Optimizing in vitro spherulation cues in the fungal pathogenCoccidioides

Coccidioides spp.are part of a group ofthermallydimorphic fungal pathogens, which grow asfilamentous cells (hyphae)in the soil and transform to a different morphology upon inhalation into the host.TheCoccidioideshost form, the spherule, is unique and highly under characterized due to both technical and biocontainment challenges.Each spherule arises from an environmental spore (arthroconidium), matures, and develops hundreds of internal endospores, which are released from the spherule upon rupture. Each endospore can then go on to form another spherule in a cycle called spherulation. One of the foremosttechnicalchallenges has been reliably growing spherules in culture and consistently inducing endospore release withoutthe generation ofhyphae. Here, wepresentoptimization ofin vitro spherule growth and endospore release, by closely controlling starting cell density in the culture, usingfreshly-harvestedarthroconidia, and decreasing the concentration of multiple salts in spherulation media. We developed a minimal media to test spherule growth on various carbon and nitrogen sources andwent on to define a critical role forTamolin both early spherule formation and dispersal of the outer spherule wall, which accumulatesinto a dramatic large film inTamol’sabsence. Finally, we examined how the conditions under which arthroconidia are generated influence their transcriptome and subsequent development into spherules, demonstrating that this is an important variable to control when designing spherulation experiments. Together, our data reveal multiple strategies to optimize in vitro spherulation growth, enabling characterization ofthis virulence-relevant morphology. We generated spores (arthroconidia) and then aged them for 0-7 weeks in PBS at 4 degrees C and characterized their transcriptome after storage in these conditions. Additionally, we generated spores (arthroconidia) by growing hyphal cells for 4 weeks at either 25 degree C, 30 degrees C, or 37 degrees C and then determined their transcriptome by RNA-seq (3 replicates from each spore stock, each spore stock was generated 2 separate times).