Affinity purification, combined with mass spectrometry (AP-MS) is considered a pivotal technique in protein-protein interaction studies enabling systematic detection at near physiological conditions. The addition of a quantitative proteomic method, like SILAC metabolic labeling, allows the elimination of non-specifically bound contaminants which greatly increases the confidence of the identified interaction partners. Compared to eukaryotic cells, the SILAC labeling of bacteria has specificities that must be considered. The protocol presented here describes the labeling of bacterial cultures with stable isotope-labeled amino acids, purification of an affinity-tagged protein, and sample preparation for MS measurement. Finally, we discuss the analysis and interpretation of MS data to identify and select the specific partners interacting with the protein of interest. As an example, this workflow is applied to the discovery of potential interaction partners of glyceraldehyde-3-phosphate dehydrogenase in the gram-negative bacterium Francisella tularensis.

• Keywords
• Affinity purification, Bacteria, LC-MS/MS, Protein-protein interactions, SILAC,
• MeSH
• Bacteria metabolism   MeSH
• Chromatography, Affinity MeSH
• Mass Spectrometry methods   MeSH
• Isotope Labeling methods   MeSH
• Proteins * chemistry   MeSH
• Proteomics * methods   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Proteins * MeSH

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is known for its multifunctionality in several pathogenic bacteria. Our previously reported data suggest that the GAPDH homologue of Francisella tularensis, GapA, might also be involved in other processes beyond metabolism. In the present study, we explored GapA's potential implication in pathogenic processes at the host cell level. Using immunoelectron microscopy, we demonstrated the localization of this bacterial protein inside infected macrophages and its peripheral distribution in bacterial cells increasing with infection time. A quantitative proteomic approach based on stable isotope labeling of amino acids in cell culture (SILAC) combined with pull-down assay enabled the identification of several of GapA's potential interacting partners within the host cell proteome. Two of these partners were further confirmed by alternative methods. We also investigated the impact of gapA deletion on the transcription of selected cytokine genes and the activation of the main signaling pathways. Our results show that ∆gapA-induced transcription of genes encoding several cytokines whose expressions were not affected in cells infected with a fully virulent wild-type strain. That might be caused, at least in part, by the detected differences in ERK/MAPK signaling activation. The experimental observations together demonstrate that the F. tularensis GAPDH homologue is directly implicated in multiple host cellular processes and, thereby, that it participates in several molecular mechanisms of pathogenesis.

• Keywords
• Francisella, glyceraldehyde-3-phosphate dehydrogenase, infection, interacting partners, multitasking, pleiotropy, secretion,
• MeSH
• Cytokines metabolism   MeSH
• Gene Expression MeSH
• Francisella tularensis * genetics   metabolism   MeSH
• Glyceraldehyde-3-Phosphate Dehydrogenases genetics   metabolism   MeSH
• Proteomics MeSH
• Virulence genetics   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Cytokines MeSH
• Glyceraldehyde-3-Phosphate Dehydrogenases MeSH