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Valley fever (coccidioidomycosis) is an endemic fungal pneumonia of the North and South American deserts. The causative agents of Valley fever are the dimorphic fungi Coccidioides immitis and C. posadasii , which grow as mycelia in the environment and as spherules within the lungs of vulnerable hosts. Current diagnostics for Valley fever are severely lacking due to poor sensitivity and invasiveness, contributing to a 23-day median time to diagnosis, and therefore, new diagnostic tools are needed. We are working toward the development of a breath-based diagnostic for coccidioidomycosis, and in this initial study, we characterized the volatile metabolomes (or volatilomes) of in vitro cultures of Coccidioides Using solid-phase microextraction (SPME) and comprehensive two-dimensional gas chromatography coupled to time of flight mass spectrometry (GC×GC-TOFMS), we characterized the volatile organic compounds (VOCs) produced by six strains of each species during mycelial or spherule growth. We detected a total of 353 VOCs that were at least 2-fold more abundant in a Coccidioides culture than in medium controls and found that the volatile metabolome of Coccidioides is more dependent on the growth phase (spherules versus mycelia) than on the species. The volatile profiles of C. immitis and C. posadasii have strong similarities, indicating that a single suite of Valley fever breath biomarkers can be developed to detect both species. IMPORTANCE Coccidioidomycosis, or Valley fever, causes up to 30% of community-acquired pneumonias in highly populated areas of the U.S. desert southwest where the disease is endemic. The infection is difficult to diagnose by standard serological and histopathological methods, which delays appropriate treatment. Therefore, we are working toward the development of breath-based diagnostics for Valley fever. In this study, we characterized the volatile metabolomes (or volatilomes) of six strains each of Coccidioides immitis and C. posadasii , the dimorphic fungal species that cause Valley fever. By analyzing the volatilomes during the two modes of growth of the fungus-mycelia and spherules-we observed that the life cycle plays a significant role in the volatiles produced by Coccidioides In contrast, we observed no significant differences in the C. immitis versus C. posadasii volatilomes. These data suggest that life cycle, rather than species, should guide the selection of putative biomarkers for a Valley fever breath test., (Copyright © 2021 Higgins Keppler et al.)

Coccidioides spp. are important pneumonia-causing pathogens of the American southwest, but little is known about their glycobiology and how their glycosylations differ from other pneumonia-causing fungi. There is mounting preliminary evidence to suggest genus or even species-specific glycosylations in the fungal kingdom due to the presence of unique carbohydrate-active enzymes (CAZymes) in fungal genomes (Deshpande et al. 2008, Glycobiology, 18(8), 626-637; Karkowska-Kuleta and Kozik 2015, Acta Biochim Pol., 62(3), 339-351). If Coccidioides spp.-specific glycans can be identified, it may be possible to exploit these differences to develop more specific diagnostic approaches and more effective therapeutics. Herein, we i) mined Coccidioides spp. and other pathogenic fungal genomes to identify CAZymes specific for Coccidioides spp., ii) proteomically determined the Coccidioides spp. "CAZome" produced in vivo and in vitro, and iii) utilized glycomics to differentiate Coccidioides genus-specific N-glycans from other pathogenic fungi. As far as we are aware, this is the first proteomic and glycomic comparison of the N-glycomes and CAZomes of different fungal genera during infection in human hosts., (© The Author(s) 2019. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Calcineurin is important for fungal virulence and a potential antifungal target, but compounds targeting calcineurin, such as FK506, are immunosuppressive. Here we report the crystal structures of calcineurin catalytic (CnA) and regulatory (CnB) subunits complexed with FK506 and the FK506-binding protein (FKBP12) from human fungal pathogens (Aspergillus fumigatus, Candida albicans, Cryptococcus neoformans and Coccidioides immitis). Fungal calcineurin complexes are similar to the mammalian complex, but comparison of fungal and human FKBP12 (hFKBP12) reveals conformational differences in the 40s and 80s loops. NMR analysis, molecular dynamic simulations, and mutations of the A. fumigatus CnA/CnB-FK506-FKBP12-complex identify a Phe88 residue, not conserved in hFKBP12, as critical for binding and inhibition of fungal calcineurin. These differences enable us to develop a less immunosuppressive FK506 analog, APX879, with an acetohydrazine substitution of the C22-carbonyl of FK506. APX879 exhibits reduced immunosuppressive activity and retains broad-spectrum antifungal activity and efficacy in a murine model of invasive fungal infection.

