RESUMEN
In response to temperature, Blastomyces dermatitidis converts between yeast and mold forms. Knowledge of the mechanism(s) underlying this response to temperature remains limited. In B. dermatitidis, we identified a GATA transcription factor, SREB, important for the transition to mold. Null mutants (SREBΔ) fail to fully complete the conversion to mold and cannot properly regulate siderophore biosynthesis. To capture the transcriptional response regulated by SREB early in the phase transition (0-48 hours), gene expression microarrays were used to compare SREB∆ to an isogenic wild type isolate. Analysis of the time course microarray data demonstrated SREB functioned as a transcriptional regulator at 37°C and 22°C. Bioinformatic and biochemical analyses indicated SREB was involved in diverse biological processes including iron homeostasis, biosynthesis of triacylglycerol and ergosterol, and lipid droplet formation. Integration of microarray data, bioinformatics, and chromatin immunoprecipitation identified a subset of genes directly bound and regulated by SREB in vivo in yeast (37°C) and during the phase transition to mold (22°C). This included genes involved with siderophore biosynthesis and uptake, iron homeostasis, and genes unrelated to iron assimilation. Functional analysis suggested that lipid droplets were actively metabolized during the phase transition and lipid metabolism may contribute to filamentous growth at 22°C. Chromatin immunoprecipitation, RNA interference, and overexpression analyses suggested that SREB was in a negative regulatory circuit with the bZIP transcription factor encoded by HAPX. Both SREB and HAPX affected morphogenesis at 22°C; however, large changes in transcript abundance by gene deletion for SREB or strong overexpression for HAPX were required to alter the phase transition.
Asunto(s)
Blastomyces/metabolismo , Factores de Transcripción GATA/metabolismo , Homeostasis/fisiología , Hierro/metabolismo , Metabolismo de los Lípidos/fisiología , Hongos/metabolismo , Regulación Fúngica de la Expresión Génica/genética , Genes Fúngicos/genética , Metabolismo de los Lípidos/genéticaRESUMEN
Blastomyces adhesin-1 (BAD-1) is a 120-kD surface protein on B. dermatitidis yeast. We show here that BAD-1 contains 41 tandem repeats and that deleting even half of them impairs fungal pathogenicity. According to NMR, the repeats form tightly folded 17-amino acid loops constrained by a disulfide bond linking conserved cysteines. Each loop contains a highly conserved WxxWxxW motif found in thrombospondin-1 (TSP-1) type 1 heparin-binding repeats. BAD-1 binds heparin specifically and saturably, and is competitively inhibited by soluble heparin, but not related glycosaminoglycans. According to SPR analysis, the affinity of BAD-1 for heparin is 33 nM±14 nM. Putative heparin-binding motifs are found both at the N-terminus and within each tandem repeat loop. Like TSP-1, BAD-1 blocks activation of T cells in a manner requiring the heparan sulfate-modified surface molecule CD47, and impairs effector functions. The tandem repeats of BAD-1 thus confer pathogenicity, harbor motifs that bind heparin, and suppress T-cell activation via a CD47-dependent mechanism, mimicking mammalian TSP-1.
Asunto(s)
Antígenos Fúngicos/metabolismo , Antígenos de Superficie/metabolismo , Blastomyces/inmunología , Proteínas Fúngicas/metabolismo , Heparina/metabolismo , Activación de Linfocitos , Linfocitos T/inmunología , Animales , Antígenos Fúngicos/química , Antígenos Fúngicos/genética , Antígenos de Superficie/química , Antígenos de Superficie/genética , Blastomyces/química , Blastomyces/metabolismo , Blastomyces/patogenicidad , Blastomicosis/inmunología , Blastomicosis/metabolismo , Blastomicosis/microbiología , Antígeno CD47/química , Antígeno CD47/genética , Antígeno CD47/metabolismo , Células Cultivadas , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Humanos , Células Jurkat , Masculino , Ratones Endogámicos BALB C , Ratones Transgénicos , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Linfocitos T/citología , Linfocitos T/metabolismo , Secuencias Repetidas en Tándem , Trombospondina 1/química , Trombospondina 1/metabolismo , VirulenciaRESUMEN
Blastomyces dermatitidis, the etiologic agent of blastomycosis, belongs to a group of thermally dimorphic fungi that change between mold (22°C) and yeast (37°C) in response to temperature. The contribution of structural proteins such as septins to this phase transition in these fungi remains poorly understood. Septins are GTPases that serve as a scaffold for proteins involved with cytokinesis, cell polarity, and cell morphology. In this study, we use a GFP sentinel RNA interference system to investigate the impact of CDC3, CDC10, CDC12, and ASPE on the morphology and phase transition of B. dermatitidis. Targeting CDC3, CDC10, and CDC12 by RNA interference resulted in yeast with aberrant morphology at 37°C with defects in cytokinesis. Downshifting the temperature to 22°C promoted the conversion to the mold phase, but did not abrogate the morphologic defects. CDC3, CDC10, and CDC12 knockdown strains grew as mold with curved, thickened hyphae. Knocking down ASPE transcript did not alter morphology of yeast at 37°C or mold at 22°C. Following an increase in temperature from 22°C to 37°C, all septin knockdown strains were able to revert to yeast. In conclusion, CDC3, CDC10, and CDC12 septin- encoding genes are required for proper morphology of yeast and hyphae, but are dispensable for the phase transition.
