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Bacterial LPX motif-harboring virulence factors constitute a species-spanning family of cell-penetrating effectors

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Abstract

Effector proteins are key virulence factors of pathogenic bacteria that target and subvert the functions of essential host defense mechanisms. Typically, these proteins are delivered into infected host cells via the type III secretion system (T3SS). Recently, however, several effector proteins have been found to enter host cells in a T3SS-independent manner thereby widening the potential range of these virulence factors. Prototypes of such bacteria-derived cell-penetrating effectors (CPEs) are the Yersinia enterocolitica-derived YopM as well as the Salmonella typhimurium effector SspH1. Here, we investigated specifically the group of bacterial LPX effector proteins comprising the Shigella IpaH proteins, which constitute a subtype of the leucine-rich repeat protein family and share significant homologies in sequence and structure. With particular emphasis on the Shigella-effector IpaH9.8, uptake into eukaryotic cell lines was shown. Recombinant IpaH9.8 (rIpaH9.8) is internalized via endocytic mechanisms and follows the endo-lysosomal pathway before escaping into the cytosol. The N-terminal alpha-helical domain of IpaH9.8 was identified as the protein transduction domain required for its CPE ability as well as for being able to deliver other proteinaceous cargo. rIpaH9.8 is functional as an ubiquitin E3 ligase and targets NEMO for poly-ubiquitination upon cell penetration. Strikingly, we could also detect other recombinant LPX effector proteins from Shigella and Salmonella intracellularly when applied to eukaryotic cells. In this study, we provide further evidence for the general concept of T3SS-independent translocation by identifying novel cell-penetrating features of these LPX effectors revealing an abundant species-spanning family of CPE.

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References

  1. Abby SS, Rocha EP (2012) The non-flagellar type III secretion system evolved from the bacterial flagellum and diversified into host-cell adapted systems. PLoS Genet 8:e1002983. https://doi.org/10.1371/journal.pgen.1002983

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Albani JR (2009) Fluorescence origin of 6, P-toluidinyl-naphthalene-2-sulfonate (TNS) bound to proteins. J Fluoresc 19:399–408. https://doi.org/10.1007/s10895-008-0426-y

    Article  CAS  PubMed  Google Scholar 

  3. Ashida H, Kim M, Sasakawa C (2014) Exploitation of the host ubiquitin system by human bacterial pathogens. Nat Rev Microbiol 12:399–413. https://doi.org/10.1038/nrmicro3259

    Article  CAS  PubMed  Google Scholar 

  4. Ashida H, Kim M, Schmidt-Supprian M, Ma A, Ogawa M, Sasakawa C (2010) A bacterial E3 ubiquitin ligase IpaH9.8 targets NEMO/IKKgamma to dampen the host NF-kappaB-mediated inflammatory response. Nat Cell Biol 12(66–73):1–9. https://doi.org/10.1038/ncb2006

    Google Scholar 

  5. Ashida H, Nakano H, Sasakawa C (2013) Shigella IpaH0722 E3 ubiquitin ligase effector targets TRAF2 to inhibit PKC-NF-kappaB activity in invaded epithelial cells. PLoS Pathog 9:e1003409. https://doi.org/10.1371/journal.ppat.1003409

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Bahrani FK, Sansonetti PJ, Parsot C (1997) Secretion of Ipa proteins by Shigella flexneri: inducer molecules and kinetics of activation. Infect Immun 65:4005–4010

    CAS  PubMed  PubMed Central  Google Scholar 

  7. Bernal-Bayard J, Cardenal-Munoz E, Ramos-Morales F (2010) The Salmonella type III secretion effector, Salmonella leucine-rich repeat protein (SlrP), targets the human chaperone ERdj3. J Biol Chem 285:16360–16368. https://doi.org/10.1074/jbc.m110.100669

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Bhavsar AP, Brown NF, Stoepel J, Wiermer M, Martin DD, Hsu KJ, Imami K, Ross CJ, Hayden MR, Foster LJ, Li X, Hieter P, Finlay BB (2013) The Salmonella type III effector SspH2 specifically exploits the NLR co-chaperone activity of SGT1 to subvert immunity. PLoS Pathog 9:e1003518. https://doi.org/10.1371/journal.ppat.1003518

