Abstract
Identification of protein-protein interactions of bacterial effectors and cellular targets during infection is at the core to understand how bacteria manipulate the infected host to overcome the immune response. Potential interacting proteins might be identified by genetic methods, i.e., two hybrid screens and could be verified by co-immunoprecipitation. The tandem affinity purification (TAP) method allows an unbiased screen of potential interaction partners of bacterial effectors in a physiological approach: target cells can be infected with a bacterial strain harboring the TAP-tagged bacterial effector protein which is translocated in the host similar as under physiological infection conditions. No transfection and overexpression of the bacterial protein in the eukaryotic host are needed. Therefore, also host target cells not easy to transfect can be analyzed by this method. Moreover, the two consecutive affinity tags Calmodulin-Binding-Peptide (CBP) and Streptavidin-Binding-Peptide (SBP) fused to the translocated bacterial protein allow an outstanding clear purification of protein complexes formed between the bacterial protein of interest and host cell proteins with less occurrence of contaminants. Mass spectrometry allows an unbiased identification of interacting eukaryotic proteins.
Keywords: Yersinia, Affinity purification, Interaction, Effector protein, Type Three secretion system, TTSS
Background
The interaction of bacterial effectors with eukaryotic proteins during infection is fundamental to understand the processes initiated by the bacteria to manipulate the host cell responses in order to establish infection. The type three secretion system (TTSS) is a needle-like structure found in several gram-negative bacteria that facilities direct secretion of bacterial effector proteins into the host cell. The tandem affinity purification (TAP) of TAP-tagged bacterial effector proteins secreted directly into the host cell via the TTSS provides the invaluable possibility to identify eukaryotic proteins interacting with bacterial effector proteins in a physiological approach. No transfection and overexpression of the bacterial protein in the eukaryotic host is needed once the tandem affinity tag labeled bacterial protein is translocated in the host cell cytoplasm during infection with the manipulated bacteria. The use of two affinity tags increases the specificity of the protein-protein interaction and ensures a low level background of nonspecific interactions. This method can be adapted to a wide range of gram-positive or gram-negative bacterial species and various host target cell types. We have utilized this assay to identify new interaction partners of the Yersinia effector protein YopM. In an approach with TAP-tagged YopM being translocated into the macrophage cell line J774A.1 via the natural type three secretion system of Yersinia we verified the interaction of YopM with host kinases PKN2 and RSK1. Moreover, we detected association with additional PKN and RSK isoforms and identified a new interaction partner of YopM, the DEAD box Helicase DDX3. This assay can be adapted to other bacterial species and host cell types.
Materials and Reagents
Equipment
Procedure
Data analysis
Data analysis of the obtained peptide mass fingerprints of MALDI-TOF analysis can be performed with the Mascot search algorithm version 2.2 (Matrix Sciences, London, UK).
Notes
Recipes
Acknowledgments
This protocol is an extended version of the one described in the original papers (Hentschke et al., 2010; Berneking et al., 2016). Details of the cloning procedure and identification of interacting proteins of the Yersinia effector protein YopM can be found in the original papers. We thank the UKE Core Facility Mass Spectrometry and Fritz Buck for help with experimental setup and data analysis. This work was supported by grants from the Deutsche Forschungsgemeinschaft DFG (HE 5875/1-1) to MH and MS. LB was supported by the Deutsche Forschungsgemeinschaft (GRK-1459).
Competing interests
The authors declare that there is no conflict of interest.
References
If you have any questions/comments about this protocol, you are highly recommended to post here. We will invite the authors of this protocol as well as some of its users to address your questions/comments. To make it easier for them to help you, you are encouraged to post your data including images for the troubleshooting.