The aims of this study are to provide protein-based evidence upon which to reannotate the genome of Coccidiodes posadasii, one of two closely related species of Coccidioides, a dimorphic fungal pathogen that causes coccidioidomycosis, also called Valley fever. Proteins present in lysates and filtrates of in vitro grown mycelia and parasitic phase spherules from C. posadasii strain Silveira are analyzed using a GeLC-MS/MS method. Acquired spectra are processed with a proteogenomics workflow comprising a Silveira proteome database, a six-frame translation of the Silveira genome and an ab initio gene prediction tool prior to validation against published ESTs. This study provides evidence for 837 genes expressed at the protein level, of which 169 proteins (20.2%) are putative proteins and 103 (12.3%) are not annotated in the Silveira genome. Additionally, 275 novel peptides are derived from intragenic regions of the genome and 13 from intergenic regions, resulting in 172 gene refinements. Additionally, we are the first group to report translationally active retrotransposon elements in a Coccidioides spp. Our study reveals that the currently annotated genome of C. posadasii str. Silveira needs refinement, which is likely to be the case for many nonmodel organisms., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Aims: This study aimed to evaluate the in vitro activity of miltefosine and levamisole against strains of Coccidioides posadasii in the filamentous phase and strains of Histoplasma capsulatum in filamentous and yeast phases., Methods and Results: Strains of C. posadasii in the filamentous phase (n = 22) and strains of H. capsulatum in filamentous (n = 40) and yeast phases (n = 13) were, respectively, submitted to broth macrodilution and broth microdilution methods, as described by the Clinical and Laboratory Standards Institute, to determine the minimum inhibitory concentration (MIC) and the minimum fungicidal concentration (MFC) of miltefosine and levamisole. The effect of the drugs on cell membrane permeability under osmotic stress conditions and total ergosterol production were also assessed, along with quantification of extravasated molecules. The results show the inhibitory effect of levamisole and miltefosine against C. posadasii and H. capsulatum and the effect of these drugs on ergosterol synthesis and the permeability of the plasma membrane using subinhibitory concentrations against strains subjected to osmotic stress. Levamisole was also able to cause the release of nucleic acids., Conclusions: Miltefosine and levamisole are capable of inhibiting the in vitro growth of C. posadasii and H. capsulatum, probably by altering the permeability of the cellular membrane., Significance and Impact of the Study: This work presents alternatives for the treatment of histoplasmosis and coccidioidomycosis, raising the possibility of the use of miltefosine and levamisole as adjuvants in antifungal therapy, providing perspectives for the design of in vivo studies., (© 2015 The Society for Applied Microbiology.)

Coccidioides is the causative agent of a potentially life-threatening respiratory disease of humans. A feature of this mycosis is that pH measurements of the microenvironment of pulmonary abscesses are consistently alkaline due to ammonia production during the parasitic cycle. We previously showed that enzymatically active urease is partly responsible for elevated concentrations of extracellular ammonia at sites of lung infection and contributes to both localized host tissue damage and exacerbation of the respiratory disease in BALB/c mice. Disruption of the urease gene (URE) of Coccidioides posadasii only partially reduced the amount of ammonia detected during in vitro growth of the parasitic phase, suggesting that other ammonia-producing pathways exist that may also contribute to the virulence of this pathogen. Ureidoglycolate hydrolase (Ugh) expressed by bacteria, fungi and higher plants catalyzes the hydrolysis of ureidoglycolate to yield glyoxylate and the release CO2 and ammonia. This enzymatic pathway is absent in mice and humans. Ureidoglycolate hydrolase gene deletions were conducted in a wild type (WT) isolate of C. posadasii as well as the previously generated Δure knock-out strain. Restorations of UGH in the mutant stains were performed to generate and evaluate the respective revertants. The double mutant revealed a marked decrease in the amount of extracellular ammonia without loss of reproductive competence in vitro compared to both the WT and Δure parental strains. BALB/c mice challenged intranasally with the Δugh/Δure mutant showed 90% survival after 30 days, decreased fungal burden, and well-organized pulmonary granulomas. We conclude that loss of both Ugh and Ure activity significantly reduced the virulence of this fungal pathogen., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Coccidioidomycosis is a systemic mycosis caused by the dimorphic fungi Coccidioides spp. The treatment for chronic and/or disseminated coccidioidomycosis can be prolonged and complicated. Therefore, the search for new drugs is necessary. Farnesol is a precursor in the sterol biosynthesis pathway that has been shown to present antifungal activity. Thus, the objective of this study was to evaluate the in vitro antifungal activity of farnesol alone and in combination with antifungal agents against clinical and environmental strains of Coccidioides posadasii as well as to determine their effect on the synthesis of ergosterol and on cell permeability. This study employed the broth macrodilution method to determine the MIC of farnesol against 18 strains of C. posadasii. Quantification of ergosterol was performed with 10 strains of C. posadasii after exposure to subinhibitory concentrations of farnesol. Finally, the activity of farnesol was evaluated in the presence of osmotic stress, induced by the addition of NaCl to the culture medium, during the susceptibility tests. The results showed that farnesol exhibited low MICs (ranging from 0.00171 to 0.01369 mg/liter) against all tested strains. The combination of farnesol with the antifungals showed synergistic effects (fractional inhibitory concentration index [FICI] ≤ 0.5). As for the ergosterol quantification, it was observed that exposure to subinhibitory concentrations of farnesol decreased the amount of ergosterol extracted from the fungal cells. Furthermore, farnesol also showed lower MIC values when the strains were subjected to osmotic stress, indicating the action of this compound on the fungal membrane. Thus, due to the high in vitro antifungal activity, this work brings perspectives for the performance of in vivo studies to further elucidate the effects of farnesol on the host cells.

Aspergillus species are responsible for most cases of fatal mold infections in immunocompromised patients, particularly in those receiving hematopoietic stem cell transplants. Experimental vaccines in mouse models have demonstrated a promising avenue of approach for the prevention of aspergillosis, as well as infections caused by other fungal pathogens, such as Coccidioides, the etiological agent of valley fever (coccidioidomycosis). Here, we investigated the hyphal proteomes of Aspergillus fumigatus and Coccidioides posadasii via quantitative MS(E) mass spectrometry with the objective of developing a vaccine that cross-protects against these and other species of fungi. Several homologous proteins with highly conserved sequences were identified and quantified in A. fumigatus and C. posadasii. Many abundant proteins from the cell wall of A. fumigatus present themselves as possible cross-protective vaccine candidates, due to the high degree of sequence homology to other medically relevant fungal proteins and low homologies to human or murine proteins., (© 2012 New York Academy of Sciences.)

