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Abstract
BackgroundPathogenic yersiniae inject several effector proteins (Yops) into host cells, which subverts immune functions and enables the bacteria to survive within the host organism. YopM, whose deletion in enteropathogenic yersiniae results in a dramatic loss of virulence, has previously been shown to form a complex with and activate the multifunctional kinases PKN2 and RSK1 in transfected cells.Methodology/Principal FindingsIn a near physiological approach with double-affinity-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 PKN2 and RSK1 and detected association with additional PKN and RSK isoforms. In transfected and infected cells YopM induced sustained phosphorylation of RSK at its activation sites serine-380 and serine-221 even in the absence of signalling from its upstream kinase ERK1/2, suggesting inhibition of dephosphorylation. ATP-depletion and in vitro assays using purified components directly confirmed that YopM shields RSK isoforms from phosphatase activity towards serines 380 and 221.Conclusions/SignificanceOur study suggests that during Yersinia infection YopM induces sustained activation of RSK by blocking dephosphorylation of its activatory phosphorylation sites. This may represent a novel mode of action of a bacterial virulence factor.
Highlights
Yersiniae are gram-negative bacteria which belong to the family of the Enterobacteriaceae
Anti-flag was from Sigma and used in the dilution 1:3000, anti-myc clone 9E10 was from Cell Signaling and used in the dilution 1:1000, anti-HA high affinity was from Roche and used in the dilution 1:1000, anti-phospho-S380RSK was from Cell signaling and used in the dilution 1:1000, antiphospho-S221RSK was from R&D systems and used in the dilution 1:2000, anti-RSK1 was from Santa Cruz Bitotechnology and used in the dilution 1:1000, anti-ERK1/2 was from Cell Signaling and used in the dilution 1:1000, anti phospho-ERK1/2 was from Cell Signaling and used in the dilution 1:1000, antiYopM was a gift from Jurgen Heesemann and used in the dilution 1:3000
In order to verify these interaction partners under lifelike infection conditions and to potentially discover novel ones, we translocated affinity tagged YopM into the macrophage cell line J774A.1 via the type III-secretion system (TTSS) of Yersinia. For this purpose the coding region of the Yersinia enterocolitica Serotype O:8 strain WA-314 YopM gene was fused N-terminally to a Tandemaffinity-Purification tag (TAP-tag), which consists of two separate affinity tags, one calmodulin binding peptide (CBP) and one streptavidin binding peptide (SBP), which can be eluted from their affinity matrices with EGTA and Biotin, respectively
Summary
Yersiniae are gram-negative bacteria which belong to the family of the Enterobacteriaceae. Like many other gram negative bacteria yersiniae utilize a so called type III-secretion system (TTSS) to inject proteins into host cells [1]. The injected virulence proteins (‘‘Yersinia outer proteins’’, Yops) subvert the immune cells, which permits extracellular persistence and proliferation of Yersinia. Six different Yops, YopE, YopT, YopO/YpkA, YopH, YopP/YopJ and YopM, are translocated and attack diverse aspects of cellular immunity which disturbs immune cell function at different levels [4,5]. YopE, YopT and YopO/YopkA modulate small GTP-binding proteins of the Rho-family, which take part in the regulation of the actin cytoskeleton. Pathogenic yersiniae inject several effector proteins (Yops) into host cells, which subverts immune functions and enables the bacteria to survive within the host organism. YopM, whose deletion in enteropathogenic yersiniae results in a dramatic loss of virulence, has previously been shown to form a complex with and activate the multifunctional kinases PKN2 and RSK1 in transfected cells
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