The pandemic of coronavirus disease 2019 (COVID-19) has reached nearly 240 million cases, caused nearly 5 million deaths worldwide as of October 2021, and has raised an urgent need for the development of novel drugs and therapeutics to prevent the spread and pathogenesis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). To achieve this goal, an animal model that recapitulates the features of human COVID-19 disease progress and pathogenesis is greatly needed.

The disease San Joaquin Valley Fever (coccidioidomycosis) is caused by the inhalation of Coccidioides arthroconidia. In vivo, arthroconidia transform into pathogenic structures termed spherules. Exposure to the host milieu triggers spherule development; however, the molecular mechanisms responsible for the morphological shift are not well characterized. This study compared the morphogenesis of five strains of both species of Coccidioides in two media types to improve the in vitro model of dimorphism that can be easily reproduced, and is amenable to tissue culture. We also sought to establish a modern record of the morphological switch among commonly used lab strains through a detailed account of growth under various conditions. Spherules from five strains were grown in standard (Converse) and experimental media (RPMI-sph). Strain behavior was quantified by median spherule size and spherule concentration, beginning 3 days after inoculation and followed for 10 days of growth. There were significant differences observed among Coccidioides immitis and C. posadasii strains, as well as differences between the in vitro systems.

Coccidioides immitis and C. posadasii are soil dwelling dimorphic fungi found in North and South America. Inhalation of aerosolized asexual conidia can result in asymptomatic, acute, or chronic respiratory infection. In the United States there are approximately 350,000 new infections per year. The Coccidioides genus is the only known fungal pathogen to make specialized parasitic spherules, which contain endospores that are released into the host upon spherule rupture. The molecular determinants involved in this key step of infection remain largely elusive as 49% of genes are hypothetical with unknown function. An attenuated mutant strain C. posadasii cts2/ ard1/ cts3 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 in the mutant. We compared the transcriptome and volatile metabolome of the mutant cts2/ ard1/ cts3 to the wild-type C735. First, the global transcriptome was compared for both isolates using RNA sequencing. The raw reads were aligned to the reference genome using TOPHAT2 and analyzed using the Cufflinks package. Genes of interest were screened in an in vivo model 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 show opposite expression patterns 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 endospore development and/or spherule rupture in the wild-type strain. Of these genes, 14 were specific to the Coccidioides genus. We also found that the wild-type and mutant strains differed

Coccidioides immitis and C. posadasii. These fungi are thermally dimorphic, cycling between mycelia and arthroconidia in the environment and converting into spherules and endospores within a host. Coccidioides can cause a broad spectrum of disease that can be difficult to treat. There has been a steady increase in disease, with an estimated 350,000 new infections per year in the United States. With the increase in disease and difficulty in treatment, there is an unmet need to increase research in basic biology and identify new treatments, diagnostics, and vaccine candidates. Here, we describe protocols required in any Coccidioides laboratory, such as growing, harvesting, and storing the different stages of this dimorphic fungal pathogen.