RESUMEN
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.
Asunto(s)
Coccidioides/crecimiento & desarrollo , Coccidioides/metabolismo , Estadios del Ciclo de Vida , Metaboloma , Compuestos Orgánicos Volátiles/análisis , Biomarcadores/metabolismo , Pruebas Respiratorias/métodos , Coccidioides/clasificación , Coccidioidomicosis/microbiología , Medios de Cultivo/química , Humanos , Micelio/crecimiento & desarrollo , Micelio/metabolismoRESUMEN
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.
Asunto(s)
Coccidioides/enzimología , Coccidioidomicosis/diagnóstico , Proteínas Fúngicas/análisis , Polisacáridos/análisis , Coccidioides/aislamiento & purificación , Coccidioides/metabolismo , Coccidioidomicosis/metabolismo , Coccidioidomicosis/microbiología , Proteínas Fúngicas/biosíntesis , Proteínas Fúngicas/genética , Glicómica , Glicosilación , Humanos , Polisacáridos/metabolismo , ProteómicaRESUMEN
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.
Asunto(s)
Antifúngicos/farmacología , Aspergillus fumigatus/metabolismo , Inhibidores de la Calcineurina/farmacología , Calcineurina/metabolismo , Cryptococcus neoformans/metabolismo , Proteína 1A de Unión a Tacrolimus/metabolismo , Tacrolimus/farmacología , Animales , Aspergilosis/tratamiento farmacológico , Aspergilosis/microbiología , Aspergillus fumigatus/efectos de los fármacos , Sitios de Unión , Candida albicans/efectos de los fármacos , Candida albicans/metabolismo , Células Cultivadas , Coccidioides/efectos de los fármacos , Coccidioides/metabolismo , Criptococosis/tratamiento farmacológico , Criptococosis/microbiología , Cryptococcus neoformans/efectos de los fármacos , Cristalografía por Rayos X , Descubrimiento de Drogas/métodos , Femenino , Masculino , Ratones , Ratones Endogámicos A , Ratones Endogámicos C57BL , Simulación de Dinámica Molecular , Tacrolimus/metabolismoRESUMEN
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.
Asunto(s)
Coccidioides/genética , Coccidioides/metabolismo , Anotación de Secuencia Molecular , Fragmentos de Péptidos/análisis , Fragmentos de Péptidos/genética , Proteogenómica/métodos , Proteoma/metabolismo , Coccidioidomicosis/microbiología , Biología Computacional , Fragmentos de Péptidos/química , Espectrometría de Masas en TándemRESUMEN
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.
Asunto(s)
Antifúngicos/farmacología , Coccidioides/efectos de los fármacos , Ergosterol/biosíntesis , Histoplasma/crecimiento & desarrollo , Levamisol/farmacología , Fosforilcolina/análogos & derivados , Permeabilidad de la Membrana Celular/efectos de los fármacos , Coccidioides/crecimiento & desarrollo , Coccidioides/metabolismo , Histoplasma/efectos de los fármacos , Histoplasma/metabolismo , Pruebas de Sensibilidad Microbiana , Fosforilcolina/farmacologíaRESUMEN
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.
Asunto(s)
Amidina-Liasas/metabolismo , Amoníaco/metabolismo , Coccidioides/metabolismo , Coccidioides/patogenicidad , Coccidioidomicosis/patología , Enfermedades Pulmonares Fúngicas/patología , Ureasa/metabolismo , Amidina-Liasas/genética , Animales , Coccidioides/enzimología , Coccidioides/genética , Coccidioidomicosis/microbiología , Modelos Animales de Enfermedad , Técnicas de Inactivación de Genes , Humanos , Enfermedades Pulmonares Fúngicas/microbiología , Ratones , Ratones Endogámicos BALB C , Análisis de Supervivencia , Ureasa/genética , VirulenciaRESUMEN
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.
Asunto(s)
Antifúngicos/farmacología , Coccidioides/efectos de los fármacos , Ergosterol/antagonistas & inhibidores , Farnesol/farmacología , Fluconazol/farmacología , Permeabilidad de la Membrana Celular/efectos de los fármacos , Coccidioides/crecimiento & desarrollo , Coccidioides/metabolismo , Sinergismo Farmacológico , Quimioterapia Combinada , Ergosterol/biosíntesis , Pruebas de Sensibilidad Microbiana , Concentración Osmolar , Presión Osmótica , Cloruro de Sodio/químicaRESUMEN
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.
Asunto(s)
Aspergillus/metabolismo , Coccidioides/metabolismo , Proteínas Fúngicas/inmunología , Vacunas Fúngicas/inmunología , Proteoma , Aspergillus/inmunología , Coccidioides/inmunología , Espectrometría de MasasRESUMEN
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.
Asunto(s)
Antígenos Fúngicos/análisis , Micosis/diagnóstico , Micosis/metabolismo , Análisis por Matrices de Proteínas/métodos , Coccidioides/metabolismo , Coccidioides/patogenicidad , Humanos , Micosis/microbiologíaRESUMEN
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.