Asunto(s)
Blastomyces/genética , Proteínas Fúngicas/metabolismo , Hifa/citología , Septinas/metabolismo , Levaduras/citología , Blastomyces/citología , Blastomyces/metabolismo , Proteínas Fúngicas/genética , Técnicas de Silenciamiento del Gen , Proteínas Fluorescentes Verdes , Hifa/genética , Interferencia de ARN , Reacción en Cadena en Tiempo Real de la Polimerasa , Septinas/genética , Temperatura , Levaduras/genéticaRESUMEN
Blastomyces dermatitidis belongs to a group of human pathogenic fungi that exhibit thermal dimorphism. At 22 degrees C, these fungi grow as mold that produce conidia or infectious particles, whereas at 37 degrees C they convert to budding yeast. The ability to switch between these forms is essential for virulence in mammals and may enable these organisms to survive in the soil. To identify genes that regulate this phase transition, we used Agrobacterium tumefaciens to mutagenize B. dermatitidis conidia and screened transformants for defects in morphogenesis. We found that the GATA transcription factor SREB governs multiple fates in B. dermatitidis: phase transition from yeast to mold, cell growth at 22 degrees C, and biosynthesis of siderophores under iron-replete conditions. Insertional and null mutants fail to convert to mold, do not accumulate significant biomass at 22 degrees C, and are unable to suppress siderophore biosynthesis under iron-replete conditions. The defect in morphogenesis in the SREB mutant was independent of exogenous iron concentration, suggesting that SREB promotes the phase transition by altering the expression of genes that are unrelated to siderophore biosynthesis. Using bioinformatic and gene expression analyses, we identified candidate genes with upstream GATA sites whose expression is altered in the null mutant that may be direct or indirect targets of SREB and promote the phase transition. We conclude that SREB functions as a transcription factor that promotes morphogenesis and regulates siderophore biosynthesis. To our knowledge, this is the first gene identified that promotes the conversion from yeast to mold in the dimorphic fungi, and may shed light on environmental persistence of these pathogens.
Asunto(s)
Blastomyces/genética , Blastomyces/metabolismo , Factores de Transcripción GATA/genética , Factores de Transcripción GATA/metabolismo , Morfogénesis/genética , Sideróforos/biosíntesis , Secuencia de Aminoácidos , Secuencia de Bases , Northern Blotting , Southern Blotting , Hongos/genética , Hongos/metabolismo , Expresión Génica , Regulación Fúngica de la Expresión Génica , Genes Fúngicos , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Temperatura , Levaduras/genética , Levaduras/metabolismoRESUMEN
The ability of pathogens to evade host antimicrobial mechanisms is crucial to their virulence. The dimorphic fungal pathogen Blastomyces dermatitidis can infect immunocompetent patients, producing a primary pulmonary infection that can later disseminate to other organs. B. dermatitidis possesses a remarkable ability to resist killing by alveolar macrophages. To date, no mechanism to explain this resistance has been described. Here, we focus on macrophage production of the toxic molecule nitric oxide as a potential target of subversion by B. dermatitidis yeast cells. We report that B. dermatitidis yeast cells reduce nitric oxide levels in the supernatants of activated alveolar macrophages. This reduction is not due to detoxification of nitric oxide, but rather to suppression of macrophage nitric oxide production. We show that B. dermatitidis yeast cells do not block upregulation of macrophage inducible nitric oxide synthase (iNOS) expression or limit iNOS access to its arginine substrate. Instead, B. dermatitidis yeast cells appear to inhibit iNOS enzymatic activity. Further investigation into the genetic basis of this potential virulence mechanism could lead to the identification of novel antifungal drug targets.
