Shigella flexneri is an enteroinvasive bacterium responsible for causing bacterial dysentery. This infection is the result of a complex delivery system — known as the type III apparatus — that injects various proteins into intestinal epithelia to evade an immune response from the human host.1 Mechanisms involved in S. flexneri infection have been well documented in previous studies, including the importance of protein phosphorylation involved in infection.2 As a result of previous studies, Schmutz et al. (2013) hypothesize that the complexities of host signaling during infection with S. flexneri are controlled by protein phosphorylation.3 Schmutz et al. were able to perform an unbiased analysis of protein phosphorylation during and after infection with S. flexneri using a label-free, quantitative phosphoproteomics approach.
HeLa cells infected with S. flexneri were used for the phosphoproteomic analysis and allowed to incubate for 15, 30, 60 and 120 minutes at a multiplicity of infection of 40. Cells were lysed, enriched and prepared for liquid chromatography and tandem mass spectrometry on a dual-pressure LTQ Orbitrap mass spectrometer with a nanoelectrospray ion source (Thermo Scientific), followed by collision-induced dissociation.
The researchers compared the results of the mass spectrometry analysis to a recently published phosphoproteomics analysis of Salmonella typhimurium infection.4 A total of 3,234 phosphopeptides corresponding to 3,109 phospho-sites and 1,183 phosphorylated proteins were identified, and 14.3% of the phosphopeptides showed a statistically significant change in phosphorylation after S. flexneri infection. Proteins contained primarily single and dual phosphorylated peptides and were located on serine and threonine residues.
To determine what pathways were involved, gene ontology analysis was performed using the Database for Annotation Visualization and Integrated Discovery (DAVID). This analysis revealed that phosphorylated proteins were involved in many different cellular processes, including transcription, signal transduction, intracellular trafficking cell cycle, and assembly components of the cytoskeleton. Using another database called KEGG, Schmutz et al. identified the mTOR signaling pathway as the most over-represented pathway in the phosphoproteome of S. flexneri. This pathway contains a central serine/threonine protein kinase that regulates cell growth and metabolism, autophagy, the actin cytoskeleton, cell proliferation, cell survival, protein synthesis and transcription within S. flexneri cells.5
Schmutz et al. were also able to investigate the downstream effects of the effector protein OspF, which is secreted to moderate the inflammatory response in infected host cells and signals protein phosphorylation. OspF was found to affect the phosphorylation of more than 300 proteins, which was a much greater impact than the researchers had anticipated. Taken as a whole, the data presented in these experiments by Schmutz et al. contain new insights into complex host–pathogen interactions and could be used to identify potential drug targets for S. flexneri and other bacterial infections.
1. Schroeder, G.N., and Hilbi, H. (2008) “Molecular Pathogenesis of Shigella spp.: Controlling Host Cell Signaling, Invasion, and Death by Type III Secretion,” Clinical Microbiology Reviews, 21(1) (pp. 134–56).
2. Marteyn, B., Gazi, A., and Sansonetti, P. (2012) “Shigella: A model of virulence regulation in vivo,” Gut Microbes, 3(2) (pp. 104–20).
3. Schmutz, C., et al. (2013, July) “Systems-level overview of host protein phosphorylation during Shigella flexneri infection revealed by phosphoproteomics,” Molecular and Cellular Proteomics, [Epub ahead of print], doi: 10.1074/mcp.M113.029918.
4. Rogers, L.D., et al. (2011) “Phosphoproteomic Analysis of Salmonella-Infected Cells Identifies Key Kinase Regulators and SopB-Dependent Host Phosphorylation Events,” Science Signaling, 4(191), rs9, doi: 10.1126/scisignal.2001668.
5. Wullschleger, S., Loewith, R., and Hall, M.N. (2006) “TOR Signaling in Growth and Metabolism,” Cell, 124(3) (pp. 471–84).