Using a Systems Biology Approach to Develop a Better Understanding of the Fungal Response to Cell Wall Stress

Abstract

This study focuses on the fungi Aspergillus nidulans and the Cell Wall Integrity (CWI) signaling pathway. MpkA is the final kinase in this pathway. To better understand the role of MpkA under ideal, steady-state growth conditions, a single endpoint phosphoproteomic study was carried out to compare changes in protein phosphorylation and gene expression between a mpkA deletion mutant and the isogenic control strain, suggesting that MpkA is crucial for maintaining cell wall strength as well as other important functions. Next, a dynamic phosphoproteomic approach was used to characterize the normal cellular response to cell wall stress. A multi-omic approach was used to capture dynamic changes in both phosphorylation and transcription over time using phosphoproteomics and transcriptomic studies. A multivariate adaptive regression splines (MARS) analysis was used to model the dynamic behavior in both datasets in response to micafungin treatment. This approach identified 431 dynamic phosphorylation sites and 1810 dynamic transcripts after cell wall perturbation. The kinases that exhibited dynamic behavior were further characterized by testing kinase deletion strains. These strains were tested to determine the impact specific kinase deletions on micafungin sensitivity. This revealed novel connections and crosstalk mediated by the CWI pathway with the endocytic pathway and the septation initiation network (SIN). Using the same dynamic approach, the MpkA deletion mutant’s response to micafungin was characterized. Novel approaches to the dynamic analysis increased the identification of dynamic p-sites in the control dataset to ~1900 phosphosites. In the deletion mutant, this approach identified 188 dynamic phosphosites. This implicates MpkA as an indirect regulator of 1735 phosphosites. These phosphosites are located on 902 proteins including a significant number of actin and septation related proteins. The crosstalk between the CWI and SIN pathways reveals an important role for septation in the survival of wall. To characterize this response and understand resource allocation in the cell and the response to cell wall stress, a novel live cell imaging system was developed to capture highly resolved timecourse images. This system leveraged the Lifeact reporter to characterize actin localization, dynamics, and early growth and development in A. nidulans.