21 patients who had HIV infection and active coccidioidomycosis, no differences were found with regard to age, race, risk factor for HIV infection, or initial CD4 lymphocyte count. In summary, persistently positive coccidioidal serological tests in HIV-infected patients appear to represent true coccidioidal infection and are associated with a significant risk of the development of active coccidioidomycosis.

Adult↗
PubMed1995

Coccidioidomycosis among visitors to a Coccidioides immitis-endemic area: an outbreak in a military reserve unit.

An outbreak of coccidioidomycosis occurred in a US Marine reserve unit based in Tennessee after a 3-week training exercise in California that involved substantial exposure to soil and dust. Interviews and serologic testing were done on three occasions (6, 11, and 15 weeks) after the men returned from California, and spherulin skin tests were done at 6 months. Of 27 men, 8 (30%) had evidence of recent coccidioidal infection. Of these, 7 (88%) had an illness consistent with coccidioidomycosis that, despite medical evaluation, was diagnosed incorrectly in 5 men (71%). Diagnosis of coccidioidal pneumonia outside an area in which Coccidioides immitis is endemic is unlikely unless the health care provider is aware that the patient traveled recently. Detection of coccidioidomycosis could be facilitated if organizations that regularly send people to C. immitis-endemic regions were to inform these persons about the risks of infection.

Adult↗
PubMedDate not supplied

Pulmonary coccidioidal mycetoma.

Cavitary pulmonary disease due to coccidioidomycosis is common; however, development of both the underlying cavity and the fungus ball due to Coccidioides immitis is rare and has been reported on only five occasions. We report a case of coccidioidal mycetoma formation as a late sequelae of primary pulmonary coccidioidal pneumonia. Therapy with 2 gm of amphotericin B was unsuccessful and, because of recurrent hemoptysis, the patient required lobar resection. Endosporulating spherules, as well as septate mycelia with arthrospore formation, were found on histologic examination. The medical and surgical management, as well as the potential of such lesions to produce human to human transmission, is discussed.

Coccidioides↗
PubMed2006

Temporal expression of inflammatory mediators in brain basilar artery vasculitis and cerebrospinal fluid of rabbits with coccidioidal meningitis.

Strokes due to transmural vasculitis associated with coccidioidal meningitis result in significant morbidity and mortality. The immunological and inflammatory processes responsible are poorly understood. To determine the inflammatory mediators, i.e. cytokines, chemokines, iNOS, matrix metalloproteinase-9 (MMP-9), that possibly contribute to vasculitis, temporal mRNA expression in brain basilar artery samples and MMP-9 protein in the CSF of male NZW rabbits infected intracisternally with 6.5 x 10(4) arthroconidia of Coccidioides immitis were assessed. Five infected and 3 sham-injected rabbits at each time point were euthanized 4, 9, 14 and 20 days post infection. All infected rabbits had neurological abnormalities and severe vasculitis in the basilar arteries on days 9-20. In basilar arteries of infected animals versus controls, mRNAs encoding for IL-6, iNOS, IFN-gamma, IL-2, MCP-1, IL-1beta, IL-10, TNF-alpha, CCR-1, MMP-9, TGF-beta, as well as MMP-9 protein in CSF, were found to be significantly up-regulated. Thus, this study identified inflammatory mediators associated with CNS vasculitis and meningitis due to C. immitis infection. Assessment of the individual contribution of each mediator to vasculitis may offer novel approaches to the treatment of coccidioidal CNS infection. This study also provides unique methodology for immunology studies in a rabbit model.

Animals↗
PubMed2006

Comparison of itraconazole and fluconazole treatments in a murine model of coccidioidal meningitis.

Coccidioidal meningitis (CM) is a devastating disease that requires long-term therapy and for which there is little hope of a cure. A model was used to compare the efficacies of itraconazole and fluconazole. CD-1 mice were infected intrathecally with 30 to 36 viable arthroconidia of Coccidioides. Oral therapy with cyclodextrin (control) or itraconazole or fluconazole at 10, 25, or 50 mg/kg of body weight twice daily (BID) was given for 12 days, from day 3 of infection. Treatment with both antifungals at all doses prolonged survival compared with that of the control treatment (P < 0.01 to 0.0001). At 50 mg/kg, itraconazole and fluconazole were equivalent, whereas itraconazole at 10 or 25 mg/kg prolonged survival compared to that achieved with fluconazole at these dosages (P < 0.05 and 0.01, respectively). Early histologic analysis (10 days of treatment) with 50 mg/kg BID itraconazole or fluconazole showed suppression of CM in all five animals per group; in quantitative cultures, three of three animals from each group had no detectable infection in the brain, spinal cord, or a site of secondary infection, the lungs. In contrast, four of seven controls showed mild to severe meningitis, with arteritis detected in three animals. In a short-term organ clearance study, 5 days of treatment with 10 or 50 mg/kg BID itraconazole or fluconazole reduced the tissue burdens in the brain and spinal cord compared to the tissue burdens in the controls (P < 0.02 to 0.0003). Fluconazole at 10 mg/kg did not reduce the fungal burden in secondary sites, the lungs and kidneys, whereas this itraconazole dose was more effective in clearing the fungi from both organs (P < 0.05 and P < 0.001, respectively). At 50 mg/kg, itraconazole and fluconazole were equivalent in clearing the fungi from the brain and kidney, but itraconazole was superior to fluconazole in clearing the fungi from the spinal cord and lungs (P < 0.05). Thus, both itraconazole and fluconazole were effective at controlling CM, but neither eliminated Coccidioides from tissues. Overall, itraconazole was more efficacious on an mg/kg basis; at high doses they were similarly effective.

