Cellular and Molecular Life Sciences

, Volume 70, Issue 24, pp 4809–4823 | Cite as

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
Research Article
First Online:
Received:
Revised:
Accepted:

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.

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 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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Notes

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.

Supplementary material

18_2013_1413_MOESM1_ESM.tif (508 kb)
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 (1 mb)
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 (7.7 mb)
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)

References

  1. 1.
    Forsberg A, Viitanen AM, Skurnik M, Wolf-Watz H (1991) The surface-located YopN protein is involved in calcium signal transduction in Yersinia pseudotuberculosis. Mol Microbiol 5:977–986PubMedCrossRefGoogle Scholar
  2. 2.
    Straley SC, Bowmer WS (1986) Virulence genes regulated at the transcriptional level by Ca2 + in Yersinia pestis include structural genes for outer membrane proteins. Infect Immun 51:445–454PubMedGoogle Scholar
  3. 3.
    Boland A, Havaux S, Cornelis GR (1998) Heterogeneity of the Yersinia YopM protein. Microb Pathog 25:343–348PubMedCrossRefGoogle Scholar
  4. 4.
    Skrzypek E, Cowan C, Straley SC (1998) Targeting of the Yersinia pestis YopM protein into HeLa cells and intracellular trafficking to the nucleus. Mol Microbiol 30:1051–1065PubMedCrossRefGoogle Scholar
  5. 5.
    Benabdillah R, Mota LJ, Lützelschwab S, Demoinet E, Cornelis GR (2004) Identification of a nuclear targeting signal in YopM from Yersinia spp. Microb Pathog 36:247–261PubMedCrossRefGoogle Scholar
  6. 6.
    Kerschen EJ, Cohen DA, Kaplan AM, Straley SC (2004) The plague virulence protein YopM targets the innate immune response by causing a global depletion of NK cells. Infect Immun 72:4589–4602PubMedCrossRefGoogle Scholar
  7. 7.
    Ye Z, Uittenbogaard AM, Cohen DA, Kaplan AM, Ambati J et al (2011) Distinct CCR2(+) Gr1(+) cells control growth of the Yersinia pestis ΔyopM mutant in liver and spleen during systemic plague. Infect Immun 79:674–687PubMedCrossRefGoogle Scholar
  8. 8.
    Rüter C, Buss C, Scharnert J, Heusipp G, Schmidt MA (2010) A newly identified bacterial cell-penetrating peptide that reduces the transcription of pro-inflammatory cytokines. J Cell Sci 123:2190–2198PubMedCrossRefGoogle Scholar
  9. 9.
    Kilk K, Langel U (2005) Cellular delivery of peptide nucleic acid by cell-penetrating peptides. Methods Mol Biol 298:131–141PubMedGoogle Scholar
  10. 10.
    Holm T, Andaloussi SE, Langel U (2011) Comparison of CPP uptake methods. Methods Mol Biol 683:207–217PubMedCrossRefGoogle Scholar
  11. 11.
    Richard JP, Melikov K, Brooks H, Prevot P, Lebleu B et al (2005) Cellular uptake of unconjugated TAT peptide involves clathrin-dependent endocytosis and heparan sulfate receptors. J Biol Chem 280:15300–15306PubMedCrossRefGoogle Scholar
  12. 12.
    Fischer R, Fotin-Mleczek M, Hufnagel H, Brock R (2005) Break on through to the other side-biophysics and cell biology shed light on cell-penetrating peptides. Chem BioChem 6:2126–2142Google Scholar
  13. 13.
    Kaplan IM, Wadia JS, Dowdy SF (2005) Cationic TAT peptide transduction domain enters cells by macropinocytosis. J Control Release 102:247–253PubMedCrossRefGoogle Scholar
  14. 14.
    Wadia JS, Stan RV, Dowdy SF (2004) Transducible TAT-HA fusogenic peptide enhances escape of TAT-fusion proteins after lipid raft macropinocytosis. Nat Med 10:310–315PubMedCrossRefGoogle Scholar
  15. 15.
    Säälik P, Padari K, Niinep A, Lorents A, Hansen M et al (2009) Protein delivery with transportans is mediated by caveolae rather than flotillin-dependent pathways. Bioconjug Chem 20:877–887PubMedCrossRefGoogle Scholar
  16. 16.
    Duchardt F, Fotin-Mleczek M, Schwarz H, Fischer R, Brock R (2007) A comprehensive model for the cellular uptake of cationic cell-penetrating peptides. Traffic 8:848–866PubMedCrossRefGoogle Scholar
  17. 17.
    Tünnemann G, Martin RM, Haupt S, Patsch C, Edenhofer F et al (2006) Cargo-dependent mode of uptake and bioavailability of TAT-containing proteins and peptides in living cells. FASEB J 20:1775–1784PubMedCrossRefGoogle Scholar
  18. 18.
    Heusipp G, Spekker K, Brast S, Fälker S, Schmidt MA (2006) YopM of Yersinia enterocolitica specifically interacts with alpha1-antitrypsin without affecting the anti-protease activity. Microbiology 152:1327–1335PubMedCrossRefGoogle Scholar
