Investigating How the Pathogenic Yeast Cryptococcus neoformans Effects Gene Expression in Host Macrophages

Abstract

The pathogenic yeast Cryptococcus neoformans (Cn) is the cause of death of nearly 181,000 people annually. When Cn is inhaled, it is first detected by alveolar macrophages, innate phagocytic cells that engulf and attempt to destroy Cn. However, post-ingestion a range of different outcomes are possible. For an example, the macrophage may kill the ingested yeast, the yeast may replicate within and eventually escape from the macrophage, or the yeast may persist within the macrophage in a dormant state. To investigate how Cn infection may affect the fate of host macrophages, our lab uses RNAseq-based transcriptome profiling to determine how phagosomal Cn influences gene expression in host cells. These experiments utilize in vitro cultures of Cn infected murine macrophages. Our preliminary data has suggested that the metabolic activity of the Cn may compromise our transcriptome data by artificially altering the expression of glucose-regulated genes, such as thioredoxin interacting protein (TXNIP), which may indirectly impact the activity of important transcriptional regulators including p53. To circumvent this problem, I used a nutrient replenishment strategy, replacing the growth media in the macrophage: Cn cultures at experimentally determined time intervals. The effectiveness of this strategy was determined by measuring the protein products of glucose-responsive genes by western blotting. Glucose concentration was measured via glucose oxidase assays by withdrawing medium from samples at our experimentally determined time intervals. This work revealed that the depletion of glucose in Cn:macrophage cultures could be circumvented by replenishing the media at 6-hour intervals for the duration of 24 hours. We assert that this modified Cn:macrophage culture system is suitable for use in future transcriptome profiling experiments that will help us to accurately determine how Cninfection alters gene expression in host macrophages.