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Bonifacino JS, Rojas R (2006) Retrograde transport from endosomes to the trans-Golgi network. Nat Rev Mol Cell Biol 7:568–579

    Article  CAS  PubMed  Google Scholar 

  10. Chen GJ, Qiu N, Karrer C, Caspers P, Page MG (2000) Restriction site-free insertion of PCR products directionally into vectors. BioTechniq 28:498–500 (504–505)

    CAS  Google Scholar 

  11. Cirl C, Wieser A, Yadav M, Duerr S, Schubert S, Fischer H, Stappert D, Wantia N, Rodriguez N, Wagner H, Svanborg C, Miethke T (2008) Subversion of Toll-like receptor signaling by a unique family of bacterial Toll/interleukin-1 receptor domain-containing proteins. Nat Med 14:399–406. https://doi.org/10.1038/nm1734

    Article  CAS  PubMed  Google Scholar 

  12. Coburn B, Sekirov I, Finlay BB (2007) Type III secretion systems and disease. Clin Microbiol Rev 20:535–549

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. de Jong MF, Liu Z, Chen D, Alto NM (2016) Shigella flexneri suppresses NF-kappaB activation by inhibiting linear ubiquitin chain ligation. Nat Microbiol 1:16084. https://doi.org/10.1038/nmicrobiol.2016.84

    Article  PubMed  PubMed Central  Google Scholar 

  14. Edgar RC (2004) MUSCLE: a multiple sequence alignment method with reduced time and space complexity. BMC Bioinf 5:113. https://doi.org/10.1186/1471-2105-5-113

    Article  Google Scholar 

  15. Evdokimov AG, Anderson DE, Routzahn KM, Waugh DS (2001) Unusual molecular architecture of the Yersinia pestis cytotoxin YopM: a leucine-rich repeat protein with the shortest repeating unit. J Mol Biol 312:807–821. https://doi.org/10.1006/jmbi.2001.4973

    Article  CAS  PubMed  Google Scholar 

  16. 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. Chembiochem 6:2126–2142. https://doi.org/10.1002/cbic.200500044

    Article  CAS  PubMed  Google Scholar 

  17. Fischer R, Kohler K, Fotin-Mleczek M, Brock R (2004) A stepwise dissection of the intracellular fate of cationic cell-penetrating peptides. J Biol Chem 279:12625–12635. https://doi.org/10.1074/jbc.m311461200

    Article  CAS  PubMed  Google Scholar 

  18. Gofman Y, Haliloglu T, Ben-Tal N (2012) Monte Carlo simulations of peptide-membrane interactions with the MCPep web server. Nucleic Acids Res 40:W358–W363. https://doi.org/10.1093/nar/gks577

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Gomarasca M, Martins TFC, Greune L, Hardwidge PR, Schmidt MA, Rüter C (2017) Bacterium-derived cell-penetrating peptides deliver gentamicin to kill intracellular pathogens. Antimicrob Agents Chemother. https://doi.org/10.1128/aac.02545-16

    PubMed  PubMed Central  Google Scholar 

  20. Hentschke M, Berneking L, Belmar Campos C, Buck F, Ruckdeschel K, Aepfelbacher M (2010) Yersinia virulence factor YopM induces sustained RSK activation by interfering with dephosphorylation. PLoS One. https://doi.org/10.1371/journal.pone.0013165

    PubMed  PubMed Central  Google Scholar 

  21. Hjerpe R, Aillet F, Lopitz-Otsoa F, Lang V, England P, Rodriguez MS (2009) Efficient protection and isolation of ubiquitylated proteins using tandem ubiquitin-binding entities. EMBO Rep 10:1250–1258. https://doi.org/10.1038/embor.2009.192

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Höfling S, Scharnert J, Cromme C, Bertrand J, Pap T, Schmidt MA, Rüter C (2014) 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. Virulence 5:761–771. https://doi.org/10.4161/viru.29062

    Article  PubMed  PubMed Central  Google Scholar 

  23. Humphries WH 4th, Payne CK (2012) Imaging lysosomal enzyme activity in live cells using self-quenched substrates. Anal Biochem 424:178–183. https://doi.org/10.1016/j.ab.2012.02.033

