Proper Care and Feeding of Coccidioides: A Laboratorian's Guide to Cultivating the Dimorphic Stages of C. immitis and C. posadasii

Mead, Heather; Dyke, Marley C. Caballero Van; Barker, Bridget

doi:10.1002/cpmc.113

Cited by 26 publications

(26 citation statements)

References 57 publications

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“…The morphological transition between mycelia and spherule forms of C. immitis is dependent on sensing the host environment, and this transition can be recapitulated in the laboratory by changing the temperature and other growth conditions. C. immitis grow in saprobic form at 22–30 °C; culturing at 37–42 °C with 5–20% CO 2 in Converse media is used to convert arthroconidia to spherules [ 5 , 6 ]. Utilizing these conditions, whole genome-level transcriptional profiling studies of saprobic and parasitic forms have been performed [ 7 ].…”

Section: Introductionmentioning

confidence: 99%

Transcriptional Analysis of Coccidioides immitis Mycelia and Spherules by RNA Sequencing

Carlin

Beyhan

Peña

et al. 2021

JoF

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Coccidioides immitis and C. posadasii are dimorphic fungi that transform from mycelia with internal arthroconidia in the soil to a tissue form known as a spherule in mammals. This process can be recapitulated in vitro by increasing the temperature, CO2 and changing other culture conditions. In this study, we have analyzed changes in gene expression in mycelia and young and mature spherules. Genes that were highly upregulated in young spherules include a spherule surface protein and iron and copper membrane transporters. Genes that are unique to Coccidioides spp. are also overrepresented in this group, suggesting that they may be important for spherule differentiation. Enriched GO terms in young spherule upregulated genes include oxidation-reduction, response to stress and membrane proteins. Downregulated genes are enriched for transcription factors, especially helix–loop–helix and C2H2 type zinc finger domain-containing proteins, which is consistent with the dramatic change in transcriptional profile. Almost all genes that are upregulated in young spherules remain upregulated in mature spherules, but a small number of genes are differentially expressed in those two stages of spherule development. Mature spherules express more Hsp31 and amylase and less tyrosinase than young spherules. Some expression of transposons was detected and most of the differentially expressed transposons were upregulated in spherules.

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Section: Introductionmentioning

confidence: 99%

Transcriptional Analysis of Coccidioides immitis Mycelia and Spherules by RNA Sequencing

Carlin

Beyhan

Peña

et al. 2021

JoF

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“…Fungal growth conditions were performed as described previously ( 18 , 43 ) and are summarized here in brief. For mycelial growth, a 50-ml vented falcon tube containing 10 ml of RPMI medium (filter-sterilized RPMI 1640, 10% fetal bovine serum) was inoculated with a 1-cm by 1-cm 2× glucose yeast extract (GYE) agar plug for each strain.…”

Section: Methodsmentioning

confidence: 99%

“…Cultures were grown on a shaking incubator at 150 rpm at 30°C for 96 h. For spherule cultures, a 50-ml vented falcon tube containing 10 ml of RPMI medium was inoculated to a final concentration of 1.0 × 10 5 arthroconidia/ml in 1× phosphate-buffered saline (PBS). Arthroconidia were grown and harvested as previously described ( 18 , 43 ). Strains RMSCC2343 and RMSCC3505 did not produce enough conidia to achieve 1.0 × 10 5 arthroconidia/ml and were inoculated at 7.0 × 10 4 and 4.0 × 10 4 arthroconidia/ml, respectively.…”

Section: Methodsmentioning

confidence: 99%

“…In this study, we have cultured six strains of each species and used solid-phase microextraction (SPME) and comprehensive two-dimensional gas chromatography–time of flight mass spectrometry (GC×GC-TOFMS) to characterize the volatile organic compounds (VOCs) produced during mycelial or spherule growth. Although Coccidioides almost exclusively grows as spherules in the host, many in vitro studies on the pathogenesis and diagnosis of coccidioidomycosis have been performed using mycelial cultures, as the saprobic life cycle has historically been easier to cultivate and maintain ( 18 ). We expected to find significant differences in the VOCs between species and between life cycles but also posited that there would be a subset of VOCs that are common to Coccidioides spp.…”

Section: Introductionmentioning

confidence: 99%

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Life Cycle Dominates the Volatilome Character of Dimorphic Fungus Coccidioides spp.

Keppler

Mead

Barker

et al. 2021

mSphere

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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.

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“…Arthroconidia suspension was stored at 4 • C for up to 3 months. Complete protocol used was from Mead et al [16].…”

Section: Fungal Strain and Culturing For Methods Arthroconidia Harvestmentioning

confidence: 99%

Macrophage and Dendritic Cell Activation and Polarization in Response to Coccidioidesposadasii Infection

Diep

Tejeda-Garibay

Miranda

et al. 2021

JoF

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Coccidioidomycosis is a fungal, respiratory disease caused by Coccidioides immitis and Coccidioides posadasii. The host immune responses that define disease outcome during infection are largely unknown, although T helper responses are required. Adaptive immunity is influenced by innate immunity as antigen-presenting cells activate and educate adaptive responses. Macrophage and dendritic cell (DC) recognition of pathogen surface molecules are critical for Coccidioides clearance. We characterize the broad innate immune responses to Coccidioides by analyzing macrophage and dendritic cell responses to Coccidioides arthroconidia using avirulent, vaccine Coccidioides strain NR-166 (Δcts2/Δard1/Δcts3), developed from parental virulent strain C735. We developed a novel flow cytometry-based method to analyze macrophage phagocytosis to complement traditional image-scoring methods. Our study found that macrophage polarization is blocked at M0 phase and activation reduced, while DCs polarize into proinflammatory DC1s, but not anti-inflammatory DC2, following interaction with Coccidioides. However, DCs exhibit a contact-dependent reduced activation to Coccidioides as defined by co-expression of MHC-II and CD86. In vivo, only modest DC1/DC2 recruitment and activation was observed with avirulent Coccidioides infection. In conclusion, the vaccine Coccidioides strain recruited a mixed DC population in vivo, while in vitro data suggest active innate immune cell inhibition by Coccidioides.

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Proper Care and Feeding of Coccidioides: A Laboratorian's Guide to Cultivating the Dimorphic Stages of C. immitis and C. posadasii

Cited by 26 publications

References 57 publications

Transcriptional Analysis of Coccidioides immitis Mycelia and Spherules by RNA Sequencing

Transcriptional Analysis of Coccidioides immitis Mycelia and Spherules by RNA Sequencing

Life Cycle Dominates the Volatilome Character of Dimorphic Fungus Coccidioides spp.

Macrophage and Dendritic Cell Activation and Polarization in Response to Coccidioidesposadasii Infection

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