Asunto(s)
Blastomyces/metabolismo , Blastomicosis/metabolismo , Macrófagos Alveolares/enzimología , Óxido Nítrico Sintasa de Tipo II/metabolismo , Óxido Nítrico/biosíntesis , Animales , Western Blotting , Línea Celular , Interacciones Huésped-Patógeno , Macrófagos Alveolares/metabolismo , Macrófagos Alveolares/microbiología , Ratones , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Inteins are intervening sequences that are transcribed and translated with flanking host protein sequences and then self-excised by protein splicing. Bi-functional inteins also contain a homing endonuclease responsible for their genetic mobility. The PRP8 intein, the most widespread among fungi, occurs in important pathogens such as Histoplasma capsulatum and Paracoccidioides brasiliensis, from the Ajellomycetaceae family. Herein, we describe the bi-functional PRP8 intein in two other Ajellomycetacean pathogens, Blastomyces dermatitidis and Emmonsia parva. Sequence analysis and experimental evidence suggest that the homing endonuclease from PbrPRP8 is inactive. The splicing activity of the PRP8 intein from the B. dermatitidis, E. parva and P. brasiliensis species complex was demonstrated in a non-native protein context in Escherichia coli. Since the PRP8 intein is located in a functionally essential nuclear protein, it can be considered a promising therapeutic target for anti-fungal drugs, because inhibition of intein splicing should inhibit proliferation of intein-containing pathogens.
Asunto(s)
Blastomyces/enzimología , Chrysosporium/enzimología , Endonucleasas/genética , Endonucleasas/metabolismo , Inteínas/genética , Empalme de Proteína , Secuencia de Aminoácidos , Blastomyces/genética , Blastomyces/metabolismo , Chrysosporium/genética , Chrysosporium/metabolismo , Análisis por Conglomerados , Escherichia coli/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Datos de Secuencia Molecular , Filogenia , Alineación de Secuencia , Análisis de SecuenciaRESUMEN
Blastomycosis is the fungal disease caused by thermally dimorphic fungi in the genus Blastomyces, with B dermatitidis complex causing most cases. It is considered hyperendemic in areas adjacent to the Great Lakes and along the St. Lawrence, Mississippi, and Ohio rivers, but definitive geographic distribution of blastomycoses remains obscure. Clinical presentation is variable. Disseminated blastomycosis with extrapulmonary manifestations is more common in immunosuppressed individuals. Culture positivity is required for definitive diagnosis, but compatible histology is often sufficient for presumptive diagnosis and initiation of treatment. Treatment should be provided to all symptomatic cases to prevent progression or recurrence.
Asunto(s)
Antifúngicos/uso terapéutico , Blastomyces/patogenicidad , Blastomicosis/tratamiento farmacológico , Anfotericina B/farmacología , Anfotericina B/uso terapéutico , Antifúngicos/farmacología , Blastomyces/crecimiento & desarrollo , Blastomyces/metabolismo , Blastomicosis/diagnóstico , Blastomicosis/epidemiología , Enfermedades Endémicas , Humanos , Itraconazol/uso terapéuticoRESUMEN
Fluorescence-based techniques enable researchers to monitor physiologic processes, specifically fungal cell viability and death, during cellular encounters with the mammalian immune system with single event resolution. By incorporating two independent fluorescent probes in fungal organisms either prior to, or ensuing experimental infection in mice or in cultured leukocytes, it is possible to distinguish and quantify live and killed fungal cells to interrogate genetic, pharmacologic, and cellular determinants that shape host-fungal cell outcomes. This chapter reviews the techniques and applications of fluorescent fungal reporters of viability, with emphasis on the North American endemic dimorphic fungus, Blastomyces dermatitidis.
