Project description:The aims of this study were to present modifications to the annotations of the genome of C. 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 were analyzed using a GeLC-MS/MS method.
Project description:Coccidioides immitis (C. immitis) is a dimorphic fungus that causes disease in mammals including human beings. It grows as a mycelium in the soil but differentiates into a pathogenic structure called a spherule in the host. We compared the transcriptome of C. immitis mycelia and day 2 and day 8 spherules grown in vitro using a custom custom oligonucleotide microarray from Nimblegen.
Project description:Coccidioides immitis (C. immitis) is a dimorphic fungus that causes disease in mammals including human beings. It grows as a mycelium in the soil but differentiates into a pathogenic structure called a spherule in the host. We compared the transcriptome of C. immitis mycelia and day 2 and day 8 spherules grown in vitro using a custom custom oligonucleotide microarray from Nimblegen. C. immitis RS strain isolated from infected mice was grown on agar. Arthroconidia was harvested and inoculated into mycelial spores. RNA was extracted from spores at different days post-inoculation and hybridized to a custom Nimblegen array, in order to identify differentially expressed genes between mycelia and spherules.
Project description:Coccidioides spp. are fungal pathogens endemic to Southern California, Arizona, and other desert regions where they cause a spectrum of symptoms known as Valley Fever in otherwise healthy individuals. In the soil, Coccidioides grows in a hyphal form that produces vegetative spores (arthroconidia). When arthroconidia are inhaled by mammals they undergo an elaborate developmental transition consisting of germination, multiple rounds of nuclear division, and segmentation to form large spherules filled with vegetative endospores. Very little is understood about the molecular basis of spherule formation. Here we characterize the role of the conserved transcription factor Ryp1 in Coccidioides spherule formation. We show that Coccidioides ryp1 mutants are unable to form mature spherules, instead arresting at an early stage of spherulation. We analyze the transcriptional profile of wild-type and Δryp1 mutant cells under hyphal and spherule-promoting conditions, thereby defining a set of hyphal- or spherule-enriched transcripts that are dependent on Ryp1 for their expression. Ryp1-dependent transcripts include key virulence factors such as SOWgp, which encodes the spherule outer wall glycoprotein. In the mouse model of coccidioidomycosis, we find that the Δryp1 mutant is completely avirulent, indicating that Ryp1-dependent pathways are essential for the ability of Coccidioides to cause disease. Vaccination of C57BL/6 mice with live Δryp1 spores does not provide any protection from lethal C. posadasii intranasal infection, consistent with our findings that the Δryp1 mutant fails to make mature spherules. Taken together, this work identifies the first transcriptional regulator that drives mature spherulation and virulence in Coccidioides.
Project description:The CPS1 gene was identified as a virulence factor in the maize pathogen, Cochliobolus heterostrophus. Hypothesizing that the homologous gene in Coccidioides posadasii (Cp) could be important for virulence, we created a deletion mutant, Îcps1, which was unable to cause disease in three strains of mice (C57BL/6, BALB/c, or the severely immunodeficient NOD-scid,γcnull [NSG]). Only a single colony was recovered from one of 60 C57BL/6 mice following intranasal infections of up to 4400 spores. Following administration of very high doses (10,000 to 2.5 x 10^7 spores) to NSG and BALB/c mice, spherules were observed in lung sections at time points from day 3 to day 10 post-infection, but nearly all appeared degraded with infrequent endosporulation. Although the role of CPS1 in virulence is not understood, phenotypic alterations and transcription differences of at least 33 genes in Îcps1 vs. Cp is consistent with both metabolic and regulatory functions for the gene. The in vitro phenotype of Îcps1 showed slower growth of mycelia with delayed and lower spore production compared to Cp, and in vitro spherules were smaller. Vaccination of C57BL/6 or BALB/c mice with live Îcps1 spores either intranasally, intraperitoneally or subcutaneously resulted in over 95% survival with mean residual lung fungal burdens <1000 colony-forming units from an otherwise lethal Cp intranasal infection. Considering its apparently complete attenuation of virulence and the high degree of resistance to Cp infection when used as a vaccine, Îcps1 is a promising vaccine candidate for preventing coccidioidomycosis in humans or other animals. Wild type and CPS1 deletion mutant strains of Coccidioides posadasii strain Silveira spherules grown for 48 hours in Converse medium at 38 degrees celsius in duplicate. RNAseq was performed on an Illumina HiSeq2000 (2x100 paired end).
Project description: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 spherules within the lungs of vulnerable hosts. The 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 and comprehensive two-dimensional gas chromatography coupled to time-of-flight mass spectrometry (GC×GC–TOFMS), we characterized the VOCs produced by six strains of each species during mycelial or spherule growth. We detected a total of 353 VOCs that were at least two-fold more abundant in a Coccidioides culture versus medium controls and found the volatile metabolome of Coccidioides is more dependent on growth phase (spherule 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.