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Kelley LA, Mezulis S, Yates CM, Wass MN, Sternberg MJ (2015) The Phyre2 web portal for protein modeling, prediction and analysis. Nat Protoc 10:845–858. https://doi.org/10.1038/nprot.2015.053

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Keszei AF, Tang X, McCormick C, Zeqiraj E, Rohde JR, Tyers M, Sicheri F (2014) Structure of an SspH1-PKN1 complex reveals the basis for host substrate recognition and mechanism of activation for a bacterial E3 ubiquitin ligase. Mol Cell Biol 34:362–373. https://doi.org/10.1128/mcb.01360-13

    Article  PubMed  PubMed Central  Google Scholar 

  26. Kobe B, Kajava AV (2001) The leucine-rich repeat as a protein recognition motif. Curr Opin Struct Biol 11:725–732

    Article  CAS  PubMed  Google Scholar 

  27. Langel Ü (2005) Handbook of cell-penetrating peptides. CRC Press, Taylor & Francis Group, Boca Raton

    Google Scholar 

  28. Lee SH, Galán JE (2004) Salmonella type III secretion-associated chaperones confer secretion-pathway specificity. Mol Microbiol 51:483–495. https://doi.org/10.1046/j.1365-2958.2003.03840.x

    Article  CAS  PubMed  Google Scholar 

  29. Liu X, Lu L, Liu X, Liu X, Pan C, Feng E, Wang D, Niu C, Zhu L, Wang H (2016) Proteomic analysis of Shigella virulence effectors secreted under different conditions. J Microbiol Biotechnol. https://doi.org/10.4014/jmb.1603.03015

    Google Scholar 

  30. Lubos M, Norkowski S, Stolle A, Langel Ü, Schmidt MA, Rüter C (2014) Analysis of T3SS-independent autonomous internalization of the bacterial effector protein SspH1 from Salmonella typhimurium. Inflamm Cell Signal 1:1–10. https://doi.org/10.14800/ics.423

    Google Scholar 

  31. Mattoo S, Lee YM, Dixon JE (2007) Interactions of bacterial effector proteins with host proteins. Curr Opin Immunol 19:392–401

    Article  CAS  PubMed  Google Scholar 

  32. McGuffin LJ, Bryson K, Jones DT (2000) The PSIPRED protein structure prediction server. Bioinformatics 16:404–405

    Article  CAS  PubMed  Google Scholar 

  33. Miao EA, Scherer CA, Tsolis RM, Kingsley RA, Adams LG, Baumler AJ, Miller SI (1999) Salmonella typhimurium leucine-rich repeat proteins are targeted to the SPI1 and SPI2 type III secretion systems. Mol Microbiol 34:850–864. https://doi.org/10.1046/j.1365-2958.1999.01651.x

    Article  CAS  PubMed  Google Scholar 

  34. Michgehl S, Heusipp G, Greune L, Rüter C, Schmidt MA (2006) Esp-independent functional integration of the translocated intimin receptor (Tir) of enteropathogenic Escherichia coli (EPEC) into host cell membranes. Cell Microbiol 8:625–633

    Article  CAS  PubMed  Google Scholar 

  35. Mitchell DJ, Kim DT, Steinman L, Fathman CG, Rothbard JB (2000) Polyarginine enters cells more efficiently than other polycationic homopolymers. J Pept Res 56:318–325

    Article  CAS  PubMed  Google Scholar 

  36. Nemeth J, Straley SC (1997) Effect of Yersinia pestis YopM on experimental plague. Infect Immun 65:924–930

    CAS  PubMed  PubMed Central  Google Scholar 

  37. Okuda J, Toyotome T, Kataoka N, Ohno M, Abe H, Shimura Y, Seyedarabi A, Pickersgill R, Sasakawa C (2005) Shigella effector IpaH9.8 binds to a splicing factor U2AF(35) to modulate host immune responses. Biochem Biophys Res Commun 333:531–539

    Article  CAS  PubMed  Google Scholar 

  38. Popovic D, Vucic D, Dikic I (2014) Ubiquitination in disease pathogenesis and treatment. Nat Med 20:1242–1253. https://doi.org/10.1038/nm.3739