A protein microarray containing fungal antigens (the "mycoarray") has been set up to provide rapid and appropriate serodiagnosis of primitive endemic mycoses, an important cause of morbidity and mortality in an increasingly high number of patients. The mycoarray consists of three antigen extracts (histoplasmin, coccidioidin and Coccidioides "TP") and antibody dilution curves were spotted on microarray slides. The arrays were processed with coccidioidomycosis and histoplasmosis patients� sera or with control sera and the occurring immunocomplexes were detected by indirect immunofluorescence. In agreement with clinical and microbiological diagnosis, the results distinguished between histoplasmosis and coccidioidomycosis patients. In addition, the assay could clearly discriminate between IgM and IgG antibody reactivity. No reactivity was ever observed in the arrays processed with negative control sera. Therefore, this pilot study demonstrates that the "mycoarray" is sensitive and specific enough to discriminate between healthy individuals and patients with histoplasmosis or coccidioidomycosis. Because of miniaturization and multiparametricity, the new assay cuts costs and processing time. Thus, once clinically validated and implemented as a large-scale array, the "mycoarray" will be ready to be applied to the daily clinical practice.

Search of the protein database with the aflatoxin pathway polyketide synthase (PKS) revealed putative PKSs in the pathogenic fungi Coccidioides immitis and Coccidioides posadasii that could require partnerships with a pair of fatty acid synthase (FAS) subunits for the biosynthesis of fatty acid-polyketide hybrid metabolites. A starter unit:acyl-carrier protein transacylase (SAT) domain was discovered in the nonreducing PKS. This domain is thought to accept the fatty acid product from the FAS to initiate polyketide synthesis. We expressed the C. immitis SAT domain in Escherichia coli and showed that this domain, unlike that from the aflatoxin pathway PKS, transferred octanoyl-CoA four times faster than hexanoyl-CoA. The SAT domain also formed a covalent octanoyl intermediate and transferred this group to a free-standing ACP domain. Our results suggest that C. immitis/posadasii, both human fungal pathogens, contain a FAS/PKS cluster with functional similarity to the aflatoxin cluster found in Aspergillus species. Dissection of the PKS and determination of in vitro SAT domain specificity provides a tool to uncover the growing number of similar sequenced pathways in fungi, and to guide elucidation of the fatty acid-polyketide hybrid metabolites that they produce.

Molecular studies of the genome of the fungus Coccidioides have demonstrated two nearly identical, but well-identified species, Coccidioides immitis and C. posadasii, known as "California" and "non-California" species, respectively. The objective of this study was to determine, through molecular methods, whether both species of Coccidioides are present in Mexican patients with coccidioidomycosis and to estimate, their geographical distribution in Mexico. We analyzed 56 clinical isolates of Coccidioides spp. from Mexican patients. Molecular identification of each strain was done by means of real time PCR using TaqMan(R) probes to amplify single nucleotide polymorphisms (SNPs) in four target sequences, loci, named proline 157, proline 174, hexokinase 149 and glucose-synthase 192. SNP analysis identified two of the 56 isolates as Coccidioides immitis and the remaining 54 as C. posadasii. The dual probe assay that included proline 157, proline 174 and glucose-synthase 192 gave consistent results on SNP differentiation between the two species. In contrast, the template matching hexokinase 149 gave negative results for any species in 34 samples. Our results did not show geographical overlap of the species, and they also confirmed that C. posadasii is the most frequent species in Mexico. A vast majority of C. posadasii strains were localized in the north-central region of the country.

Parenchymal brain involvement from disseminated coccidioidomycosis occurs rarely and there are few documented pediatric cases. We report a four-month-old male infant with a cerebellar lesion seen in the brain on computed tomography (CT). Coccidioides immitis (C. immitis) grew on bronchoscopic fluid samples and serum titers to C. immitis were 1:1024. Antifungal treatment was initiated and after 3 months, CT scans demonstrated brain mass resolution and serum titers were decreased.

Coccidioidomycosis is a fungal infection endemic to the southwestern United States. Typically a respiratory illness, coccidioidomycosis can rarely present as extrapulmonary infection. Skeletal coccidioidomycosis occurs in 20% to 50% of disseminated infections. Skeletal coccidioidomycosis is a chronic and progressive infection that eventually results in bone destruction and loss of function and often involves adjacent structures, such as joints, muscles, and tendons and other soft tissues. Sinus tract formation may occur. This infection may be multifocal. Although radiographs, white blood cell count scans, and other imaging methods identify and define relevant abnormalities, histopathologic examination with culture of the involved bone is the only means to confirm the diagnosis. Serologic testing is adjunctive, and complement fixation titers can be evaluated serially to assess response to treatment. A number of studies addressing the efficacy of various antifungal agents have been performed, and the results of these studies as they pertain to skeletal coccidioidomycosis are summarized herein. Among the various studies, response rates ranged from 23% to 100%, but relapse was common. A combination of medical therapy-often, itraconazole or fluconazole-and surgical débridement is often needed to control skeletal coccidioidomycosis. Early diagnosis and treatment are critical to avoid long-term problems with chronically infected bones and joints. Anatomical issues, diagnostic studies, and data related to treatment of this form of extrapulmonary coccidioidomycosis are reviewed in this article.