Asunto(s)
Blastomyces/genética , Regulación Fúngica de la Expresión Génica , Genes Reporteros , Leucocitos/microbiología , Proteínas Luminiscentes/genética , Pulmón/microbiología , Microscopía Fluorescente , Animales , Blastomyces/inmunología , Blastomyces/metabolismo , Citometría de Flujo , Interacciones Huésped-Patógeno , Humanos , Leucocitos/inmunología , Leucocitos/metabolismo , Proteínas Luminiscentes/biosíntesis , Pulmón/inmunología , Pulmón/metabolismo , Viabilidad Microbiana , Proteína Fluorescente RojaRESUMEN
The precise microecology of Blastomyces dermatitidis is unknown, but the fungus has been associated with nitrogenous waste products and rapidly changing environmental conditions. Ammonia accumulates in certain microenvironments, is toxic to most fungi, but may not be identified in processed soil samples. Ammonia tolerance of B. dermatitidis was investigated with two strains recovered in Wisconsin, one from a dog and the other from an environmental source. The samples were grown on phosphate and HEPES buffered agar media supplemented with mineral salts, low (1 g/l) and high (20 g/l) dextrose and increasing amounts of ammonium sulfate, at pH 7-8.2, in gas-impermeable bags at 20 degrees C. Moderate mold growth and sporulation of the strains were observed at low glucose concentration and calculated ammonia concentrations of 4.2 mmol/l when plates were inoculated with either mold or yeast forms. Three recent B. dermatitidis human clinical isolates also exhibited similar growth on this media, and 4/5 strains tolerated ammonia concentrations of 42-62 mmol/l. Growth of virtually all soil fungi from 206 aqueous slurries of fresh and frozen soil from the northern USA and Canada was inhibited at ammonia concentrations of 2.1-4.2 mmol/l. The ability of B. dermatitidis to survive and grow in organic carbon-poor, high ammonia microenvironments may be important to the competitive success of this fungus. These findings may have implications for other dimorphic fungi.
Asunto(s)
Amoníaco/metabolismo , Blastomyces/crecimiento & desarrollo , Blastomyces/metabolismo , Microbiología del Suelo , Animales , Blastomyces/citología , Blastomicosis , Medios de Cultivo/química , Medios de Cultivo/metabolismo , Perros , Glucosa/metabolismo , Humanos , Modelos BiológicosRESUMEN
Serum factors, including mannose binding lectins (MBL), influence innate responses to microbes. Little is known about the effects of serum factors or MBL on the interaction of Blastomyces dermatitidis, a pulmonary fungal pathogen, with macrophages or on tumor necrosis factor alpha (TNF-alpha) production. Since macrophage production of TNF-alpha is an important innate immune response, we examined a mouse peritoneal macrophage (PM) cell line (RAW) and resident PM from CD-1 mice to study TNF-alpha production by PM stimulated with heat-killed (HK) or live B. dermatitidis yeast cells. Mouse serum and heat-inactivated mouse serum inhibited TNF-alpha production 94% when macrophages were stimulated by B. dermatitidis, whereas mouse immunoglobulin G (IgG) did not have this effect. HK B. dermatitidis incubated with serum and then washed also failed to stimulate significant TNF-alpha production by PM. By the sandwich immunofluorescent antibody (IFA) method with anti-mouse MBL (MBL-A or -C), we showed that serum MBL bound to B. dermatitidis. When serum was absorbed with HK B. dermatitidis or live B. dermatitidis, absorbed serum failed to significantly inhibit TNF-alpha production by RAW cells plus B. dermatitidis, and immunoblotting showed that absorbed serum was depleted of MBL-C. If serum was absorbed with live B. dermatitidis, unbound serum was eluted, and bound serum factor(s) (BS) was released with guanidine buffer, BS inhibited TNF-alpha production by PM plus B. dermatitidis in a concentration-dependent manner. BS contained MBL-C, which bound B. dermatitidis, as shown by IFA assay. 1,3-beta-Glucan stimulated TNF-alpha production by PM, and this was inhibited by mouse serum. Treatment of B. dermatitidis with anti-1,3-beta-glucan antibody inhibited TNF-alpha production by PM. With anti-1,3-beta-glucan antibody, we showed by IFA assay that B. dermatitidis contained 1,3-beta-glucan. In an IFA study with B. dermatitidis, serum with an anti-mouse IgG conjugate did not result in fluorescence, yet serum blocked IFA staining of B. dermatitidis by anti-1,3-beta-glucan IgG antibody. This indicated that non-IgG serum factors binding to B. dermatitidis prevented access to 1,3-beta-glucan by anti-1,3-beta-glucan antibody. These results suggest that the mechanism of inhibition of the innate proinflammatory immune response of PM to B. dermatitidis is mediated by serum MBL binding to B. dermatitidis at 1,3-beta-glucan sites or sterically masking 1,3-beta-glucan sites, thus preventing 1,3-beta-glucan stimulation of PM for TNF-alpha production.