    Article  CAS  PubMed  Google Scholar 

  39. Qian Z, Dougherty PG, Pei D (2015) Monitoring the cytosolic entry of cell-penetrating peptides using a pH-sensitive fluorophore. Chem Commun (Camb) 51:2162–2165. https://doi.org/10.1039/c4cc09441g

    Article  CAS  Google Scholar 

  40. Quezada CM, Hicks SW, Galán JE, Stebbins CE (2009) A family of Salmonella virulence factors functions as a distinct class of autoregulated E3 ubiquitin ligases. Proc Natl Acad Sci USA 106:4864–4869. https://doi.org/10.1073/pnas.0811058106

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Rohde JR, Breitkreutz A, Chenal A, Sansonetti PJ, Parsot C (2007) Type III secretion effectors of the IpaH family are E3 ubiquitin ligases. Cell Host Microbe 1:77–83

    Article  CAS  PubMed  Google Scholar 

  42. 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 123:2190–2198. https://doi.org/10.1242/jcs.063016

    Article  PubMed  Google Scholar 

  43. Rüter C, Hardwidge PR (2014) ‘Drugs from bugs’: bacterial effector proteins as promising biological (immune-) therapeutics. FEMS Microbiol Lett 351:126–132. https://doi.org/10.1111/1574-6968.12333

    Article  PubMed  Google Scholar 

  44. Rüter C, Schmidt MA (2017) Cell-penetrating bacterial effector proteins: better tools than targets. Trends Biotechnol 35:109–120

    Article  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  46. Scheibner F, Marillonnet S, Büttner D (2017) The TAL effector AvrBs3 from Xanthomonas campestris pv. vesicatoria contains multiple export signals and can enter plant cells in the absence of the type III secretion translocon. Front Microbiol 8:2180

    Article  PubMed  PubMed Central  Google Scholar 

  47. Seyedarabi A, Sullivan JA, Sasakawa C, Pickersgill RW (2010) A disulfide driven domain swap switches off the activity of Shigella IpaH9.8 E3 ligase. FEBS Lett 584:4163–4168. https://doi.org/10.1016/j.febslet.2010.09.006

    Article  CAS  PubMed  Google Scholar 

  48. Stewart KM, Horton KL, Kelley SO (2008) Cell-penetrating peptides as delivery vehicles for biology and medicine. Org Biomol Chem 6:2242–2255. https://doi.org/10.1039/b719950c

    Article  CAS  PubMed  Google Scholar 

  49. Stolle AS, Norkowski S, Körner B, Schmitz J, Lüken L, Frankenberg M, Rüter C, Schmidt MA (2017) T3SS-independent uptake of the short-trip toxin-related recombinant NleC effector of enteropathogenic Escherichia coli leads to NF-kappaB p65 cleavage. Front Cell Infect Microbiol 7:119. https://doi.org/10.3389/fcimb.2017.00119

    Article  PubMed  PubMed Central  Google Scholar 

  50. Suzuki S, Mimuro H, Kim M, Ogawa M, Ashida H, Toyotome T, Franchi L, Suzuki M, Sanada T, Suzuki T, Tsutsui H, Nunez G, Sasakawa C (2014) Shigella IpaH7.8 E3 ubiquitin ligase targets glomulin and activates inflammasomes to demolish macrophages. Proc Natl Acad Sci USA 111:E4254–E4263. https://doi.org/10.1073/pnas.1324021111

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Thorén PE, Persson D, Isakson P, Goksör M, Önfelt A, Nordén B (2003) Uptake of analogs of penetratin, Tat(48-60) and oligoarginine in live cells. Biochem Biophys Res Commun 307:100–107

    Article  PubMed  Google Scholar 

  52. Toyotome T, Suzuki T, Kuwae A, Nonaka T, Fukuda H, Imajoh-Ohmi S, Toyofuku T, Hori M, Sasakawa C (2001) Shigella protein IpaH(9.8) is secreted from bacteria within mammalian cells and transported to the nucleus. J Biol Chem 276:32071–32079. https://doi.org/10.1074/jbc.m101882200

    Article  CAS  PubMed  Google Scholar 

  53. van den Ent F, Lowe J (2006) RF cloning: a restriction-free method for inserting target genes into plasmids. J Biochem Biophys Methods 67:67–74

