ChIP-seq analysis of SREB during yeast phase growth of Blastomyces dermatitidis at 37oC
ABSTRACT: ChIP-seq analysis was used to identify B. dermatitidis genes bound by the GATA transcription factor encoded by SREB during growth as yeast at 37oC SREB was engineered to contain an in-frame 3x-hemagglutinin (HA) epitope tag at the C-terminus. The SREB-3xHA construct was under control of its native promoter and contained the 3-untranslated region. Using Agrobacterium tumefaciens, B. dermatitidis ATCC 26199 was transformed with the SREB-3xHA construct (referred to a SREB-3xHA strain in this document). The SREB-3xHA construct was functional because retransformation of SREBΔ with the construct complented the null mutant. Chromatin was extracted and sheared from ATCC 26199 and SREB-3xHA yeast grown in liquid Histoplasma macrophage medium (HMM) containing 10 μM iron sulfate (FeSO4) at 37oC. ATCC 26199 was the untagged control strain.
Project description:ChIP-seq analysis was used to identify B. dermatitidis genes bound by the GATA transcription factor encoded by SREB during growth as yeast at 37oC SREB was engineered to contain an in-frame 3x-hemagglutinin (HA) epitope tag at the C-terminus. The SREB-3xHA construct was under control of its native promoter and contained the 3-untranslated region. Using Agrobacterium tumefaciens, B. dermatitidis ATCC 26199 was transformed with the SREB-3xHA construct (referred to a SREB-3xHA strain in this document). The SREB-3xHA construct was functional because retransformation of SREB? with the construct complented the null mutant. Chromatin was extracted and sheared from ATCC 26199 and SREB-3xHA yeast grown in liquid Histoplasma macrophage medium (HMM) containing 10 ?M iron sulfate (FeSO4) at 37oC. ATCC 26199 was the untagged control strain.
Project description:For ChIPseq analyses JB1 and UVO151 (JB1 Pcrg:clp1) were transformed with a Cib1-3xHA fusion construct was expressed under control of the endogenous promoter. The fusion construct was integrated at the endogenous locus replacing the native Cib1 gene.
Project description:Thermally dimorphic human fungal pathogens undergo a reversible program of cellular differentiation in response to their environment that is essential for infectivity and pathogenicity. In the soil, these organisms grow as highly polarized, multicellular hyphal filaments that produce infectious particles. When inhaled by a mammalian host, these cells switch to a unicellular yeast form that causes disease even in healthy hosts. Temperature is considered to be the primary environmental cue that promotes reversible cellular differentiation; however, a shift to a lower temperature in vitro induces filamentous growth in an inefficient and asynchronous manner. In a search for other signals that regulate morphogenesis, we considered the monosaccharide N-acetylglucosamine (GlcNAc), which is a major component of microbial cell walls and is ubiquitous in the environment. GlcNAc was a potent and specific inducer of the yeast-to-filament transition in two thermally dimorphic fungi, Histoplasma capsulatum and Blastomyces dermatitidis. Micromolar concentrations of GlcNAc induced a robust morphological transition of H. capsulatum after temperature shift, indicating that fungal cells sense GlcNAc to promote filamentation. The synchronous morphologic transition stimulated by low temperature and GlcNAc allowed us to examine the temporal regulation of the transcriptome during morphogenesis to reveal candidate genes involved in establishing the filamentous growth program. Through this analysis, we identified two genes encoding GlcNAc transporters, NGT1 and NGT2, that were necessary for H. capsulatum cells to robustly filament in response to GlcNAc. Unexpectedly, NGT1 and NGT2 were important for efficient H. capsulatum yeast-to-filament conversion in standard glucose medium, suggesting that Ngt1 and Ngt2 monitor endogenous levels of GlcNAc to control multicellular filamentous growth in response to temperature. Overall, our work indicates that GlcNAc functions as a highly conserved cue of morphogenesis in fungi, which further enhances the significance of this ubiquitous sugar in cellular signaling in eukaryotes. For each time-course sample, cDNA was coupled to Cy5 and a reference cDNA pool was made by combining RNA from t = 0 and all late time course samples, which was coupled to Cy3. For end point microarray experiments (i.e., established yeast samples compared to established filamentous samples), G217B yeast cDNA was coupled to Cy5 and filament cDNA was coupled to Cy3.
