How host-like signals drive gene expression and gene expression drives capsule expansion in Cryptococcus neoformans

Cryptococcus neoformans is an opportunistic fungal pathogen with a polysaccharide capsule that becomes greatly enlarged in the mammalian host and during in vitro growth under host-like conditions. To understand how individual environmental signals affect capsule size and gene expression, we grew cells in all combinations of five signals implicated in capsule size and systematically measured cell and capsule sizes. We also sampled these cultures over time and performed RNA-Seq in quadruplicate, yielding 881 RNA-Seq samples. Analysis of the resulting data sets showed that capsule induction in tissue culture medium, typically used to represent host-like conditions, requires the presence of either CO2 or exogenous cyclic AMP (cAMP). Surprisingly, adding either of these pushes overall gene expression in the opposite direction from tissue culture media alone, even though both are required for capsule development. Another unexpected finding was that rich medium blocks capsule growth completely. Statistical analysis further revealed many genes whose expression is associated with capsule thickness; deletion of one of these significantly reduced capsule size. Beyond illuminating capsule induction, our massive, uniformly collected dataset will be a significant resource for the research community. IMPORTANCECryptococcus neoformans is an opportunistic yeast that kills [~]150,000 people each year. This major impact on human health makes it imperative to understand the basic biology of C. neoformans and the factors that mediate its virulence. One key virulence factor is a polysaccharide capsule that expands greatly during infection. To help define capsule synthesis and fungal biology, we provided cells with many different combinations of host-like signals and sampled the cultures over time for transcriptional analysis. The resulting time resolved data set is by far the largest gene expression resource ever produced for C. neoformans (881 RNA-seq samples), further enriched by accompanying capsule images and measurements. It revealed surprising findings, including that rich medium suppresses capsule size regardless of other signals. This landmark data resource will be enormously valuable to the research community as it continues to define the relationships between environmental signals and cryptococcal gene expression, biology, and virulence.