Asunto(s)
Blastomyces/inmunología , Macrófagos Peritoneales/inmunología , Macrófagos Peritoneales/microbiología , Lectinas de Unión a Manosa/inmunología , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Animales , Blastomyces/química , Blastomyces/metabolismo , Línea Celular , Células Cultivadas , Masculino , Lectinas de Unión a Manosa/metabolismo , Ratones , Ratones Endogámicos C3H , Unión Proteica , Proteoglicanos , beta-Glucanos/inmunología , beta-Glucanos/metabolismoRESUMEN
Blastomyces dermatitidis is a human fungal pathogen of the lung that can lead to disseminated disease in healthy and immunocompromised individuals. Genetic analysis of this fungus is hampered by the relative inefficiency of traditional recombination-based gene-targeting approaches. Here, we demonstrate the feasibility of applying CRISPR/Cas9-mediated gene editing to Blastomyces, including to simultaneously target multiple genes. We created targeting plasmid vectors expressing Cas9 and either one or two single guide RNAs and introduced these plasmids into Blastomyces via Agrobacterium gene transfer. We succeeded in disrupting several fungal genes, including PRA1 and ZRT1, which are involved in scavenging and uptake of zinc from the extracellular environment. Single-gene-targeting efficiencies varied by locus (median, 60% across four loci) but were approximately 100-fold greater than traditional methods of Blastomyces gene disruption. Simultaneous dual-gene targeting proceeded with efficiencies similar to those of single-gene-targeting frequencies for the respective targets. CRISPR/Cas9 disruption of PRA1 or ZRT1 had a variable impact on growth under zinc-limiting conditions, showing reduced growth at early time points in low-passage-number cultures and growth similar to wild-type levels by later passage. Individual impairment of PRA1 or ZRT1 resulted in a reduction of the fungal burden in a mouse model of Blastomyces infection by a factor of ~1 log (range, up to 3 logs), and combined disruption of both genes had no additional impact on the fungal burden. These results underscore the utility of CRISPR/Cas9 for efficient gene disruption in dimorphic fungi and reveal a role for zinc metabolism in Blastomyces fitness in vivoIMPORTANCEBlastomyces is a human fungal pathogen that can cause serious, even fatal, lung infections. Genetic analysis of this fungus is possible but inefficient. We applied a recently developed gene editing technology, CRISPR/Cas9, to dramatically improve the efficiency with which gene disruptions are introduced into Blastomyces We used this system to disrupt genes involved in zinc uptake and found that this reduced the fitness of the fungus upon infection.
