Quantifying Substrate Protein Secretion via the Type III Secretion System of the Bacterial Flagellum.
Bacterial flagellum
Carbonyl cyanide m-chlorophenylhydrazone (CCCP)
Fluorescence microscopy
Ionophore
Protein export
Proton motive force
Split NanoLuc luciferase
T3SS
Type III secretion system
Valinomycin
ΔpH gradient
ΔΨ gradient
Journal
Methods in molecular biology (Clifton, N.J.)
ISSN: 1940-6029
Titre abrégé: Methods Mol Biol
Pays: United States
ID NLM: 9214969
Informations de publication
Date de publication:
2024
2024
Historique:
medline:
7
11
2023
pubmed:
6
11
2023
entrez:
6
11
2023
Statut:
ppublish
Résumé
Protein transport across the cytoplasmic membrane is coupled to energy derived from ATP hydrolysis or the proton motive force. A sophisticated, multi-component type III secretion system (T3SS) exports substrate proteins of both the bacterial flagellum and virulence-associated injectisome system of many Gram-negative pathogens. The T3SS is primarily a proton motive force-driven protein exporter. Here, we describe a method to investigate the export of substrate proteins of the flagellar T3SS into the culture supernatant under conditions that manipulate the proton motive force. Further, we describe methods to precisely quantify flagellar protein export into the culture supernatant using a split NanoLuc luciferase, and how fluorescence labeling of the extracellular flagellar filament can bring insights into the protein export rate of individual flagellar T3SS.
Identifiants
pubmed: 37930553
doi: 10.1007/978-1-0716-3445-5_36
doi:
Substances chimiques
Type III Secretion Systems
0
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
577-592Informations de copyright
© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.
Références
Wickner W, Schekman R (2005) Protein translocation across biological membranes. Science 310:1452–1456
doi: 10.1126/science.1113752
pubmed: 16322447
Halte M, Erhardt M (2021) Protein export via the type III secretion system of the bacterial flagellum. Biomol Ther 11:186
Minamino T (2014) Protein export through the bacterial flagellar type III export pathway. Biochim Biophys Acta 1843:1642–1648
doi: 10.1016/j.bbamcr.2013.09.005
pubmed: 24064315
Diepold A, Wagner S (2014) Assembly of the bacterial type III secretion machinery. FEMS Microbiol Rev 38:802–822
doi: 10.1111/1574-6976.12061
pubmed: 24484471
Erhardt M, Namba K, Hughes KT (2010) Bacterial nanomachines: the flagellum and type III injectisome. Cold Spring Harb Perspect Biol 2:a000299
doi: 10.1101/cshperspect.a000299
pubmed: 20926516
pmcid: 2964186
Lee PC, Zmina SE, Stopford CM, Toska J, Rietsch A (2014) Control of type III secretion activity and substrate specificity by the cytoplasmic regulator PcrG. Proc Natl Acad Sci U S A 111:E2027–E2036
pubmed: 24778208
pmcid: 4024851
Paul K, Erhardt M, Hirano T, Blair DF, Hughes KT (2008) Energy source of flagellar type III secretion. Nature 451:489–492
doi: 10.1038/nature06497
pubmed: 18216859
Minamino T, Namba K (2008) Distinct roles of the FliI ATPase and proton motive force in bacterial flagellar protein export. Nature 451:485–488
doi: 10.1038/nature06449
pubmed: 18216858
Wilharm G, Lehmann V, Krauss K, Lehnert B, Richter S, Ruckdeschel K, Heesemann J, Trülzsch K (2004) Yersinia enterocolitica type III secretion depends on the proton motive force but not on the flagellar motor components MotA and MotB. Infect Immun 72:4004–4009
doi: 10.1128/IAI.72.7.4004-4009.2004
pubmed: 15213145
pmcid: 427454
Hüsing S, Halte M, van Look U, Guse A, Gálvez EJC, Charpentier E, Blair DF, Erhardt M, Renault TT (2021) Control of membrane barrier during bacterial type-III protein secretion. Nat Commun 12:3999
doi: 10.1038/s41467-021-24226-1
pubmed: 34183670
pmcid: 8239009
Ward E, Renault TT, Kim EA, Erhardt M, Hughes KT, Blair DF (2018) Type-III secretion pore formed by flagellar protein FliP. Mol Microbiol 107:94–103
doi: 10.1111/mmi.13870
pubmed: 29076571
