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Autonomous translocation and intracellular trafficking of the cell-penetrating and immune-suppressive effector protein YopM

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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 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.

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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 endosome

LYS:

Lysosome

MITO:

Mitochondria

MVB:

Multi-vesicular-bodies

nCCV:

Non-clathrin-coated vesicles

N:

Nucleus

NE:

Nuclear envelope

NF:

Nuclear fraction

PM:

Plasma membrane

PNS:

Post-nuclear supernatant

PTD:

Protein transduction domain

SE:

Sorting endosome

TC:

Transport carrier

TGN:

Trans-Golgi network

YopM:

Yersinia outer protein M

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Acknowledgments

This study was supported in part by the Graduate Program “Cell Dynamics and Disease (CEDAD)” of the Westfälische Wilhelms-Universität Münster (9817300 to J. S.) and by the Deutsche Forschungsgemeinschaft (Graduiertenkolleg GRK 1409) (209657 to M. A. S. and M.-L. L.) and by a grant of the Innovative Medizinische Forschung (IMF) (I-RÜ11106 to C. R.). This study is part of the PhD thesis of J. S. and M.-L. L.

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Authors and Affiliations

  1. Center for Molecular Biology of Inflammation (ZMBE), Institute of Infectiology, Von-Esmarch-Str. 56, 48149, Münster, Germany

    Julia Scharnert, Lilo Greune, Marie-Luise Lubos, M. Alexander Schmidt & Christian Rüter

  2. Electron Microscopy Facility, Max-Planck-Institute for Molecular Biomedicine, Röntgenstr. 20, 48149, Münster, Germany

    Dagmar Zeuschner

Authors
  1. Julia Scharnert
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  2. Lilo Greune
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  3. Dagmar Zeuschner
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  4. Marie-Luise Lubos
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  5. M. Alexander Schmidt
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  6. Christian Rüter
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Corresponding author

Correspondence to Christian Rüter.

Additional information

M. A. Schmidt and C. Rüter contributed equally to this work.

Electronic supplementary material

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18_2013_1413_MOESM1_ESM.tif

Fig. S1 Non-penetrative YopM87-C lacking the PTD of YopM is not taken up at 4°C. Immunoblot of HeLa membrane fractionation after incubation with YopM87-C at 4°C and 37°C. Membranes were separated using a discontinuous sucrose flotation gradient. The purity of the fractions was assessed by using marker proteins for different cellular compartments. At 37°C YopM87-C localized in the endosomal and heavy membrane fraction, whereas at 4°C no YopM87-C was detectable in these fraction. E, endosomes; HM, heavy membranes; PNS, post nuclear supernatant; PDI, protein disulfide isomerase; TfR, transferrin receptor (TIFF 507 kb)

18_2013_1413_MOESM2_ESM.tif

Fig. S2 Removal of cell surface proteins by proteinase K does not abolish membrane translocation of YopM. HeLa cells were treated with 500 μg/ml proteinase K for 15 min prior to YopM (25 μg/ml) incubation. a, b FACS analysis of HeLa cells incubated with YopM-Cy3 for a total of 1 h (a). Successful removal of cell surface proteins (e.g. TfR) was tested by measuring internalization of Alexa 488-labeled transferrin (b). Samples were taken at indicated time points and trypan blue (final concentration 0.2 %) was added directly before measurement to quench extracellular fluorescence. The number of analyzed cells is plotted against the mean fluorescence intensity measured for each cell. Furthermore, overlay of histogram plots from cells incubated for 15 min with YopM-Cy3 or TF-Alexa 488 after removal of surface proteins by proteinase K are depicted. c Immunoblot of subcellular fractionations of HeLa cells incubated with YopM at 37°C for 1 h. In order to assess their purity, fractions were analyzed using antibodies against α-Tubulin and LSD1. YopM partition was assessed using a YopM-specific polyclonal antibody. CF, cytosolic fraction; LSD1, Lysine-specific demethylase 1; NF, nuclear fraction; PK, proteinase K; TfR, transferrin receptor (TIFF 1043 kb)

18_2013_1413_MOESM3_ESM.tif

Fig. S3 Low temperature does not effect plasma membrane integrity of HeLa cells. To analyze membrane integrity of HeLa cells at 4°C cells were incubated with propidium iodide (1 μg/ml) on ice for a total of 1 h with or without YopM-Alexa 488 (25 μg/ml). The number of analyzed cells is plotted against the mean fluorescence intensity of propium iodide measured for each cell (TIFF 7895 kb)

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Scharnert, J., Greune, L., Zeuschner, D. et al. Autonomous translocation and intracellular trafficking of the cell-penetrating and immune-suppressive effector protein YopM. Cell. Mol. Life Sci. 70, 4809–4823 (2013). https://doi.org/10.1007/s00018-013-1413-2

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