Project description:Blastomyces dermatitidis is a dimorphic fungal pathogen that converts from mycelia or conidia to a host-adapted yeast morphotype upon infection. Conversion to the yeast form is accompanied by the production of the virulence factor BAD1. Yeast-phase-specific expression of BAD1 is transcriptionally regulated, and its promoter shares homology with that of the yeast-phase-specific gene YPS3 of Histoplasma capsulatum. Serial truncations of the BAD1 upstream region were fused to the lacZ reporter to define functional areas in the promoter. Examination of PBAD1-lacZ fusions in B. dermatitidis indicated that BAD1 transcription is upregulated in the yeast phase. The 63-nucleotide box A region conserved in the YPS3 upstream region was shown to be an essential component of the minimal BAD1 promoter. A matched PYPS3-lacZ construct indicated that this same region was needed for minimal YPS3 promoter activity in B. dermatitidis transformants. Reporter activity in H. capsulatum transformants similarly showed a requirement for box A in the minimal BAD1 promoter. Several putative transcription factor binding sites were identified within box A of BAD1. Replacement of two of these predicted sites within box A--a cAMP responsive element and a Myb binding site--sharply reduced transcriptional activity, indicating that these regions are critical in dictating the yeast-phase-specific expression of this crucial virulence determinant of B. dermatitidis.
Project description:The black-pigmented fungus Exophiala dermatitidis is considered to be a harmless colonizer of the airways of cystic fibrosis (CF) patients. The aim of this study was to establish the recovery rate of E. dermatitidis in respiratory specimens from CF patients, transplant recipients, and subjects with other respiratory disorders in Sweden. Second, we wished to determine if particular clinical traits were associated with E. dermatitidis colonization of the airways and the antifungal susceptibility profiles of Exophiala strains. Sputum and bronchoalveolar lavage samples (n = 492) derived from 275 patients were investigated. E. dermatitidis was isolated in respiratory specimens from 19% (18/97) of the CF patients but in none of the other patient categories. All isolates were recovered after 6 to 25 days of incubation on erythritol-chloramphenicol agar (ECA) medium. Morphological and genetic analyses confirmed species identity. Pancreatic insufficiency was positively associated with the presence of E. dermatitidis in sputum samples (P = 0.0198). Antifungal susceptibility tests demonstrated that voriconazole and posaconazole had the lowest MICs against E. dermatitidis. In conclusion, E. dermatitidis is a frequent colonizer of the respiratory tract in CF patients in Sweden and appears to be associated with more advanced disease. Whether E. dermatitidis is pathogenic remains to be elucidated.
Project description:Exophiala dermatitidis is a dematiaceous fungus that is increasingly being identified as a cause of fungal infection especially in patients with immunodeficiency. To date, however, the factors predisposing E. dermatitidis and its optimal treatments have not been fully addressed. Here, we report the first patient with untreated multiple myeloma who developed E. dermatitidis pulmonary infection. We also review recent clinical reports describing the features of E. dermatitidis infection.
