Spherule remodeling and endospore development in the fungal pathogen, Coccidioides posadasii
Coccidioides immitis and C. posadasii are soil dwelling dimorphic fungi found in North and South America. Inhalation of environmentally produced conidia results in infection. Coccidioides spp. make specialized parasitic spherules, which contain endospores that are released into the host upon spherule rupture. The molecular determinants involved in this key developmental step of infection remain largely elusive. IN this study, we utilize an attenuated mutant strain Δcts2/Δard1/Δcts3 in which chitinase genes 2 and 3 were deleted was previously created for vaccine development. This strain does not complete endospore development, which prevents completion of the parasitic lifecycle. We sought to identify pathways active in the wild-type strain during spherule remodeling and endospore formation that have been affected by gene deletion. We compared the transcriptome and volatile metabolome of the mutant Δcts2/Δard1/Δcts3 to the wild-type C735 strain. First, the global transcriptome was compared for both isolates using RNA sequencing. The raw reads were aligned to the reference genome and transcript expression analyzed. Expression of genes of interest was assessed in vivo using NanoString technology. Using solid phase microextraction (SPME) and comprehensive two-dimensional gas chromatography – time-of-flight mass spectrometry (GC×GC-TOFMS), volatile organic compounds (VOCs) were collected and analyzed. Our RNA-seq analyses reveal approximately 280 significantly differentially regulated transcripts that are either absent or improperly up or down-regulated in the mutant compared to the parent strain. This suggests that these genes are tied to networks impacted by deletion and may be critical for proper development. Of these genes, 14 were specific to the Coccidioides spp. Finally, the wild-type and mutant strains differed significantly in their production versus consumption of metabolites, with the mutant displaying increased nutrient scavenging. Overall, our results provide a set of key genes that are active during endospore formation, and demonstrate that this bioinformatics approach can define logical targets for future functional studies.