Manipulation of pro-inflammatory cytokine production by the bacterial cell-penetrating effector protein YopM is independent of its interaction with host cell kinases RSK1 and PRK2

• Published in: Virulence 2014-10, Vol.5 (7), p.761-771
• Main Authors: Höfling, Sabrina, Scharnert, Julia, Cromme, Christoph, Bertrand, Jessica, Pap, Thomas, Schmidt, M Alexander, Rüter, Christian
• Format: Article
• Language: English
• Subjects: anti-inflammatory; bacterial cell-penetrating proteins; Bacterial Outer Membrane Proteins - chemistry; Bacterial Outer Membrane Proteins - genetics; Bacterial Outer Membrane Proteins - metabolism; Gene Expression Regulation; HeLa Cells; Host-Pathogen Interactions; Humans; Immunoprecipitation; pro-inflammatory cytokines; Protein Binding; Protein Interaction Domains and Motifs; Protein Kinase C - metabolism; protein kinases; Real-Time Polymerase Chain Reaction; Recombinant Proteins - metabolism; Ribosomal Protein S6 Kinases, 90-kDa - metabolism; Signal Transduction; Tumor Necrosis Factor-alpha - biosynthesis; Tumor Necrosis Factor-alpha - genetics; Yersinia enterocolitica - metabolism; Yersinia enterocolitica - pathogenicity; Yersinia enterocolitica - physiology; Yersinia outer protein; YopM
• ISSN: 2150-5594; 2150-5608
• DOI: 10.4161/viru.29062

The effector protein Yersinia outer protein M (YopM) of Yersinia enterocolitica has previously been identified and characterized as the first bacterial cell-penetrating protein (CPP). We found that recombinant YopM (rYopM) enters different eukaryotic cell types and downregulates the expression of several pro-inflammatory cytokines (e.g., tumor necrosis factor-α [TNF-α]) after autonomous translocation. After infection with Y. enterocolitica or transfection of host cells, YopM interacts with isoforms of the two kinases ribosomal S6 protein kinase (RSK) and protein kinase C-related kinase (PRK). This interaction caused sustained RSK activation due to interference with dephosphorylation.   Here we demonstrate by co-immunoprecipitation that rYopM interacts with RSK and PRK following cell-penetration. We show that autonomously translocated rYopM forms a trimeric complex with different RSK and PRK isoforms. Furthermore, we constructed a series of truncated versions of rYopM to map the domain required for the formation of the complex. The C-terminus of rYopM was identified to be essential for the interaction with RSK1, whereas any deletion in rYopM's leucin-rich repeat domains abrogated PRK2 binding. Moreover, we found that the interaction of cell-penetrating rYopM with RSK led to enhanced autophosphorylation of this kinase at serine 380. Finally, we investigated whether downstream signaling of the trimeric rYopM-RSK/PRK complex modulates the expression of pro-inflammatory TNF-α. Here, we could exclude that interaction with RSK1 and PRK2 is essential for the anti-inflammatory effects of rYopM.