Abstract
The Dot/Icm type IV secretion system (T4SS) is essential for the pathogenesis of Legionella species and translocates a multitude of effector proteins into host cells. The identification of host cell targets of these effectors is often critical to unravel their roles in controlling the host. Here we describe a method to characterize the protein complexes associated with effectors in infected host cells. To achieve this, Legionella expressing an effector of interest fused to a Bio-tag, a combination of hexahistidine tags and a specific recognition sequence for the biotin ligase BirA, are used to infect host cells expressing BirA, which leads to biotinylation of the translocated effector. Following chemical cross-linking, effector interactomes are isolated by tandem affinity purification employing metal affinity and NeutrAvidin resins and identified by western blotting or mass spectrometry.
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References
Zhu W, Banga S, Tan Y et al (2011) Comprehensive identification of protein substrates of the Dot/Icm type IV transporter of Legionella pneumophila. PLoS One 6:e17638. https://doi.org/10.1371/journal.pone.0017638
Kubori T, Hyakutake A, Nagai H (2008) Legionella translocates an E3 ubiquitin ligase that has multiple U-boxes with distinct functions. Mol Microbiol 67:1307–1319. https://doi.org/10.1111/j.1365-2958.2008.06124.x
Luo Z-Q, Isberg RR (2004) Multiple substrates of the Legionella pneumophila Dot/Icm system identified by interbacterial protein transfer. Proc Natl Acad Sci U S A 101:841–846. https://doi.org/10.1073/pnas.0304916101
Huang L, Boyd D, Amyot WM et al (2011) The E Block motif is associated with Legionella pneumophila translocated substrates. Cell Microbiol 13:227–245. https://doi.org/10.1111/j.1462-5822.2010.01531.x
Lifshitz Z, Burstein D, Peeri M et al (2013) Computational modeling and experimental validation of the Legionella and Coxiella virulence-related type-IVB secretion signal. Proc Natl Acad Sci 110:E707–E715. https://doi.org/10.1073/pnas.1215278110
Burstein D, Zusman T, Degtyar E et al (2009) Genome-scale identification of Legionella pneumophila effectors using a machine learning approach. PLoS Pathog 5. https://doi.org/10.1371/journal.ppat.1000508
De Felipe KS, Pampou S, Jovanovic OS et al (2005) Evidence for acquisition of Legionella type IV secretion substrates via interdomain horizontal gene transfer. J Bacteriol 187:7716–7726. https://doi.org/10.1128/JB.187.22.7716-7726.2005
De Felipe KS, Glover RT, Charpentier X et al (2008) Legionella eukaryotic-like type IV substrates interfere with organelle trafficking. PLoS Pathog 4:e1000117. https://doi.org/10.1371/journal.ppat.1000117
Nagai H, Kagan JC, Zhu X et al (2002) A bacterial guanine nucleotide exchange factor activates ARF on Legionella phagosomes. Science 295(80):679. https://doi.org/10.1126/science.1067025
Finsel I, Hilbi H (2015) Formation of a pathogen vacuole according to Legionella pneumophila : how to kill one bird with many stones. Cell Microbiol 17:935–950. https://doi.org/10.1111/cmi.12450
Horwitz MA (1983) Formation of a novel phagosome by the Legionnaires’ disease bacterium (Legionella pneumophila) in human monocytes. J Exp Med 158:1319–1331. https://doi.org/10.1084/jem.158.4.1319
Escoll P, Rolando M, Gomez-Valero L, Buchrieser C (2013) From amoeba to macrophages: exploring the molecular mechanisms of Legionella pneumophila infection in both hosts. Curr Top Microbiol Immunol 376:1–34. https://doi.org/10.1007/82-2013-351
So EC, Mattheis C, Tate EW et al (2015) Creating a customized intracellular niche: subversion of host cell signaling by Legionella type IV secretion system effectors. Can J Microbiol 635:617–635. https://doi.org/10.1139/cjm-2015-0166
