Infections with the opportunistic fungal pathogen Aspergillus fumigatus continue to be associated with a poor outcome. Major obstacles to effective treatment include the rapid growth of the organism in the host environment and the limited ability of current diagnostic methods to identify the infection, resulting in impaired therapeutic efficacy and a high level of mortality. In this grant we propose to explore the use of translation state array analysis (TSAA) to increase our understanding of the metabolic reprogramming that is fundamental to the adaptation of this organism to growth at 37oC. Our overarching hypothesis is that the growth of A. fumigatus at 37oC can be distinguished from growth at 25oC by the selective translation of specific mRNAs. This will be tested using TSAA, a microarray based technology that evaluates mRNA association with the translational machinery on a genome wide scale.
Aim I will use TSAA to test the hypothesis that growth at 37oC induces the translation of a specific subset of mRNAs. TSAA combines ribosome fractionation by sucrose gradient centrifugation with DNA array technology to measure the translational efficiency of individual mRNAs. A. fumigatus will be cultured at 25oC and 37oC, and hyphal extracts will be fractionated on a sucrose gradient. Since ribosome loading onto each mRNA is proportional to the rate of synthesis of the protein product, the fractionated gradient will be separated into two pools;one containing mRNAs with abundant ribosomes (representing well translated mRNA) and one containing mRNAs with few ribosomes (representing under translated mRNAs). RNA from the two pools will then be used to interrogate A. fumigatus micro-arrays, and the translation state ratio of each mRNA will be used as an indicator of how well an mRNA is translated at each temperature.
Aim II will validate candidate genes by demonstrating that their mRNAs undergo changes in ribosome loading in a temperature dependent manner, and that their encoded products are modulated by temperature and can be detected in vivo. Proteins that are up-regulated at 37oC culture are expected to contribute to the rapid growth and overall fitness of the organism at this temperature, which has the potential to identify novel drug targets or the development of new diagnostics. PROJECT NARRATIVE: Major obstacles to the effective treatment of aspergillosis include the rapid growth of the organism in the host and the limited ability of current diagnostic methods to identify the infection, resulting in a high level of mortality. The goal of this study is to use a recently developed technique, translation state array analysis, to identify mRNAs that are preferentially translated into protein at 37oC, with the long term goal of identifying proteins that could serve as novel therapeutic and/or diagnostic targets.
| Krishnan, Karthik; Askew, David S (2014) Endoplasmic reticulum stress and fungal pathogenesis. Fungal Biol Rev 28:29-35 |
| Krishnan, Karthik; Ren, Zhaowei; Losada, Liliana et al. (2014) Polysome profiling reveals broad translatome remodeling during endoplasmic reticulum (ER) stress in the pathogenic fungus Aspergillus fumigatus. BMC Genomics 15:159 |