    Article  PubMed  Google Scholar 

  54. Varkouhi AK, Scholte M, Storm G, Haisma HJ (2011) Endosomal escape pathways for delivery of biologicals. J Control Release 151:220–228. https://doi.org/10.1016/j.jconrel.2010.11.004

    Article  CAS  PubMed  Google Scholar 

  55. Wang F, Jiang Z, Li Y, He X, Zhao J, Yang X, Zhu L, Yin Z, Li X, Wang X, Liu W, Shang W, Yang Z, Wang S, Zhen Q, Zhang Z, Yu Y, Zhong H, Ye Q, Huang L, Yuan J (2013) Shigella flexneri T3SS effector IpaH4.5 modulates the host inflammatory response via interaction with NF-kappaB p65 protein. Cell Microbiol 15:474–485. https://doi.org/10.1111/cmi.12052

    Article  CAS  PubMed  Google Scholar 

  56. Waterhouse AM, Procter JB, Martin DM, Clamp M, Barton GJ (2009) Jalview Version 2—a multiple sequence alignment editor and analysis workbench. Bioinformatics 25:1189–1191. https://doi.org/10.1093/bioinformatics/btp033

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Wei C, Wang Y, Du Z, Guan K, Cao Y, Yang H, Zhou P, Wu F, Chen J, Wang P, Zheng Z, Zhang P, Zhang Y, Ma S, Yang R, Zhong H, He X (2016) The Yersinia type III secretion effector YopM is an E3 ubiquitin ligase that induced necrotic cell death by targeting NLRP3. Cell Death Dis 7:e2519. https://doi.org/10.1038/cddis.2016.413

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Yadav M, Zhang J, Fischer H, Huang W, Lutay N, Cirl C, Lum J, Miethke T, Svanborg C (2010) Inhibition of TIR domain signaling by TcpC: MyD88-dependent and independent effects on Escherichia coli virulence. PLoS Pathog 6:e1001120. https://doi.org/10.1371/journal.ppat.1001120

    Article  PubMed  PubMed Central  Google Scholar 

  59. Young BM, Young GM (2002) YplA is exported by the Ysc, Ysa, and flagellar type III secretion systems of Yersinia enterocolitica. J Bacteriol 184:1324–1334

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. Zheng Z, Wei C, Guan K, Yuan Y, Zhang Y, Ma S, Cao Y, Wang F, Zhong H, He X (2016) Bacterial E3 ubiquitin ligase IpaH4.5 of Shigella flexneri targets TBK1 to dampen the host antibacterial response. J Immunol 196:1199–1208. https://doi.org/10.4049/jimmunol.1501045

    Article  CAS  PubMed  Google Scholar 

  61. Zhu Y, Li H, Hu L, Wang J, Zhou Y, Pang Z, Liu L, Shao F (2008) Structure of a Shigella effector reveals a new class of ubiquitin ligases. Nat Struct Mol Biol 15:1302–1308

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

We like to thank our colleagues of the Institute of Infectiology for fruitful discussions and their valuable contributions.

Funding

This study was supported by Grants from the Deutsche Forschungsgemeinschaft (DFG, RU1884/2-1 to CR, SFB1009 TP B03 to CR and MAS), by the DFG Graduiertenkolleg GRK1409, the Cells-in-Motion Cluster of Excellence (EXC 1003-CiM), and by a Grant from the Interdisciplinary Centre for Clinical Research (IZKF, Rüt2/002/16 to CR) of the Medical Faculty of the University of Münster. The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

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

    Stefanie Norkowski, Britta Körner, Lilo Greune, Anne-Sophie Stolle, Marie-Luise Lubos, M. Alexander Schmidt & Christian Rüter

  2. College of Veterinary Medicine, Kansas State University, 1710 Denison Ave, 101 Trotter Hall, Manhattan, KS, 66506-5600, USA

    Philip R. Hardwidge

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  1. Stefanie Norkowski
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Norkowski, S., Körner, B., Greune, L. et al. Bacterial LPX motif-harboring virulence factors constitute a species-spanning family of cell-penetrating effectors. Cell. Mol. Life Sci. 75, 2273–2289 (2018). https://doi.org/10.1007/s00018-017-2733-4

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