Coccidioidomycosis (Valley Fever) is a fungal infection found in the southwestern United States, northern Mexico, and some places in Central and South America. The fungi that cause it (Coccidioides immitis and Coccidioides posadasii) are normally soil dwelling, but, if disturbed, become airborne and infect the host when their spores are inhaled. It is thus natural to surmise that weather conditions, which foster the growth and dispersal of Coccidioides, must have an effect on the number of cases in the endemic areas. This article reviews our attempts to date at quantifying this relationship in Kern County, California (where C. immitis is endemic). We have examined the effect on incidence resulting from precipitation, surface temperature, and wind speed. We have performed our studies by means of a simple linear correlation analysis, and by a generalized autoregressive moving average model. Our first analysis suggests that linear correlations between climatic parameters and incidence are weak; our second analysis indicates that incidence can be predicted largely by considering only the previous history of incidence in the county-the inclusion of climate- or weather-related time sequences improves the model only to a relatively minor extent. Our work therefore suggests that incidence fluctuations (about a seasonally varying background value) are related to biological and/or anthropogenic reasons, and not so much to weather or climate anomalies.

Coccidioidomycosis is a disease of both national and worldwide importance that is most often diagnosed in nonendemic regions. The endemic region for Coccidioides spp. lies exclusively in the Western Hemisphere. Coccidioides spp. has long been identified in semiarid areas of the United States and Mexico, and endemic foci have been described in areas of Central and South America. Infection is usually the result of activities that cause the fungus to become airborne and inhaled by a susceptible host. Underlying medical diseases that affect T cell function are known to increase the risk of disseminated disease and include human immunodeficiency virus, cancer, and disease processes requiring transplantation and its subsequent immunosuppressive agents. In recent years the incidence of the coccidioidomycosis has increased in California and Arizona, which may be partially due to the massive migration of Americans to the Sunbelt states. To date the highest number of cases reported in Arizona was in 2004, when a total of 3,665 cases of coccidioidomycosis was reported, representing a 281% increase since 1997. Statistics on the prevalence and incidence of coccidioidomycosis in Latin America either are fragmentary or simply are not available.

Studies of field- and patient-derived isolates conducted over the past 75 years have provided a general picture of the population structure of Coccidioides, the cause of coccidioidomycosis. Premolecular studies provided a general outline of the geographical range, epidemiology and distribution of the fungus. Recent studies based on molecular markers have demonstrated that the genus is comprised of two genetically diverse, and genetically isolated, species: Coccidioides immitis and C. posadasii. Both species are composed of biogeographically distinct populations. Structure for two of these populations (C. immitis from central California, and C. posadasii from southern Arizona) indicates that frequent genetic recombination occurs within the entire geographic range of each population, even though sex has never been observed in the genus. Outbreaks of coccidioidomycosis are not the result of the spread of a single clonal isolate, but are caused by a diversity of genotypes. Although it is now possible to match patient isolates to populations, the lack of apparent structure within each population and the current paucity of environmental isolates limit map-based epidemiological approaches to understanding outbreaks. Therefore, a comprehensive database comprised of soil-derived isolates from across the biogeographic range of Coccidioides will improve the utility of this approach. Appropriate collection of environmental isolates will assist the investigation of remaining questions regarding the population biology of Coccidioides. The comparative genomics of representative genotypes from both species and all populations of Coccidioides will provide a thorough set of genetic markers in order to resolve the population genetics of this pathogenic fungus.

Prior to the 1950s no effective therapy for coccidioidomycosis existed. The advent of amphotericin B ushered in the therapeutic era for coccidioidomycosis. Until this time amphotericin B and its lipid congeners have been regarded as the "gold standard" of therapy for severe pulmonary and disseminated coccidioidomycosis. The availability of azoles and later triazoles for the past three decades have relegated the amphotericins into a rescue mode, used mainly in widely disseminated cases, azole intolerance, or when there are contraindications to Azoles, such as pregnancy. In meningitis the intrathecal use of amphotericin B is still used frequently by some clinicians alone or with a triazole. The newer lipid preparations, while more expensive, have significantly reduced toxicity, particularly nephropathy.

While the whole killed spherule vaccine, protective in mice and monkeys, did not prevent coccidioidal disease in humans, the 27K vaccine, a soluble derivative, retains protective activity in mice with little irritant action. Gel filtration and anion exchange fractions of thimerosal-inactivated spherules (T27K), when administered with alum adjuvant, also protect mice against lethal respiratory coccidioidal challenge. However, the superb protection afforded by T27K antigens is maintained for some 3 months, but may then diminish. This appears unrelated to the aging of the mice. Prolongation of the protective action may require addition of a different adjuvant or administration of booster doses of vaccine.