Project description:Blastomyces dermatitidis is a dimorphic fungal pathogen that primarily causes blastomycosis in the midwestern and northern United States and Canada. While the genes controlling sexual development have been known for a long time, the genes controlling sexual reproduction of B. dermatitidis (teleomorph, Ajellomyces dermatitidis) are unknown. We identified the mating-type (MAT) locus in the B. dermatitidis genome by comparative genomic approaches. The B. dermatitidis MAT locus resembles those of other dimorphic fungi, containing either an alpha-box (MAT1-1) or an HMG domain (MAT1-2) gene linked to the APN2, SLA2, and COX13 genes. However, in some strains of B. dermatitidis, the MAT locus harbors transposable elements (TEs) that make it unusually large compared to the MAT locus of other dimorphic fungi. Based on the MAT locus sequences of B. dermatitidis, we designed specific primers for PCR determination of the mating type. Two B. dermatitidis isolates of opposite mating types were cocultured on mating medium. Immature sexual structures were observed starting at 3 weeks of coculture, with coiled-hyphae-containing cleistothecia developing over the next 3 to 6 weeks. Genetic recombination was detected in potential progeny by mating-type determination, PCR-restriction fragment length polymorphism (PCR-RFLP), and random amplification of polymorphic DNA (RAPD) analyses, suggesting that a meiotic sexual cycle might have been completed. The F1 progeny were sexually fertile when tested with strains of the opposite mating type. Our studies provide a model for the evolution of the MAT locus in the dimorphic and closely related fungi and open the door to classic genetic analysis and studies on the possible roles of mating and mating type in infection and virulence.
Project description:Research into the cooperative pathogenicity of microbes in cystic fibrosis (CF) lungs is crucial for an understanding of the pathophysiology of infections and the development of novel treatment strategies. This study investigated the impact of the common CF-associated bacterial pathogen Pseudomonas aeruginosa on the black yeast Exophiala dermatitidis. It evaluated the planktonic growth, biofilm formation, morphology, and virulence of the fungus in the presence or absence of P. aeruginosa. It also determined the role of P. aeruginosa quorum-sensing (QS) molecules within these interactions, e.g., by using sterile culture filtrate and QS-deficient mutants. P. aeruginosa is known to inhibit the planktonic growth of E. dermatitidis. We found that fungal biofilm formation increased in the presence of P. aeruginosa after 24 h but is decreased significantly after 48 h. This effect was reversed when, instead of QS wild-type strains, ?lasR, and ?rhlR mutants were added to E. dermatitidis biofilm formation. The number and length of hyphae were substantially reduced when E. dermatitidis was co-cultivated with P. aeruginosa, but not when it was co-cultivated with the mutants. Experiments testing the virulence of E. dermatitidis in the greater wax moth Galleria mellonella showed a synergetic effect on larval killing when E. dermatitidis was injected together with P. aeruginosa culture filtrate. Survival rates were decreased when biofilm culture filtrate was added but not when planktonic culture filtrate was added. In summary, P. aeruginosa affects the growth, morphology, biofilm formation, and virulence of E. dermatitidis. N-acyl-L-homoserine lactone (AHL) QS molecules regulated factors that have been shown to contribute to the inhibition of the ability of E. dermatitidis to form filaments and biofilm.
Project description:Various fungi have the ability to colonize surfaces and to form biofilms. Fungal biofilm-associated infections are frequently refractory to targeted treatment because of resistance to antifungal drugs. One fungus that frequently colonises the respiratory tract of cystic fibrosis (CF) patients is the opportunistic black yeast-like fungus Exophiala dermatitidis. We investigated the biofilm-forming ability of E. dermatitidis and its susceptibility to various antiinfective agents and natural compounds. We tested 58 E. dermatitidis isolates with a biofilm assay based on crystal violet staining. In addition, we used three isolates to examine the antibiofilm activity of voriconazole, micafungin, colistin, farnesol, and the plant derivatives 1,2,3,4,6-penta-O-galloyl-b-D-glucopyranose (PGG) and epigallocatechin-3-gallate (EGCG) with an XTT reduction assay. We analysed the effect of the agents on cell to surface adhesion, biofilm formation, and the mature biofilm. The biofilms were also investigated by confocal laser scan microscopy. We found that E. dermatitidis builds biofilm in a strain-specific manner. Invasive E. dermatitidis isolates form most biomass in biofilm. The antiinfective agents and the natural compounds exhibited poor antibiofilm activity. The greatest impact of the compounds was detected when they were added prior cell adhesion. These findings suggest that prevention may be more effective than treatment of biofilm-associated E. dermatitidis infections.