Mitchell, Natalie M.

Laser capture microdissection (LCM) coupled to label-free quantitative mass spectrometry is a viable strategy to identify biomarkers from infected tissues. In this study, LCM was employed to take a “snapshot” of proteins produced in vivo during Coccidiodies spp. infection in human lungs. Proteomic analysis of LCM lung sections revealed hundreds of hosts and Coccidioidal proteins. Twenty-seven highly abundant Coccidioides spp. proteins were identified which do not share significant sequence orthology with human proteins. Three of the 27 Coccidioidal proteins are also potential Coccidoides-specific biomarkers, as they also do not share sequence homology to any other pathogenic fungus or microbe. Gene ontology analysis of the 27 biomarker candidate proteins revealed enriched hydrolase activity and increased purine and carbohydrate metabolism functions. Finally, we provide proteomic evidence that all 27 biomarker candidates are produced by the fungus when grown in vitro in a media- and growth-phase dependent manner.

Abstract Coccidioides spp. are important pneumonia-causing pathogens of the American southwest, but little is known about their glycobiology and how their glycosylations differ from other pneumonia-causing fungi. There is mounting preliminary evidence to suggest genus or even species-specific glycosylations in the fungal kingdom due to the presence of unique Carbohydrate-Active Enzymes (CAZymes) in fungal genomes (Deshpande and others 2008; Karkowska-Kuleta and Kozik 2015). If Coccidioides spp.-specific glycans can be identified, it may be possible to exploit these differences to develop more specific diagnostic approaches and more effective therapeutics. Herein we i) mined Coccidioides spp. and other pathogenic fungal genomes to identify CAZymes specific for Coccidiodes spp., ii) proteomically determined the Coccidioides spp. “CAZome” produced in vivo and in vitro, and iii) utilized glycomics to differentiate Coccidioides genus-specific N-glycans from other pathogenic fungi. As far as we are aware, this is the first proteomic and glycomic comparison of the N-glycomes and CAZomes of different fungal genera during infection in human hosts.

AbstractThe aims of this study are to provide protein‐based evidence upon which to reannotate the genome of Coccidiodes posadasii, one of two closely related species of Coccidioides, a dimorphic fungal pathogen that causes coccidioidomycosis, also called Valley fever. Proteins present in lysates and filtrates of in vitro grown mycelia and parasitic phase spherules from C. posadasii strain Silveira are analyzed using a GeLC‐MS/MS method. Acquired spectra are processed with a proteogenomics workflow comprising a Silveira proteome database, a six‐frame translation of the Silveira genome and an ab initio gene prediction tool prior to validation against published ESTs. This study provides evidence for 837 genes expressed at the protein level, of which 169 proteins (20.2%) are putative proteins and 103 (12.3%) are not annotated in the Silveira genome. Additionally, 275 novel peptides are derived from intragenic regions of the genome and 13 from intergenic regions, resulting in 172 gene refinements. Additionally, we are the first group to report translationally active retrotransposon elements in a Coccidioides spp. Our study reveals that the currently annotated genome of C. posadasii str. Silveira needs refinement, which is likely to be the case for many nonmodel organisms.