Asunto(s)
Coccidioides/genética , Coccidioides/metabolismo , Genoma Fúngico , Sintasas Poliquetidas/genética , Sintasas Poliquetidas/metabolismo , Transducción de Señal/genética , Acilcoenzima A/metabolismo , Aciltransferasas/metabolismo , Aflatoxinas/metabolismo , Sitios de Unión , Coccidioides/clasificación , Bases de Datos de Proteínas , Escherichia coli/enzimología , Escherichia coli/genética , Ácido Graso Sintasas/metabolismo , Ácidos Grasos/metabolismo , Estructura Terciaria de Proteína , Especificidad por SustratoRESUMEN
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.
Asunto(s)
Anfotericina B/uso terapéutico , Coccidioidomicosis/tratamiento farmacológico , Meningitis/tratamiento farmacológico , Antifúngicos/uso terapéutico , Coccidioides/metabolismo , Ácido Desoxicólico/uso terapéutico , Combinación de Medicamentos , Femenino , Humanos , Enfermedades Renales/etiología , Lípidos/química , Embarazo , Resultado del Tratamiento , Triazoles/uso terapéuticoRESUMEN
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.
Asunto(s)
Coccidioides/metabolismo , Coccidioides/patogenicidad , Coccidioidomicosis/microbiología , Coccidioidomicosis/patología , Regulación Fúngica de la Expresión Génica , Animales , Antígenos Fúngicos/metabolismo , Arginasa/biosíntesis , Progresión de la Enfermedad , Humanos , Sistema Inmunológico , Proteínas de la Membrana/metabolismo , Modelos Biológicos , Fagocitosis , Factores de VirulenciaRESUMEN
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.
Asunto(s)
Coccidioides/metabolismo , Coccidioidomicosis/diagnóstico , Coccidioidomicosis/epidemiología , California , Coccidioidomicosis/microbiología , Coccidioidomicosis/prevención & control , Brotes de Enfermedades , Vacunas Fúngicas , Geografía , Hospitales Militares , Humanos , Incidencia , Medicina Militar , Personal Militar , Salud Pública , Estados UnidosRESUMEN
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.
Asunto(s)
Enfermedades Óseas/diagnóstico , Enfermedades Óseas/microbiología , Enfermedades Óseas/terapia , Coccidioidomicosis/diagnóstico , Coccidioidomicosis/terapia , Antifúngicos/uso terapéutico , Huesos/microbiología , Coccidioides/metabolismo , Humanos , Articulaciones/microbiología , Sistema Musculoesquelético/microbiología , Sudoeste de Estados UnidosRESUMEN
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.
Asunto(s)
Coccidioidomicosis/diagnóstico , Coccidioidomicosis/epidemiología , América Central , Coccidioides/metabolismo , Comorbilidad , Etnicidad , Femenino , Geografía , Humanos , Inmunosupresores/uso terapéutico , Incidencia , Masculino , México , América del Sur , Linfocitos T/microbiología , Estados UnidosRESUMEN
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.
Asunto(s)
Núcleo Celular/metabolismo , Coccidioides/metabolismo , Coccidioidomicosis/genética , Proteínas Fluorescentes Verdes/metabolismo , Microscopía Fluorescente/instrumentación , Agrobacterium tumefaciens/metabolismo , Coccidioidomicosis/metabolismo , Etiquetas de Secuencia Expresada , Técnicas Genéticas , Vectores Genéticos , Humanos , Microscopía Fluorescente/métodos , Regiones Promotoras GenéticasRESUMEN
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.
Asunto(s)
Encéfalo/microbiología , Coccidioidomicosis/diagnóstico , Antifúngicos/uso terapéutico , Encéfalo/patología , Broncoscopía , Coccidioides/metabolismo , Coccidioidomicosis/diagnóstico por imagen , Edema , Humanos , Lactante , Masculino , Meningitis Fúngica/diagnóstico , Meningitis Fúngica/diagnóstico por imagen , Tomografía Computarizada por Rayos X , Resultado del TratamientoRESUMEN
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.
Asunto(s)
Coccidioides/metabolismo , Coccidioidomicosis/diagnóstico , Coccidioidomicosis/microbiología , Modelos Animales de Enfermedad , Animales , Antifúngicos/uso terapéutico , Coccidioidomicosis/fisiopatología , Perros , Vacunas Fúngicas/metabolismo , Cobayas , Humanos , Ratones , Primates , ConejosRESUMEN
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.
Asunto(s)
Coccidioides/fisiología , Microbiología del Suelo , Arizona , Coccidioides/metabolismo , Clima Desértico , Conductividad Eléctrica , Geografía , Concentración de Iones de Hidrógeno , Dióxido de Silicio , Suelo , Temperatura , Estados Unidos , Utah , Agua/metabolismoRESUMEN
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.