  19. 19.
    el Bayâ A, Linnermann R, von Olleschik-Elbheim L, Schmidt MA (1997) Pertussis toxin. Entry into cells and enzymatic activity. Adv Exp Med Biol 419:83–86PubMedCrossRefGoogle Scholar
  20. 20.
    Mari M, Bujny MV, Zeuschner D, Geerts WJ, Griffith J et al (2008) SNX1 defines an early endosomal recycling exit for sortilin and mannose 6-phosphate receptors. Traffic 9:380–393PubMedCrossRefGoogle Scholar
  21. 21.
    Langel Ü (2005) Handbook of cell-penetrating peptides. CRC Press, Taylor & Francis Group, Boca RatonGoogle Scholar
  22. 22.
    Gorvel JP, Chavrier P, Zerial M, Gruenberg J (1991) rab5 controls early endosome fusion in vitro. Cell 64:915–925PubMedCrossRefGoogle Scholar
  23. 23.
    Brown E, Verkade P (2010) The use of markers for correlative light electron microscopy. Protoplasma 244:91–97PubMedCrossRefGoogle Scholar
  24. 24.
    Hutagalung AH, Novick PJ (2011) Role of Rab GTPases in membrane traffic and cell physiology. Physiol Rev 91:119–149PubMedCrossRefGoogle Scholar
  25. 25.
    Rink J, Ghigo E, Kalaidzidis Y, Zerial M (2005) Rab conversion as a mechanism of progression from early to late endosomes. Cell 122:735–749PubMedCrossRefGoogle Scholar
  26. 26.
    Lombardi D, Soldati T, Riederer MA, Goda Y, Zerial M et al (1993) Rab9 functions in transport between late endosomes and the trans Golgi network. EMBO J 12:677–682PubMedGoogle Scholar
  27. 27.
    Goud B, Zahraoui A, Tavitian A, Saraste J (1990) Small GTP-binding protein associated with Golgi cisternae. Nature 345:553–556PubMedCrossRefGoogle Scholar
  28. 28.
    Trabulo S, Resina S, Simões S, Lebleu B, Pedroso de Lima MC (2010) A non-covalent strategy combining cationic lipids and CPPs to enhance the delivery of splice correcting oligonucleotides. J Control Release 145:149–158PubMedCrossRefGoogle Scholar
  29. 29.
    Ter-Avetisyan G, Tünnemann G, Nowak D, Nitschke M, Herrmann A et al (2009) Cell entry of arginine-rich peptides is independent of endocytosis. J Biol Chem 284:3370–3378PubMedCrossRefGoogle Scholar
  30. 30.
    Jiao CY, Delaroche D, Burlina F, Alves ID, Chassaing G et al (2009) Translocation and endocytosis for cell-penetrating peptide internalization. J Biol Chem 284:33957–33965PubMedCrossRefGoogle Scholar
  31. 31.
    Weigel PH, Oka JA (1981) Temperature dependence of endocytosis mediated by the asialoglycoprotein receptor in isolated rat hepatocytes. Evidence for two potentially rate-limiting steps. J Biol Chem 256:2615–2617PubMedGoogle Scholar
  32. 32.
    Letoha T, Gaál S, Somlai C, Czajlik A, Perczel A et al (2003) Membrane translocation of penetratin and its derivatives in different cell lines. J Mol Recognit 16:272–279PubMedCrossRefGoogle Scholar
  33. 33.
    Padari K, Säälik P, Hansen M, Koppel K, Raid R et al (2005) Cell transduction pathways of transportans. Bioconjug Chem 16:1399–1410PubMedCrossRefGoogle Scholar
  34. 34.
    Frankel AD, Pabo CO (1988) Cellular uptake of the tat protein from human immunodeficiency virus. Cell 55:1189–1193PubMedCrossRefGoogle Scholar
  35. 35.
    Green M, Loewenstein PM (1988) Autonomous functional domains of chemically synthesized human immunodeficiency virus tat trans-activator protein. Cell 55:1179–1188PubMedCrossRefGoogle Scholar
  36. 36.
    Medina-Kauwe LK (2007) Alternative endocytic mechanisms exploited by pathogens: new avenues for therapeutic delivery? Adv Drug Deliv Rev 59:798–809PubMedCrossRefGoogle Scholar
  37. 37.
    Derossi D, Calvet S, Trembleau A, Brunissen A, Chassaing G et al (1996) Cell internalization of the third helix of the Antennapedia homeodomain is receptor-independent. J Biol Chem 271:18188–18193PubMedCrossRefGoogle Scholar
  38. 38.
    Heitz F, Morris MC, Divita G (2009) Twenty years of cell-penetrating peptides: from molecular mechanisms to therapeutics. Br J Pharmacol 157:195–206PubMedCrossRefGoogle Scholar
  39. 39.
    Lesser CF, Miller SI (2001) Expression of microbial virulence proteins in Saccharomyces cerevisiae models mammalian infection. EMBO J 20:1840–1849PubMedCrossRefGoogle Scholar

Copyright information

© Springer Basel 2013

Authors and Affiliations

  • Julia Scharnert
    • 1
  • Lilo Greune
    • 1
  • Dagmar Zeuschner
    • 2
  • Marie-Luise Lubos
    • 1
  • M. Alexander Schmidt
    • 1
  • Christian Rüter
    • 1
  1. 1.Center for Molecular Biology of Inflammation (ZMBE), Institute of InfectiologyMünsterGermany
  2. 2.Electron Microscopy Facility, Max-Planck-Institute for Molecular BiomedicineMünsterGermany

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