Coccidioidomycosis is a mild to life-threatening disease in otherwise healthy humans and other mammals caused by the fungus Coccidioides spp. Understanding the development of the unique dimorphic life cycle of Coccidioides spp. and its role in pathogenesis has been an area of research focus. However, nuclear behavior during the saprobic and parasitic life cycle has not been studied intensively. In this study, green fluorescent protein (GFP) was fused to histone H1 and introduced into Coccidioides posadasii (C. posadasii) strain Silveira to monitor the nuclear behavior of the fungus during the saprobic and parasitic stages of the life cycle. We constructed an Agrobacterium tumefaciens-mediated transformation (ATMT) vector that had in its T-DNA region a hygromycin-resistance gene as well as the fused histone H1-GFP gene under the control of the histone H3 promoter of C. posadasii. More than 30 hygromycin-resistant transformants were obtained and 23 were purified to homozygosity through multiple passages of the original transformants on hygromycin-containing media. One strain (VFC1420) transformed with a single copy of the fusion histone H1-GFP gene was selected for cytological studies. Strong nuclear-localized GFP signals were observed in arthroconidia, hyphae, as well as in spherules and endospores developed in vitro. Thus GFP can be used to study the expression pattern of potential virulence genes identified in serial analysis of gene expression (SAGE) or expressed sequence tags (EST) libraries, and could be a useful tool to monitor disease development in the murine model.

Coccidioidomycosis has had an impact on the military since the discovery of the causative agent, Coccidioides immitis, over a century ago. The first reports of Coccidioides outbreaks affecting U.S. military personnel were reported by Smith and others during World War II. Since that time, numerous outbreaks and sporadic cases have occurred, affecting both the health and readiness of our armed forces. This article summarizes the impact of Coccidioides sp. on our military troops with a review of the literature, a description of the experience at a tertiary referral hospital treating the disease, and a synopsis of incidence studies conducted at bases in the southwestern United States. The substantial effect that coccidioidomycosis has had on the military supports the development of both newer and more effective treatments as well as a Valley Fever vaccine for prevention of this disease.

Experimental models of coccidioidomycosis performed using various laboratory animals have been, and remain, a critical component of elucidation and understanding of the pathogenesis and host resistance to infection with Coccidioides spp., as well as to development of more efficacious antifungal therapies. The general availability of genetically defined strains, immunological reagents, ease of handling, and costs all contribute to the use of mice as the primary laboratory animal species for models of this disease. Five types of murine models are studied and include primary pulmonary disease, intraperitoneal with dissemination, intravenous infection emulating systemic disease, and intracranial or intrathecal infection emulating meningeal disease. Each of these models has been used to examine various aspects of host resistance, pathogenesis, or antifungal therapy. Other rodent species, such as rat, have been used much less frequently. A rabbit model of meningeal disease, established by intracisternal infection, has proven to model human meningitis well. This model is useful in studies of host response, as well as in therapy studies. A variety of other animal species including dogs, primates, and guinea pigs have been used to study host response and vaccine efficacy. However, cost and increased needs of animal care and husbandry are limitations that influence the use of the larger animal species.

Coccidioides spp. appear capable of infecting all mammals and at least some reptiles. Development of disease as a result of infection is species-dependent. Dogs seem to have a susceptibility similar to that of humans, with subclinical infections, mild-to-severe primary pulmonary disease, and disseminated disease. Whereas central nervous system disease in humans is typically meningitis, brain disease in dogs and cats takes the form of granulomatous parenchymal masses. Osteomyelitis is the most common form of disseminated disease in the dog, while skin lesions predominate in the cat. Orally administered azole antifungal agents are the backbone of therapy in animals as they are in humans.

Coccidioides is a fungal respiratory pathogen of humans that can cause disease in both immunosuppressed and immunocompetent individuals. We describe here three mechanisms by which the pathogen survives in the hostile host environment: production of a dominant spherule outer wall glycoprotein (SOWgp) that modulates host immune response and results in compromised cell-mediated immunity to coccidioidal infection, depletion of SOWgp presentation on the surface of endospores, which prevents host recognition of the pathogen when the fungal cells are most vulnerable to phagocytic defenses, and induction of elevated production of host arginase I and coccidioidal urease, which contribute to tissue damage at sites of infection. Arginase I competes with inducible nitric oxide synthase (iNOS) in macrophages for the common substrate, L-arginine, and thereby reduces nitric oxide (NO) production and increases the synthesis of host orinithine and urea. Host-derived L-ornithine may promote pathogen growth and proliferation by providing a pool of the monoamine, which could be taken up and used for synthesis of polyamines via metabolic pathways of the parasitic cells. We have shown that high concentrations of Coccidioides- and host-derived urea at infection sites in the presence of urease produced and released by the pathogen, results in secretion of ammonia and contributes to alkalinization of the microenvironment. We propose that ammonia and enzymatically active urease released from spherules during the parasitic cycle of Coccidioides exacerbate the severity of coccidioidal infection by contributing to a compromised immune response to infection and damage of host tissue at foci of infection.

In the early years of transplantation in Arizona, coccidioidomycosis occurred in 7% to 9% of recipients, with a mortality rate as high as 72% in some cases. In current transplant programs, however, evolution of immunosuppression and institution of targeted prophylaxis have resulted in coccidioidal infection rates ranging from 1% to 2%. The clinical characteristics of this infection among transplant recipients range from asymptomatic to fulminant and fatal. Dissemination is common, and mortality is high (28%). Because serologic response is often absent or slow, diagnosis can be challenging and often requires invasive diagnostic procedures. Pharmacologic treatment follows the guidelines of the Infectious Diseases Society of America, but control of infection may also dictate a decrease in immunosuppressant treatment. After infection is controlled, secondary azole prophylaxis is recommended to prevent relapse. Patients with a history of coccidioidomycosis may undergo successful transplantation when disease is inactive and azole prophylaxis is instituted. The incidence of donor-derived coccidioidomycosis is not known. The risk of coccidioidal infection among transplant recipients visiting in or relocating to an endemic area is low, and routine prophylaxis for this group is not recommended.

