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Proteome dynamics during transition from exponential to stationary phase in yeast

ABSTRACT: Glycolysis is the central pathway for sugar metabolism in most living organisms. The single celled eukaryote yeast is a widely used model organism for higher eukaryotes in which the regulation of glycolysis is widely studied. S. cerevisiae is one of the few eukaryotic organisms that can efficiently grow under both aerobic and anaerobic conditions. Furthermore, this yeast switches from proliferation into a resting phase when nutrients are exhausted. However, little is known about the proteome dynamics that takes place during this transition, particularly under anaerobic conditions. Moreover, like many other organisms including humans, the genome of S. cerevisiae contains duplications which result in the expression of so called ‘isoenzymes’ in central metabolic pathways including glycolysis. Interestingly, the role of those ‘isoenzymes’ remains elusive to date. Here we describe a large-scale quantitative proteome study combining shotgun experiments using a nano-LC coupled to a QE plus Orbitrap mass spectrometer (Thermo, Germany), to capture the proteome dynamics during transition from proliferation into stationary phase, under both aerobic and anaerobic growth. Furthermore, we explore the proteome dynamics of a mutant yeast where the glycolytic isoenzymes are deleted.

INSTRUMENT(S): Q Exactive Plus

ORGANISM(S): Saccharomyces Cerevisiae (baker's Yeast)

SUBMITTER: Martin Pabst

LAB HEAD: Martin Pabst

PROVIDER: PXD031412 | Pride | 2023-04-15

REPOSITORIES: Pride

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Project description:Johne’s disease is a chronic, incurable and fatal enteric infection of ruminant species caused by Mycobacterium avium subspecies paratuberulosis (MAP) and responsible in hundreds of millions of dollars in losses for the agricultural industry. Natural infection usually begins with bacterial uptake and translocation through the epithelium of the small intestine, followed by ingestion by tissue macrophages and dissemination via the lymphatic or blood system throughout the body. To gain insights into the host innate immune responses, promoting an intracellular phenotype and adaptation of MAP within phagocytic cells, we utilized the previously developed cell culture passage model and the mass spectrometric-based quantitative proteomics approach. Using the cell culture system, which mimics an in vivo interaction of MAP with intestinal epithelium and tissue macrophages, bacteria were passed through the bovine epithelial cells and, subsequently, used for macrophage infection (termed indirect infection), while uninfected cells and macrophage infection initiated with the culture grown bacteria (termed direct infection) served as controls. Approximately 3,900 proteins were identified across all studied groups. The comparison within the subset of proteins that showed synthesis for more than two-fold in the direct infection over the uninfected control revealed an enrichment for the pro-inflammatory pathways such as the NF-κB and cytokine/chemokine signaling, positive regulation of defense response, cell activation involved in the immune response and adaptive immune system. While these responses were absent in the indirect infection, cellular pathways such as cell cycle, healing, regulation of cell adhesion, ensemble of core extracellular matrix proteins, cell surface integrins and proteins mediating the integrin signaling were remarkably high within the indirect infection. In addition to the global analysis of the macrophage proteome, in this study, we validate the proteomics data and confirm our hypothesis that MAP passage through epithelial cells can alter the way in which bacteria are able to modulate the expression and signaling of integrins in phagocytes. We demonstrate that predominant expression of integrins in the indirectly infected macrophages allows phagocytic cells to initiate stronger binding and efficient translocation through the endothelial cells, suggesting the important role of integrins in the spread of MAP infection.

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Proteome dynamics during transition from exponential to stationary phase in yeast

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