INVESTIGATING THE IMPACT OF ATTENUTATION ON DIFFERENTIAL EXPRESSION OF COCCIDIOIDES POSADASII 

Mead, H.L., Roe, C.C., Teixeira, M., Barker, B.M.,
Northern Arizona University, Pathogen and Microbiome Institute, Flagstaff, AZ USA

Introduction
Coccidioides immitis and C. posadasii are soil dwelling fungi found in regions with semi-arid alkaline soil in both North and South America. In a susceptible host, inhalation of aerosolized asexual spores can result in asymptomatic, acute or chronic respiratory infection. The Coccidioides genus possesses the ability to exist as a dimorph having a distinct environmental and parasitic lifecycle. Chitin is a major component of the cell wall in both the saprobic and parasitic lifecycle. The Coccidioides genomes contain genes from each class (I-VII) of chitin synthase with differing expression during each lifecycle. Deletion of two chitinase genes and an acireductone dioxygenase from C. posadasii C735 resulted in a mutant (Δcts2/Δard1/Δcts3) that does not complete the parasitic lifecycle. This study compared existing transcriptional data from the pathogenic parent strain C. posadasii strain C735 and mutant strain Δcts2/Δard1/Δcts3 to characterize potential chitin dependent pathways. 

Methods
Spherule cultures from strain Δcts2/Δard1/Δct3 were grown in the same conditions as the previously characterized for the parental strain. Total RNA was extracted using Trisure and mRNA was isolated using NEBNext beads. Paired-end RNA sequencing of the wild type and attenuated strains as well as whole genome sequencing was performed on an Illumina MiSeq platform. Genomic reads were de novo assembled and mRNA reads from both the wild type and the attenuated strain were aligned to the assembled reference genome using TOPHAT2. Additionally, newly generated reads from Δcts2/Δard1/Δct3 were randomly fragmented to 33bp read length to match published experimental data and aligned to the reference genome. Differential expression results for both alignments were compared. Genes related to chitin synthesis were compared for presence or absence between the two strains. Differentially expressed genes of interest will be validated by real-time RT-PCR. 

Results
The transcriptional landscape of both strains had 98.8% similarity. A scatter plot comparing the genes expressed during the parasitic lifecycle of both strains suggest a high degree of resemblance between expression profiles (Figure 1). C. posadasii wildtype strain C735 matches previously reported results. Chitin related genes, endochitinase 2, chitinase 7, chitinase 5, chitin synthase activator, have increased expression during the parasitic lifecycle. In contrast, endochitnase 1, chitinase 4, and chitinase 3 have increased expression in the saprobic lifecycle. The attenuated Δcts2/Δard1/Δcts3 shows increased expression endochitinase 2, chitinase 7, and a complement fixation chitnase in the parasitic lifecycle. The saprobic lifecycle had increased expression of chitin synthase 4, chitin synthase D and chitin synthase class V. Future work includes validation of transcript levels with real time RT-PCR and differential expression analysis.


Conclusion
The morphological switch to spherule growth, which initiates host infection, is an important part of the infection dynamic for both species of Coccidioides. The comparison of the wild type and attenuated transcription profiles will begin to elucidate pathways crucial to pathogenesis, establishment of infection, and maintenance of the parasitic life-cycle of this important human pathogen.