title: Isolation of Blastomyces dermatitidis yeast from lung tissue during murine infection for in vivo transcriptional profiling.
abstract: Blastomyces dermatitidis belongs to a group of thermally dimorphic fungi that grow as sporulating mold in the soil and convert to pathogenic yeast in the lung following inhalation of spores. Knowledge about the molecular events important for fungal adaptation and survival in the host remains limited. The development of high-throughput analytic tools such as RNA sequencing (RNA-Seq) has potential to provide novel insight on fungal pathogenesis especially if applied in vivo during infection. However, in vivo transcriptional profiling is hindered by the low abundance of fungal cells relative to mammalian tissue and difficulty in isolating fungal cells from the tissues they infect. For the purpose of obtaining B. dermatitidis RNA for in vivo transcriptional analysis by RNA-Seq, we developed a simple technique for isolating yeast from murine lung tissue. Using a two-step approach of filtration and centrifugation following lysis of murine lung cells, 91% of yeast cells causing infection were isolated from lung tissue. B. dermatitidis recovered from the lung yielded high-quality RNA with minimal murine contamination and was suitable for RNA-Seq. Approximately 87% of the sequencing reads obtained from the recovered yeast aligned with the B. dermatitidis genome. This was similar to 93% alignment for yeast grown in vitro. The use of near-freezing temperature along with short ex vivo time minimized transcriptional changes that would have otherwise occurred with higher temperature or longer processing time. In conclusion, we have developed a technique that recovers the majority of yeast causing pulmonary infection and yields high-quality fungal RNA with minimal contamination by mammalian RNA.
authors.affiliation: Department of Medicine, School of Medicine and Public Health, University of Wisconsin - Madison, 1550 Linden Drive, Microbial Sciences Building, Room 4335A, Madison, WI 53706, USA. ajvanden@wisc.edu
authors.forename: Amber J
authors.initials: AJ
authors.lastname: Marty
authors.forename: Marcel
authors.initials: M
authors.lastname: Wüthrich
authors.forename: John C
authors.initials: JC
authors.lastname: Carmen
authors.forename: Thomas D
authors.initials: TD
authors.lastname: Sullivan
authors.forename: Bruce S
authors.initials: BS
authors.lastname: Klein
authors.forename: Christina A
authors.initials: CA
authors.lastname: Cuomo
authors.forename: Gregory M
authors.initials: GM
authors.lastname: Gauthier
mesh_terms: D000818:Animals; D001758:Blastomyces / Q000235:genetics / Q000302:isolation & purification*; D001759:Blastomycosis / Q000382:microbiology*; D004195:Disease Models, Animal; D020869:Gene Expression Profiling / Q000379:methods*; D008168:Lung / Q000382:microbiology*; D051379:Mice; D009172:Mycology / Q000379:methods*; D012331:RNA, Fungal / Q000302:isolation & purification; D017423:Sequence Analysis, RNA / Q000379:methods; D013048:Specimen Handling / Q000379:methods
chemical_list: D012331:RNA, Fungal