To determine habitat attributes and processes suitable for the growth of Coccidioides, soils were collected from sites in Arizona, California, and Utah where Coccidioides is known to have been present. Humans or animals or both have been infected by Coccidioides at all of the sites. Soil variables considered in the upper 20 cm of the soil profile included pH, electrical conductivity, salinity, selected anions, texture, mineralogy, vegetation types and density, and the overall geomorphologic and ecological settings. Thermometers were buried to determine the temperature range in the upper part of the soil where Coccidioides is often found. With the exception of temperature regimes and soil textures, it is striking that none of the other variables or group of variables that might be definitive are indicative of the presence of Coccidioides. Vegetation ranges from sparse to relatively thick cover in lower Sonoran deserts, Chaparral-upper Sonoran brush and grasslands, and Mediterranean savannas and forested foothills. No particular grass, shrub, or forb is definitive. Material classified as very fine sand and silt is abundant in all of the Coccidioides-bearing soils and may be their most common shared feature. Clays are not abundant (less than 10%). All of the examined soil locations are noteworthy as generally 50% of the individuals who were exposed to the dust or were excavating dirt at the sites were infected. Coccidioides has persisted in the soil at a site in Dinosaur National Monument, Utah for 37 years and at a Tucson, Arizona site for 41 years.

Coccidioides causes coccidioidomycosis in the southwestern United States. Its clinical manifestations range from the primary asymptomatic to progressive pulmonary and extrapulmonary disease. Because of endemicity, frequent relapse, and virulent nature of Coccidioides, there is an urgent need for the development of effective therapy or vaccine. It has been recognized from studies in human patients and in murine models that the divergence in their susceptibility to Coccidioides infection is related to differences in T cell response. Dendritic cells (DCs) are most potent antigen-presenting cells that play a critical role in activating naïve T cells. On account of their unique immunostimulatory capacity, DCs have been used for the development of immunotherapy and vaccines against cancer and infectious diseases. We recently investigated the immunostimulatory potential of a DC-based vaccine in a murine model against Coccidioides posadasii (C. posadasii). Our results suggest that DCs act as a potent adjuvant and activate protective responses in mice against C. posadasii.

We report here the first documented case of ocular coccidioidomycosis in a chimpanzee (Pan troglodytes). In 1996, a 12-year-old female chimpanzee was undergoing treatment with an experimental triazole, BayR3783, for coccidioidomycosis when she was diagnosed with severe conjunctivitis in the right eye. Subsequent development of a coccidioidal granuloma of the ventral conjunctiva and anterior uvea was noted over the next several months, distorting the lens, iris, pupil, and sclera and progressing to uveitis. Treatment with BayR3783 and subconjunctival injections of triamcinolone were successful in reducing the ocular mass, but extensive damage was done to the lens and cornea. This case study provides an interesting comparison to ocular coccidioidomycosis cases observed in both humans and canines.

Coccidioidomycosis is a fungal infection common in the southwestern United States that is caused by the endemic Coccidioides species of fungus. Coccidioidal infections are generally manifested as self-limited respiratory illnesses, but affected patients rarely present with coccidioidomycosis in extrapulmonary locations. Skin and soft-tissue coccidioidomycosis may occur in 15% to 67% of patients with disseminated infection. Skin manifestations of coccidioidomycosis can either be reactive rashes, such as erythema multiforme or erythema nodosum associated with primary pulmonary infection, or they can be the result of extrapulmonary dissemination of the infection to the skin. As many as 90% of persons with disseminated infection to the skin have other extrapulmonary sites of infection, and the presence of coccidioidal skin lesions should prompt an investigation for other extrapulmonary foci of infection. Lymph nodes are a common site of extrapulmonary infection. Nearly every organ system and soft-tissue have been described as infected with Coccididioides species, but subcutaneous abscesses, phlegmon, and sinus tracts are not uncommon and often are themselves the result of coccidioidal infection in neighboring lymph nodes, bones, or joints. A biopsy of the abnormal area is the most direct way to diagnose skin and soft-tissue lesions. Fluconazole and itraconazole are preferred therapeutic agents, and surgical intervention may be required as an adjunctive measure. This article reviews the types and locations of disseminated infections, as well as diagnostic studies and treatment of this difficult-to-treat manifestation of coccidioidomycosis.

Using techniques developed to study melanization in other fungi, we demonstrate that Coccidioides posadasii arthroconidia, spherules, and endospores produce melanin or melanin-like compounds in vitro and tissue forms synthesize pigment in vivo. Since melanin is an important virulence factor in other pathogenic fungi, it may affect the pathogenesis of coccidioidomycosis.

