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Published on: October 20, 2020
MS-Based in Situ Proteomics Reveals AMPylation of Host Proteins during Bacterial Infection
Theresa Rauh1, Sophie Brameyer2, Pavel Kielkowski3
1Department of Chemistry, Chair of Organic Chemistry II, Center for Functional Protein Assemblies (CPA), Technische Universität München, Lichtenbergstraße 4, 85748 Garching, Germany.
Abstract:
Bacteria utilize versatile strategies to propagate infections within human cells, e.g., by the injection of effector proteins, which alter crucial signaling pathways. One class of such virulence-associated proteins is involved in the AMPylation of eukaryotic Rho GTPases with devastating effects on viability. In order to get an inventory of AMPylated proteins, several technologies have been developed. However, as they were designed for the analysis of cell lysates, knowledge about AMPylation targets in living cells is largely lacking. Here, we implement a chemical-proteomic method for deciphering AMPylated host proteins in situ during bacterial infection. HeLa cells treated with a previously established cell permeable pronucleotide probe (pro-N6pA) were infected with Vibrio parahaemolyticus, and modified host proteins were identified upon probe enrichment and LC-MS/MS analysis. Three already known targets of the AMPylator VopS-Rac1, RhoA, and Cdc42-could be confirmed, and several other Rho GTPases were additionally identified. These hits were validated in comparative studies with V. parahaemolyticus wild type and a mutant producing an inactive VopS (H348A). The method further allowed to decipher the sites of modification and facilitated a time-dependent analysis of AMPylation during infection. Overall, the methodology provides a reliable detection of host AMPylation in situ and thus a versatile tool in monitoring infection processes.
Insights
This study introduces a new chemical-proteomic method to identify bacterial AMPylation targets in living human cells during infection. The technique successfully detected known and new Rho GTPase targets, offering a valuable tool for studying infection dynamics.
Area of Science:
- Microbiology
- Cell Biology
- Proteomics
Background:
- Bacteria infect human cells by injecting effector proteins that disrupt signaling pathways.
- AMPylation of Rho GTPases by bacterial virulence factors severely impacts host cell viability.
- Existing methods for identifying AMPylation targets are limited to cell lysates, lacking in situ analysis.
Purpose of the Study:
- To develop and implement a chemical-proteomic method for identifying host AMPylation targets in living cells during bacterial infection.
- To analyze AMPylation events in real-time within infected host cells.
Main Methods:
- Utilized a cell-permeable pronucleotide probe (pro-N6pA) in HeLa cells infected with Vibrio parahaemolyticus.
- Employed probe enrichment and Liquid Chromatography-Mass Spectrometry/Mass Spectrometry (LC-MS/MS) for protein identification.
- Validated findings through comparative studies with wild-type and mutant bacterial strains.
Main Results:
- Successfully identified known AMPylation targets (Rac1, RhoA, Cdc42) and several other Rho GTPases in situ.
- Confirmed the role of the VopS AMPylator in modifying these host proteins.
- Determined specific sites of modification and enabled time-dependent analysis of AMPylation during infection.
Conclusions:
- The developed chemical-proteomic method reliably detects host AMPylation in situ during bacterial infection.
- This methodology serves as a versatile tool for monitoring infection processes and understanding host-pathogen interactions.
- Provides new insights into the dynamic nature of bacterial effector protein activity within living host cells.
