Cryptococcus neoformans (Cn) is a common pathogenic yeast that is found in soil and other niches worldwide. In individuals with compromised or damaged immune systems, Cn causes life-threatening cryptococcosis and fungal meningitis, leading to ~600,000 deaths per year. However, in healthy individuals, Cn is able to establish chronic infections that can last many years or even a lifetime by utilizing host macrophages as an intracellular growth niche. Quite how Cn is able to suppress the fungicidal activity while also preserving the viability of host macrophages remains an open question but is likely to require significant transcriptional reprograming of the host and pathogen. The overarching goal of our research program is to understand how Cn manipulates gene expression in host cells (and itself) to enable its long-term persistence within macrophages. Our previous work and preliminary studies have indicated that this may be achieved in part through the modulation of macrophage canonical NF-?B signaling, a pathway closely associated with macrophage maintenance, M1 polarization and pro- inflammatory function. As a step towards achieving our long-term goal, we will investigate how the intracellular growth of Cn impacts macrophage polarization and survival through altered NF-?B signaling and use unbiased transcriptome profiling to determine how the gene expression networks underlying the M1 and M2 phenotype are affected. Specifically, we will: 1) Determine how intracellular Cn deregulates NF-?B signaling and its effects on macrophage fate and function. As our preliminary data indicates that intracellular Cn accumulation promotes constitutive nuclear localization of NF-?B and inhibitory I?B? proteins, we will use a focused approach that combines live cell reporters, chromatin immunoprecipitation, and qRT-PCR to determine how this influences the expression of a panel of cell fate-regulating and M1- associated NF-?B-responsive genes. 2) Determine how the transcriptome profile of both macrophage and Cn is affected by intracellular Cn growth and how this impacts macrophage polarization. As ingested Cn is likely to affect multiple stress-responsive signaling pathways in host cells, influencing gene expression and function in more complex ways than can assessed through targeted analysis of individual genes, we will use an unbiased RNA-seq approach to investigate how Cn affects the transcriptional profile of (A) host macrophages and (B) Cn itself. By performing these experiments in the presence of different polarizing stimuli, these studies will also enable us to determine (i) how Cn infection impacts the ability of macrophages to integrate polarizing signals and (ii) how the different phagosomal environments produced by M1 and M2 polarized macrophages affect gene expression in Cn. Collectively, these studies will provide new insights into how intracellular Cn growth alters gene expression in the host and pathogen and how this might influence or be influenced by macrophage polarization.
The objective of this proposal is to determine how intracellular infection of macrophages by the pathogenic yeast, Cryptococcus neoformans (Cn) affects the ability of these innate immune cells to correctly interpret exogenous signals that regulate their anti-microbial activity. This will be achieved using a combination of live cell microscopy and RNA-seq-based transcriptional profiling with a particular focus on how Cn infection affects NF- ?B, an essential regulator of macrophage inflammatory function, fungicidal activity, and survival.