Fabiani FD, Renault TT, Peters B, Dietsche T, Gálvez EJC, Guse A, Freier K, Charpentier E, Strowig T, Franz-Wachtel M, Macek B, Wagner S, Hensel M, Erhardt M (2017) A flagellum-specific chaperone facilitates assembly of the core type III export apparatus of the bacterial flagellum. PLoS Biol 15:e2002267
doi: 10.1371/journal.pbio.2002267
pubmed: 28771474
pmcid: 5542435
Fukumura T, Makino F, Dietsche T, Kinoshita M, Kato T, Wagner S, Namba K, Imada K, Minamino T (2017) Assembly and stoichiometry of the core structure of the bacterial flagellar type III export gate complex. PLoS Biol 15:e2002281
doi: 10.1371/journal.pbio.2002281
pubmed: 28771466
pmcid: 5542437
Erhardt M, Wheatley P, Kim EA, Hirano T, Zhang Y, Sarkar MK, Hughes KT, Blair DF (2017) Mechanism of type-III protein secretion: regulation of FlhA conformation by a functionally critical charged-residue cluster. Mol Microbiol 104:234–249
doi: 10.1111/mmi.13623
pubmed: 28106310
pmcid: 5380474
Terahara N, Inoue Y, Kodera N, Morimoto YV, Uchihashi T, Imada K, Ando T, Namba K, Minamino T (2018) Insight into structural remodeling of the FlhA ring responsible for bacterial flagellar type III protein export. Science. Advances 4:eaao7054
Erhardt M, Mertens ME, Fabiani FD, Hughes KT (2014) ATPase-independent type-III protein secretion in Salmonella enterica. PLoS Genet 10:e1004800
doi: 10.1371/journal.pgen.1004800
pubmed: 25393010
pmcid: 4230889
Morimoto YV, Ito M, Hiraoka KD, Che YS, Bai F, Kami-Ike N, Namba K, Minamino T (2014) Assembly and stoichiometry of FliF and FlhA in Salmonella flagellar basal body. Mol Microbiol 91:1214–1226
doi: 10.1111/mmi.12529
pubmed: 24450479
Diepold A, Kudryashev M, Delalez NJ, Berry RM, Armitage JP (2015) Composition, formation, and regulation of the cytosolic c-ring, a dynamic component of the type III secretion injectisome. PLoS Biol 13:e1002039
doi: 10.1371/journal.pbio.1002039
pubmed: 25591178
pmcid: 4295842
Erhardt M, Hughes KT (2010) C-ring requirement in flagellar type III secretion is bypassed by FlhDC upregulation. Mol Microbiol 75:376–393
doi: 10.1111/j.1365-2958.2009.06973.x
pubmed: 19919668
McMurry JL, Murphy JW, González-Pedrajo B (2006) The FliN-FliH interaction mediates localization of flagellar export ATPase FliI to the C ring complex. Biochemistry 45:11790–11798
doi: 10.1021/bi0605890
pubmed: 17002279
Aldridge P, Hughes KT (2002) Regulation of flagellar assembly. Curr Opin Microbiol 5:160–165
doi: 10.1016/S1369-5274(02)00302-8
pubmed: 11934612
Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, Preibisch S, Rueden C, Saalfeld S, Schmid B, Tinevez J-Y, White DJ, Hartenstein V, Eliceiri K, Tomancak P, Cardona A (2012) Fiji: an open-source platform for biological-image analysis. Nat Methods 9:676–682
doi: 10.1038/nmeth.2019
pubmed: 22743772
Hall MP, Unch J, Binkowski BF, Valley MP, Butler BL, Wood MG, Otto P, Zimmerman K, Vidugiris G, Machleidt T, Robers MB, Benink HA, Eggers CT, Slater MR, Meisenheimer PL, Klaubert DH, Fan F, Encell LP, Wood KV (2012) Engineered luciferase reporter from a deep sea shrimp utilizing a novel imidazopyrazinone substrate. ACS Chem Biol 7:1848–1857
doi: 10.1021/cb3002478
pubmed: 22894855
pmcid: 3501149
Dixon AS, Schwinn MK, Hall MP, Zimmerman K, Otto P, Lubben TH, Butler BL, Binkowski BF, Machleidt T, Kirkland TA, Wood MG, Eggers CT, Encell LP, Wood KV (2016) NanoLuc complementation reporter optimized for accurate measurement of protein interactions in cells. ACS Chem Biol 11:400–408
doi: 10.1021/acschembio.5b00753
pubmed: 26569370
Minamino T, Morimoto YV, Hara N, Namba K (2011) An energy transduction mechanism used in bacterial flagellar type III protein export. Nat Commun 2:475
doi: 10.1038/ncomms1488
pubmed: 21934659
Minamino T, Imae Y, Oosawa F, Kobayashi Y, Oosawa K (2003) Effect of intracellular pH on rotational speed of bacterial flagellar motors. J Bacteriol 185:1190–1194
doi: 10.1128/JB.185.4.1190-1194.2003
pubmed: 12562788
pmcid: 142873
Renault TT, Abraham AO, Bergmiller T, Paradis G, Rainville S, Charpentier E, Guet CC, Tu Y, Namba K, Keener JP, Minamino T, Erhardt M (2017) Bacterial flagella grow through an injection-diffusion mechanism. elife 6:e23136
doi: 10.7554/eLife.23136
pubmed: 28262091
pmcid: 5386592