Previous studies have shown that dendritic cells (DC) pulsed with T27K, an antigenic preparation derived from spherules (of Coccidioides posadasii), activate peripheral blood mononuclear cells (PBMC) from nonimmune subjects as well as from patients with disseminated coccidioidomycosis. In this study, we have assessed the interaction between human DC and C. posadasii spherules in order to better understand the initial response between Coccidioides and the human host. Whole autoclaved spherules induced lymphocyte transformation in PBMC obtained from immune but not from nonimmune donors. Immature DC (iDC) bound fluorescein isothiocyanate-labeled spherules in a time- and temperature-dependent manner. This binding was blocked by the addition of mannan, suggesting mannose receptor involvement in the DC-Coccidioides interaction. Binding was subsequently associated with ingestion and intracellular processing of spherules. Coculturing of spherules with iDC was associated with the development of mature DC that were morphologically, phenotypically, and functionally similar to those induced by tumor necrosis factor alpha and prostaglandin E2. Finally, spherules incubated with iDC induced activation of PBMC from nonimmune donors. These data indicate that human DC are capable of binding, internalizing, and presenting antigens from Coccidioides spherules and suggest that DC may play a critical early role in the formation of a cellular immune response in human coccidioidomycosis.

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.

Allosamidin is a known inhibitor of class 18 chitinases. We show that allosamidin is a competitive inhibitor of the fungal chitinase CiX1 from Coccidioides immitis, with a K(i) of 60 nM. We report the X-ray structure of the complex and show that upon inhibitor binding the side-chain of Asp169 rotates to form an ion pair with the oxazolinium cation. The mechanism of action is thought to involve protonation of the leaving group by Glu171 and substrate assistance by the sugar acetamido moiety to form an oxazoline-like intermediate. We converted both amino acid residues to the corresponding amide and found that each mutation effectively abolishes enzyme activity. X-ray structures show the mutant enzymes retain the basic wild-type structure and that the loss of mutant activity is due to their altered chemical properties. The high affinity of allosamidin, and its similarity to the putative reaction intermediate, suggests it is a transition state analog. This helps validate our contention that the role of Asp169 is to electrostatically stabilize the reaction transition state.

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.

120 soil samples collected from various sites of Cairo were processed for the isolation of keratinophilic fungi by "ToKaVa" hair baiting technique. 22 species belonging to 6 genera were isolated viz.: Chrysosporium tropicum, C. indicum, C. keratinophilum, C. queenslandicum, C. merdarium, C. anamorph of Arthroderma curreyi, C. pannicola, C. lobatum, C. anamorph of Renispora flavissima, C. pseudomerdarium, Microascus mangini, Malbranchea gypsea, Ml. State of Uncicarpus reesii, Ml. State of Coccidioides immitis, Microsporum gypseum, Mr. distortum Mr. audouinii, Mr. fulvum, Trichophyton mentagrophytes, T. terrestre, T. verrucosum and Epidermophyton floccosum. The frequency of occurrence of the isolated fungi was determined. Microsporum gypseum, Chrysosporium tropicum and Chrysosporium indicum were the most frequent species recovered from soil. Most species of keratinophilic fungi were isolated from university, public garden and zoo garden. The distribution of the isolates are discussed.

Some biochemical mechanisms involved in the processes of virulence and dimorphism in fungi pathogenic for humans are reviewed. Among them, the participation of sulphydryl and disulfide groups, hormone receptors and intra- and extracellular proteinases in Histoplasma capsulatum, Paracoccidioides brasiliensis and Coccidioides immitis.

Pregnancy is a major risk factor for coccidioidal dissemination. Because rates of Coccidioides immitis growth and endospore release are stimulated in vitro by levels of unbound progesterone and 17 beta-estradiol that are achievable, in vivo, in the sera of pregnant women (i.e., 10(-9) to 10(-8) M), a specific-hormone-binding system in C. immitis was sought. Fungal cytosols were incubated with tritiated steroids plus or minus radioinert steroids to identify specific binding systems. All five strains of C. immitis tested exhibited specific saturable binding for progestin, estrogen, androgen, and (to a lesser extent) corticosterone and glucocorticoid hormone classes. Only low or inconsistent estrogen or androgen binding was found in Blastomyces dermatitidis and Torulopsis glabrata. Cryptococcus neoformans, Paracoccidioides brasiliensis, and non-albicans Candida species showed no binding. Scatchard analysis of progestin and estrogen binding in C. immitis revealed a high-capacity, low-affinity binding system that was unaffected by RNase and DNase, but 40 to 60% degraded by trypsin or heating. Ammonium sulfate precipitation resolved a high-affinity, low-capacity binding system (Kd = 1.24 X 10(-9) to 3.60 X 10(-8) M; number of binding sites = 0.014 to 0.20 pmol/mg of protein). The Kd of this system is sufficient to compete for unbound hormone in the sera of pregnant women. The high-capacity, low-affinity system may serve as a repository for hormone before its attachment to the specific binder. These studies suggest that the effects of nanomolar concentrations of sex hormones on C. immitis may be mediated by a specific cytosol protein-binding system and that stimulatory events observed in vitro may have relevance for the mechanism of coccidioidal dissemination in pregnancy.

As arthroconidia of Coccidioides immitis transform into spherules, the fungal particles progressively become more resistant to the inhibitory effects of human polymorphonuclear leukocytes (PMNLs) as measured by PMNL inhibition of fungal incorporation of N-acetylglucosamine. Similar changes were noted when leucine incorporation was measured. When H2O2, at a concentration of 2.0 mM, was substituted for PMNLs, an equivalent inhibition of arthroconidia was produced and, as with PMNL effects, was lost with spherule maturation. The results from these studies indicate that C. immitis may evade natural inhibition by PMNLs by transformation into the spherule phase.

