Abstract
Bacterial secretion systems allow the transport of proteins, called effectors, as well as external machine components in the extracellular medium or directly into target cells. Comparison of the secretome, i.e., the proteins released in the culture medium, of wild-type and mutant cells provides information on the secretion profile. In addition, mass spectrometry analyses of the culture supernatant of bacteria grown in liquid culture under secreting conditions allow the identification of secretion systems substrates. Upon identification of the substrates, the secretion profile serves as a tool to test the functionality of secretion systems. Here, we present a classical method used to concentrate the culture supernatant, based on TCA precipitation.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Costa TR, Felisberto-Rodrigues C, Meir A et al (2015) Secretion systems in Gram-negative bacteria: structural and mechanistic insights. Nat Rev Microbiol 13:343–359
Cornelis GR, Biot T, Lambert de Rouvroit C et al (1989) The Yersinia yop regulon. Mol Microbiol 3:1455–1459
Beuzon CR, Banks G, Deiwick J, Hensel M, Holden DW (1999) pH-dependent secretion of SseB, a product of the SPI-2 type III secretion system of Salmonella typhimurium. Mol Microbiol 33:806–816
Coulthurst SJ, Lilley KS, Hedley PE, Liu H, Toth IK, Salmond GP (2008) DsbA plays a critical and multifaceted role in the production of secreted virulence factors by the phytopathogen Erwinia carotovora subsp. atroseptica. J Biol Chem 283:23739–23753
Kazemi-Pour N, Condemine G, Hugouvieux-Cotte-Pattat N (2004) The secretome of the plant pathogenic bacterium Erwinia chrysanthemi. Proteomics 4:3177–3186
Sikora AE, Zielke RA, Lawrence DA, Andrews PC, Sandkvist M (2011) Proteomic analysis of the Vibrio cholerae type II secretome reveals new proteins, including three related serine proteases. J Biol Chem 286:16555–16566
Burtnick MN, Brett PJ, DeShazer D (2014) Proteomic analysis of the Burkholderia pseudomallei type II secretome reveals hydrolytic enzymes, novel proteins, and the deubiquitinase TssM. Infect Immun 82:3214–3226
Hood RD, Singh P, Hsu F, Güvener T et al (2010) A type VI secretion system of Pseudomonas aeruginosa targets a toxin to bacteria. Cell Host Microbe 7:25–37
Russell AB, Singh P, Brittnacher M et al (2012) A widespread bacterial type VI secretion effector superfamily identified using a heuristic approach. Cell Host Microbe 11:538–549
Fritsch MJ, Trunk K, Diniz JA, Guo M, Trost M, Coulthurst SJ (2013) Proteomic identification of novel secreted antibacterial toxins of the Serratia marcescens type VI secretion system. Mol Cell Proteomics 12:2735–2749
Deng W, de Hoog CL, Yu HB et al (2010) A comprehensive proteomic analysis of the type III secretome of Citrobacter rodentium. J Biol Chem 285:6790–6800
Veith PD, Chen YY, Gorasia DG et al (2014) Porphyromonas gingivalis outer membrane vesicles exclusively contain outer membrane and periplasmic proteins and carry a cargo enriched with virulence factors. J Proteome Res 13:2420–2432
Ulhuq FR, Gomes MC, Duggan GM et al (2020) A membrane-depolarizing toxin substrate of the Staphylococcus aureus type VII secretion system mediates intraspecies competition. Proc Natl Acad Sci U S A 117:20836–20847
Hwang BJ, Chu G (1996) Trichloroacetic acid precipitation by ultracentrifugation to concentrate dilute protein in viscous solution. BioTechniques 20:982–984
Ozols J (1990) Amino acid analysis. Methods Enzymol 182:587–601
Wessel D, Flügge UI (1984) A method for the quantitative recovery of protein in dilute solution in the presence of detergents and lipids. Anal Biochem 138:141–143
Pumirat P, Saetun P, Sinchaikul S, Chen ST, Korbsrisate S, Thongboonkerd V (2009) Altered secretome of Burkholderia pseudomallei induced by salt stress. Biochim Biophys Acta 1794:898–904
Caldwell RB, Lattemann CT (2004) Simple and reliable method to precipitate proteins from bacterial culture supernatant. Appl Environ Microbiol 70:610–612
Cheng AT, Ottemann KM, Yildiz FH (2015) Vibrio cholerae response regulator VxrB controls colonization and regulates the type VI secretion system. PLoS Pathog 11:e1004933
Gueguen E, Cascales E (2013) Promoter swapping unveils the role of the Citrobacter rodentium CTS1 type VI secretion system in interbacterial competition. Appl Environ Microbiol 79:32–38
Acknowledgments
Work of L.J. was supported by the Centre National de la Recherche Scientifique, the Aix-Marseille Université, and grants from the Agence Nationale de la Recherche (ANR-14-CE14-0006 and ANR-18-CE15-0013). PhD studies of N.F. were supported by the ANR-14-CE14-0006 grant.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2024 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Flaugnatti, N., Journet, L. (2024). Identification of Effectors: Precipitation of Supernatant Material. In: Journet, L., Cascales, E. (eds) Bacterial Secretion Systems . Methods in Molecular Biology, vol 2715. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3445-5_32
Download citation
DOI: https://doi.org/10.1007/978-1-0716-3445-5_32
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-3444-8
Online ISBN: 978-1-0716-3445-5
eBook Packages: Springer Protocols