Animals↗
PubMed2000

Comparative efficacies of terbinafine and fluconazole in treatment of experimental coccidioidal meningitis in a rabbit model.

A rabbit model of coccidioidal meningitis was used to compare the therapeutic efficacies of terbinafine (TBF) and fluconazole (FCZ). Hydrocortisone acetate-treated New Zealand White male rabbits were infected intracisternally with either 2.2 x 10(4) or 6.4 x 10(4) Coccidioides immitis arthroconidia. Oral treatment with polyethylene glycol 200 (PEG) twice daily (n = 8), TBF twice daily (n = 9; 200 mg/kg of body weight/day), or FCZ once daily (n = 8; 80 mg/kg/day) began on day 5 and continued for 21 days. Mean survival times were 20, 24, and 32 days for rabbits treated with PEG, TBF, and FCZ, respectively. All of the FCZ-treated animals (100%; P = 0.003), 56% of the TBF-treated animals (P = 0.4), and 25% of the PEG-treated animals survived the length of the study. Both FCZ and TBF were effective at reducing the incidence of paresis. Only FCZ was effective at reducing most neurological and systemic signs. FCZ treatments resulted in lower cerebrospinal fluid (CSF) protein concentrations and leukocyte counts and faster clearing of CSF fungal cultures compared with those for PEG-treated controls, but TBF treatments had no significant effect on these parameters. Neither drug affected CSF glucose levels. Mean serum TBF levels by bioassay were within the range of 3.5 to 6.2 microgram/ml at 1, 2, and 4 h postdosing and 0.35 to 7.0 microgram/ml at 14 h postdosing. No TBF was detected in CSF. Mean FCZ levels (24 to 25.5 h postdosing) by bioassay were 16.4 to 19.2 and 13.5 to 19.2 microgram/ml in serum and CSF, respectively. The reduction in the numbers of CFU in the spinal cord and brain was over 100-fold (P = 0.0005) in FCZ-treated animals and 2-fold (P </= 0.2) in TBF-treated animals compared with those in PEG-treated animals. Histopathologic severity (semiquantitative scoring system) was significantly attenuated by FCZ treatment (P = 0. 05) and was slightly attenuated by TBF treatment compared with that for the controls. In conclusion, TBF appeared to have a slight effect on survival, histology, and reduction of the numbers of CFU in tissue; however, these effects were not significant. FCZ was effective at controlling coccidioidal meningitis.

Animals↗
PubMed1995

Molecular and biochemical characterization of a Coccidioides immitis-specific antigen.

Results of earlier investigations have indicated that the saprobic phase of Coccidioides immitis produces a heat-stable, 19-kDa antigen with serine proteinase activity which has been suggested to be specific for this pathogenic fungus. In the present study we have determined the N-terminal and partial internal amino acid sequences of the purified, 19-kDa antigen, cloned the gene which encodes this polypeptide, and confirmed that the secreted proteinase is a Coccidioides-specific antigen (CS-Ag). Both the genomic and cDNA sequences are reported and reveal that the csa gene which encodes this antigen has no introns. A 543-bp open reading frame encodes a 181-amino-acid-containing protein with a predicted molecular mass of 19.8 kDa and an isoelectric point of 8.3. The csa gene was localized on chromosome I of three representative C. immitis clinical isolates on the basis of Southern hybridizations. Expression of the csa gene in Escherichia coli using the pET21a plasmid vector yielded a recombinant protein that was recognized in immunoblot assays by antibody raised to the purified 19-kDa CS-Ag. Secretion of the native antigen is suggested to occur by cleavage of a putative 23-residue signal peptide. The native CS-Ag showed a low degree of glycosylation. Analysis of the carbohydrate composition of the CS-Ag revealed xylose, mannose, galactose, and glucose. However, the purified antigen showed no affinity for concanavalin A. A PCR method with specificity and high sensitivity for detection of C. immitis genomic DNA, using a pair of synthetic oligonucleotide primers whose sequences were based on that of the csa gene, was developed. A 520-bp product was amplified only when C. immitis genomic DNA was used as the template. The lower limits of DNA detection using this PCR method were 1 pg of C. immitis genomic DNA by ethidium bromide staining and 100 fg after Southern hybridization. The csa gene-based PCR method for detection of C. immitis DNA is useful for culture identification and may have clinical applications for the diagnosis of coccidioidal infections.

Amino Acid Sequence↗
PubMed2000

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.

Animals↗