Qiu J, Luo ZQ (2017) Legionella and Coxiella effectors: strength in diversity and activity. Nat Rev Microbiol 15:591–605. https://doi.org/10.1038/nrmicro.2017.67
Cazalet C, Rusniok C, Brüggemann H et al (2004) Evidence in the Legionella pneumophila genome for exploitation of host cell functions and high genome plasticity. Nat Genet 36:1165–1173. https://doi.org/10.1038/ng1447
Chien M, Morozova I, Shi S et al (2004) The genomic sequence of the accidental pathogen Legionella pneumophila. Science 305:1966–1968. https://doi.org/10.1126/science.1099776
Harding CR, Mattheis C, Mousnier AA et al (2013) LtpD is a novel Legionella pneumophila effector that binds phosphatidylinositol 3-phosphate and inositol monophosphatase IMPA1. Infect Immun 81:4261–4270. https://doi.org/10.1128/IAI.01054_13
Lomma M, Dervins-Ravault D, Rolando M et al (2010) The Legionella pneumophila F-box protein Lpp2082 (AnkB) modulates ubiquitination of the host protein parvin B and promotes intracellular replication. Cell Microbiol 12:1272–1291. https://doi.org/10.1111/j.1462-5822.2010.01467.x
Machner MP, Isberg RR (2006) Targeting of host Rab GTPase function by the intravacuolar pathogen Legionella pneumophila. Dev Cell 11:47–56. https://doi.org/10.1016/j.devcel.2006.05.013
Price CT, Al-Khodor S, Al-Quadan T et al (2009) Molecular mimicry by an F-box effector of Legionella pneumophila hijacks a conserved polyubiquitination machinery within macrophages and protozoa. PLoS Pathog 5. https://doi.org/10.1371/journal.ppat.1000704
Urbanus ML, Quaile AT, Stogios PJ et al (2016) Diverse mechanisms of metaeffector activity in an intracellular bacterial pathogen, Legionella pneumophila. Mol Syst Biol 12:893. https://doi.org/10.15252/msb.20167381
Mousnier A, Schroeder GN, Stoneham CA et al (2014) A new method to determine in vivo interactomes reveals binding of the Legionella pneumophila effector PieE to multiple Rab GTPases. MBio 5:e01148. https://doi.org/10.1128/mBio.01148-14
So EC, Schroeder GN, Carson D et al (2016) The Rab-binding profiles of bacterial virulence factors during infection. J Biol Chem 291:5832–5843. https://doi.org/10.1074/jbc.M115.700930
Tagwerker C (2006) A tandem affinity tag for two-step purification under fully denaturing conditions: application in ubiquitin profiling and protein complex identification combined with in vivo cross-linking. Mol Cell Proteomics 5:737–748. https://doi.org/10.1074/mcp.M500368-MCP200
Rappsilber J, Mann M, Ishihama Y (2007) Protocol for micro-purification, enrichment, pre-fractionation and storage of peptides for proteomics using StageTips. Nat Protoc 2:1896–1906. https://doi.org/10.1038/nprot.2007.261
Boersema PJ, Raijmakers R, Lemeer S et al (2009) Multiplex peptide stable isotope dimethyl labeling for quantitative proteomics. Nat Protoc 4:484–494. https://doi.org/10.1038/nprot.2009.21
Thompson A, Schäfer J, Kuhn K et al (2003) Tandem mass tags: a novel quantification strategy for comparative analysis of complex protein mixtures by MS/MS. Anal Chem 75:1895–1904. https://doi.org/10.1021/ac0262560
Acknowledgment
This research and manuscript were enabled by Wellcome Trust and Medical Research Council UK grants (MR/L018225/1) for GF, AM, ECS, GNS, as well as additional institutional funding for GNS from Queen’s University Belfast and MRF/Asthma UK Research Grant (MRFAUK-2015-311) funding for AM.
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So, E.C., Mousnier, A., Frankel, G., Schroeder, G.N. (2019). Determination of In Vivo Interactomes of Dot/Icm Type IV Secretion System Effectors by Tandem Affinity Purification. In: Buchrieser, C., Hilbi, H. (eds) Legionella. Methods in Molecular Biology, vol 1921. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-9048-1_19
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DOI: https://doi.org/10.1007/978-1-4939-9048-1_19
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