The elastin digestion assay was examined to determine if it would facilitate the identification of Coccidioides immitis when non-pathogenic fungi resembling C. immitis are encountered. Fungal isolants tested have anamorphs that closely resemble the macroscopic or microscopic morphology of C. immitis. Elastin hydrolysis was measured by elastin-agar plate assays. Approximately 80% of the isolants hydrolyzed elastin; thus, the elastin digestion assay as a differential test appears to have little value.

Coccidioides immitis cytosol contains a specific estrogen binder with kinetic characteristics (Kd = 2.1 X 10(-8) to 3.7 X 10(-8) M; n = 0.5 to 1.56 pmol/mg of protein) appropriate for interaction with levels of estrogen encountered during pregnancy. The Kd of a separate androgen binder (ca. 10(-7) M) is probably too low to allow interaction with physiological levels of testosterone.

Polymorphonuclear neutrophils (PMNs) possess phagocytic and fungicidal activity against Coccidioides immitis that declines during maturation from arthroconidia to round cells, is lost throughout spherule maturation, and returns when endospores are released from ruptured spherules. Studies of PMN chemiluminescence, iodination, and degranulation give similar results. Phagocytosis of forms other than spherules is strain dependent and enhanced by immune serum. The absence of adequate PMN-spherule interaction may be attributed to the production of an extracellular fibrillar matrix, glycoprotein in composition, that restricts intimate PMN-spherule contact. When the spherule ruptures, PMNs enter to phagocytose endospores that are themselves invested by a matrix derived from the inner spherule wall. The immunochemical relationship between the outer matrix and the inner matrix remains to be discovered. Nevertheless, presence of the outer matrix may help to explain the long-standing histopathologic observation that PMNs fail to attack spherules until they release their endospores.

The authors studied the ultrastructure of the Coccidiodes immitis spherules in the sections of the abdominal cavity foci and pneumonic foci of Cricetus auratus W. and in albino mice infected intraperitoneally with a lethal dose of the mycelium fungus phase. It was found that the spherules of coccidiod fungus represented multinuclear cells with a well-organized protoplast included into a complicated supermembrane structure--the cell wall. Endospores (which copied the spherule structure in miniature) formed as a result of division of the cytoplasm by entire sections into polygonal segments with the subsequent separation of the latter. Due to the rupture of the cell wall of mature sporulating spherules the endospores are released into the surrounding tissue and are attacked there and phagocytized by polynuclears and macrophages. Phygocytosis of the fungi is mostly incomplete.

Human polymorphonuclear leukocytes (PMNLs), when added to arthroconidial suspensions of Coccidioides immitis, markedly inhibit fungal incorporation of the cell-wall precursor N-acetylglucosamine. This effect does not require serum but is facilitated by a heat-labile serum component(s), probably by promoting PMNL attachment to the arthroconidia. Inhibition is entirely reversible within 24 hr. In parallel with this finding, PMNLs exhibited virtually no killing of arthroconidia and (as determined by electron microscopy) did not produce damage to the fungal ultrastructure. PMNLs from a patient with chronic granulomatous disease suppressed fungal incorporation of N-acetyl-glucosamine by only 0%-22% under various conditions, as compared with 54%-85% suppression by PMNLs from normal donors. Our studies demonstrate that PMNLs can influence the metabolism of C immitis and raise the possibility that their presence in coccidioidal histopathology may be an important immediate host defense in limiting progression early after infection.

A proteinase inhibitor was isolated from the cytosol and cell wall of the fungal respiratory pathogen Coccidioides immitis. The inhibitor was purified to apparent homogeneity by acid precipitation, gel filtration and reverse-phase h.p.l.c. It has an Mr of about 5000 under reducing conditions, as revealed by SDS/polyacrylamide-gel electrophoresis, and is both heat-stable and tolerant to low pH. The inhibitor can efficiently block activity of a 36,000-Mr serine proteinase previously isolated from the same cell-wall fraction of C. immitis, and the two molecules react in a 1:1 stoichiometry. The dissociation constant (Ki) of the enzyme-inhibitor complex is 2.3 x 10(-8) M. We suggest that the low-Mr inhibitor may play a role in regulation of the activity of the cell-wall-associated 36,000-Mr proteinase during sporulation of C. immitis.

Of various carbohydrates and amino acids tested, glucose, mannose, fructose, and glutamate were the most efficient substrates metabolized by the endospores and spherules of Coccidioides immitis.

Documentation is offered for the identification of 3-O-methyl-mannose as one of several neutral sugars found in defatted arthrospore and mycelial cell walls of Coccidioides immitis.

Lones, George W. (National Institute of Allergy and Infectious Diseases, U.S. Public Health Service, Bethesda, Md.), and Carl Peacock. Metabolism of mannitol by Coccidioides immitis. J. Bacteriol. 87:1114-1117. 1964.-Strain M-11 of Coccidioides immitis was found to utilize mannitol for growth in the mycelial form but not in the spherule form. Cell-free extracts of both forms, grown on glucose, were capable of reducing nicotinamide adenine dinucleotide with mannitol-1-PO(4) but not with mannitol. The extracts accomplished a rapid oxidation of reduced nicotinamide adenine dinucleotide by fructose-6-PO(4), the expected product of mannitol-1-PO(4) oxidation. Fructose was inactive. Paper electrophoresis and chromatography with several solvent systems demonstrated a substance in extracts of both mycelium and spherules having a migration consistent with that of mannitol.