Autonomous translocation and intracellular trafficking of the cell-penetrating and immune-suppressive effector protein Y
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Cellular and Molecular Life Sciences
Research Article
Autonomous translocation and intracellular trafficking of the cell‑penetrating and immune‑suppressive effector protein YopM Julia Scharnert · Lilo Greune · Dagmar Zeuschner · Marie‑Luise Lubos · M. Alexander Schmidt · Christian Rüter
Received: 27 February 2013 / Revised: 10 June 2013 / Accepted: 21 June 2013 © Springer Basel 2013
Abstract Extracellular Gram-negative pathogenic bacteria target essential cytoplasmic processes of eukaryotic cells by using effector protein delivery systems such as the type III secretion system (T3SS). These secretion systems directly inject effector proteins into the host cell cytoplasm. Among the T3SS-dependent Yop proteins of pathogenic Yersinia, the function of the effector protein YopM remains enigmatic. In a recent study, we demonstrated that recombinant YopM from Yersinia enterocolitica enters host cells autonomously without the presence of bacteria and thus identified YopM as a novel bacterial cell-penetrating protein. Following entry YopM down-regulates expression of pro-inflammatory cytokines such as tumor necrosis factor α. These properties earmark YopM for further development as a novel anti-inflammatory therapeutic. To elucidate the uptake and intracellular targeting mechanisms of this bacterial cell-penetrating protein, we analyzed possible routes of internalization employing ultra-cryo electron microscopy. Our results reveal that under physiological conditions, YopM enters cells predominantly by exploiting endocytic pathways. Interestingly, YopM was detected M. A. Schmidt and C. Rüter contributed equally to this work. Electronic supplementary material The online version of this article (doi:10.1007/s00018-013-1413-2) contains supplementary material, which is available to authorized users. J. Scharnert · L. Greune · M.-L. Lubos · M. Alexander Schmidt · C. Rüter (*) Center for Molecular Biology of Inflammation (ZMBE), Institute of Infectiology, Von‑Esmarch‑Str. 56, 48149 Münster, Germany e-mail: rueterc@uni‑muenster.de D. Zeuschner Electron Microscopy Facility, Max-Planck-Institute for Molecular Biomedicine, Röntgenstr. 20, 48149 Münster, Germany
free in the cytosol and inside the nucleus. We could not observe any colocalization of YopM with secretory membranes, which excludes retrograde transport as the mechanism for cytosolic release. However, our findings indicate that direct membrane penetration and/or an endosomal escape of YopM contribute to the cytosolic and nuclear localization of the protein. Surprisingly, even when endocytosis is blocked, YopM was found to be associated with endosomes. This suggests an intracellular endosome-associated transport of YopM. Keywords Yersinia outer protein M · Effector protein · Cell-penetrating protein · Electron microscopy · Intracellular trafficking Abbreviations CCV Clathrin-coated vesicles CF Cytosolic fraction CPP Cell-penetrating protein CYT Cytosol E Endosomes EE Early endosome ER Endoplasmic reticulum GA Golgi apparatus HM Heavy membranes LE Late endosom
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