Bacterial LPX motif-harboring virulence factors constitute a species-spanning family of cell-penetrating effectors
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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.
KeywordsBacterial cell-penetrating effector proteins E3 ubiquitin ligase IpaH Endocytosis Endosomal escape LPX effector protein family
We like to thank our colleagues of the Institute of Infectiology for fruitful discussions and their valuable contributions.
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.
- 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 CrossRefPubMedPubMedCentralGoogle 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) 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 CrossRefPubMedGoogle 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 CrossRefPubMedPubMedCentralGoogle 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 CrossRefPubMedPubMedCentralGoogle Scholar
- 27.Langel Ü (2005) Handbook of cell-penetrating peptides. CRC Press, Taylor & Francis Group, Boca RatonGoogle 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 CrossRefPubMedGoogle 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 CrossRefPubMedGoogle 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 CrossRefPubMedPubMedCentralGoogle 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 CrossRefPubMedPubMedCentralGoogle 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 CrossRefPubMedGoogle 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 CrossRefPubMedGoogle 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 CrossRefPubMedPubMedCentralGoogle 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 CrossRefPubMedPubMedCentralGoogle Scholar