Asunto(s)
Blastomyces/crecimiento & desarrollo , Blastomyces/metabolismo , Edición Génica/métodos , Aptitud Genética , Zinc/metabolismo , Animales , Blastomyces/genética , Blastomicosis/microbiología , Proteína 9 Asociada a CRISPR/metabolismo , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Recuento de Colonia Microbiana , Modelos Animales de Enfermedad , Redes y Vías Metabólicas/genética , Ratones , ARN Guía de Kinetoplastida/metabolismoRESUMEN
OBJECTIVE: To evaluate the sensitivity and specificity of an enzyme immunoassay (EIA) for antibodies to a recombinant Blastomyces adhesin-1 repeat antigen (rBAD-1) to aid in the diagnosis of blastomycosis in dogs and compare the findings with results from other tests used for this purpose. DESIGN: Prospective analytic study. SAMPLE: Serum and urine from 70 dogs with and without blastomycosis. PROCEDURES: Serum and urine samples were collected from dogs with blastomycosis (n = 21), histoplasmosis (8), or nonfungal pulmonary disease (21) and from healthy control dogs living in a blastomycosis-endemic area (20). Serum was tested for antibodies against Blastomyces dermatitidis with the rBAD-1 antibody EIA and an A-antigen antibody agar gel immunodiffusion (AGID) assay. Serum and urine were tested for B dermatitidis antigen with a quantitative EIA. RESULTS: Sensitivity of the quantitative antigen EIA was 100% in serum and urine samples from dogs with blastomycosis, with specificity of 95% in urine samples from dogs with nonfungal pulmonary disease and 100% in urine samples from healthy dogs. Sensitivity of the rBAD-1 antibody EIA (95%) was significantly greater than that of the A-antigen antibody AGID assay (65%). Specificity of the antibody EIA was 88% in dogs with histoplasmosis, 95% in healthy dogs, and 100% in dogs with nonfungal pulmonary disease. CONCLUSIONS AND CLINICAL RELEVANCE: The rBAD-1 antibody EIA had greater sensitivity than the A-antigen antibody AGID assay in dogs with blastomycosis. This antibody EIA may assist in distinguishing histoplasmosis from blastomycosis. Further evaluation in a larger prospective study is needed to verify these results.
Asunto(s)
Anticuerpos Antifúngicos/inmunología , Antígenos Fúngicos/inmunología , Blastomyces/metabolismo , Blastomicosis/veterinaria , Enfermedades de los Perros/microbiología , Técnicas para Inmunoenzimas/veterinaria , Animales , Anticuerpos Antifúngicos/sangre , Anticuerpos Antifúngicos/orina , Blastomicosis/sangre , Blastomicosis/diagnóstico , Blastomicosis/orina , Enfermedades de los Perros/diagnóstico , Perros , Femenino , Técnicas para Inmunoenzimas/métodos , Masculino , Sensibilidad y EspecificidadRESUMEN
Melanin is made by several important pathogenic fungi and is implicated in the pathogenesis of a number of mycoses. This study investigates whether the thermally dimorphic fungal pathogen Blastomyces dermatitidis produces melanin. Using techniques developed to study melanization in other fungi, we demonstrate that B. dermatitidis conidia and yeast produce melanin in vitro and that yeast cells synthesize melanin or melanin-like pigment in vivo. Melanization reduced susceptibility to amphotericin B, but not to itraconazole or voriconazole. Since melanin is an important virulence factor in other pathogenic fungi, this pigment may affect the pathogenesis of blastomycosis.
Asunto(s)
Blastomyces/patogenicidad , Blastomicosis/microbiología , Enfermedades de los Perros/microbiología , Pulmón/metabolismo , Melaninas/biosíntesis , Anfotericina B/farmacología , Animales , Antifúngicos/farmacología , Blastomyces/crecimiento & desarrollo , Blastomyces/metabolismo , Perros , Humanos , Pulmón/microbiología , Pruebas de Sensibilidad MicrobianaAsunto(s)
Blastomyces/crecimiento & desarrollo , Histoplasma/crecimiento & desarrollo , Blastomyces/metabolismo , Blastomyces/patogenicidad , Cisteína/metabolismo , Ecología , Genes Fúngicos , Proteínas HSP70 de Choque Térmico/inmunología , Histoplasma/metabolismo , Histoplasma/patogenicidad , Estearoil-CoA Desaturasa/genética , VirulenciaRESUMEN
BAD-1 (Blastomyces adhesin 1), a 120-kDa protein of Blastomyces dermatitidis, functions as an adhesin, immune modulator, and essential virulence factor. Structurally, BAD-1 is composed of a short N-terminal region, a core of 30 tandem repeats critical for virulence, and a C-terminal epidermal growth factor domain that binds the protein to yeast cell surface chitin. Each of the 30 acidic residue-rich tandem repeats contains a sequence that resembles the calcium-binding loop of the EF-hand domain found in many calcium-binding proteins. Here, we investigated the binding of calcium by BAD-1 and its biological significance. Yeast washed with double distilled H2O released surface-bound BAD-1, but EGTA washes were an order of magnitude more efficient, suggesting an interaction between BAD-1 and calcium. Immobilized BAD-1 was stained with ruthenium red dye, an indicator of calcium-binding proteins. In equilibrium dialysis, BAD-1 bound 45Ca2+ with an affinity of 0.41 x 10(-5) m and a capacity of 27 calcium/mol. Mass spectrometry confirmed this capacity. Elevated [Ca2+] diminished BAD-1 solubility. Upon deletion of its C-terminal epidermal growth factor-like domain, BAD-1 resisted aggregation by elevated [Ca2+] but retained its affinity and capacity for calcium. Removing 20 copies of the tandem repeat, however, sharply reduced the capacity of BAD-1 for calcium. Growth of the bad-1 null yeast was inhibited by 5 mm EGTA, and re-expression of BAD-1 in trans or the addition of exogenous purified BAD-1 restored growth. Thus, BAD-1 is a high capacity calcium-binding protein. This property contributes to the structure and function of BAD-1, as well as to B. dermatitidis acquisition of calcium from the environment.
Asunto(s)
Blastomyces/patogenicidad , Calcio/metabolismo , Proteínas Fúngicas/metabolismo , Secuencia de Aminoácidos , Blastomyces/metabolismo , Electroforesis en Gel de Poliacrilamida , Proteínas Fúngicas/química , Modelos Moleculares , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Unión Proteica , Conformación Proteica , VirulenciaRESUMEN
The microecology of Blastomyces dermatitidis, the dimorphic etiologic agent of the potentially fatal systemic fungal infection, blastomycosis, is not well defined. Blastomyces dermatitidis may occur periodically at natural sites, perhaps aided by rotting organic material, animal droppings and physical changes. Semi-quantitative growth studies of B. dermatitidis on 2% agar plates determined the ability to utilize or tolerate a variety of substrates including simple and complex molecules as carbon source, and organic and inorganic nitrogen sources. Allantoin, creatinine, quanidoacetic acid, guanidine and cysteine may be used as sole nitrogen source. Allantoin in combination with dextrose, glycerol, lichenen, celloboise and other wood by-products support growth of B. dermatitidis at room temperature. The nutritional conversion of the fungus to the yeast form at room temperature, well demonstrated on allantoin/glycerol/yeast extract media, appears to be affected by certain inorganic compounds. The organism tolerates low to moderate levels of alpha-pinene, tannic acid, shikimic acid, veratryl alcohol, vanillic acid, and polyethyleneglycol-200. There are significant differences among isolates regarding growth on various substances at 20 degrees and 37 degrees centigrade. It appears that a variety of wood by-products and animal waste substrates, in combination, support the growth of B. dermatitidis. Their role in the ecological niche of B. dermatitidis, and the importance of nutritional dimorphism in the natural environment warrants further investigation.
Asunto(s)
Blastomyces/crecimiento & desarrollo , Microbiología del Suelo , Alantoína/metabolismo , Blastomyces/metabolismo , Blastomicosis/microbiología , EcologíaRESUMEN
Brettanomyces abstinens growing on different initial glucose concentrations showed an anaerobic inhibition of fermentation. This Custers effect decreased as the initial glucose concentration in the medium increased. Two aldehyde dehydrogenases, one NAD+-linked and the other NADP+-linked were observed. The results suggest that the NAD+-linked enzyme is involved in the production of acetic acid and is repressed by glucose. The NADP+-linked enzyme seems to be a constitutive enzyme. Acetyl-CoA synthetase activity also was not greatly affected by the growth conditions. The results support the earlier hypothesis that the Clusters effect in Brettanomyces is provoked by the reduction of NAD+ in the conversion of acetaldehyde to acetic acid.
Asunto(s)
Acetaldehído/metabolismo , Blastomyces/metabolismo , Acetato CoA Ligasa/metabolismo , Aldehído Oxidorreductasas/metabolismo , Anaerobiosis , Fermentación , Glucosa/metabolismoRESUMEN
A potent chemotactic activity for neutrophils is detectable in liquid culture filtrates of Blastomyces dermatitidis. The production of this activity is medium-dependent and culture age-dependent. The highest levels of cytotaxin were produced in filtrates of B. dermatitidis grown in tissue culture medium 199 for three or more weeks. This factor(s) stimulates directed as well as random migration. It functions directly and independently of serum. It is stable at -20 degrees C and 56 degrees C, and has a molecular weight greater than 10000 daltons. These properties define a new microbial chemotactic factor.