{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,6,17]],"date-time":"2026-06-17T01:23:10Z","timestamp":1781659390047,"version":"3.54.5"},"reference-count":61,"publisher":"MDPI AG","issue":"11","license":[{"start":{"date-parts":[[2012,11,16]],"date-time":"2012-11-16T00:00:00Z","timestamp":1353024000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/3.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>F\u00f6rster resonance energy transfer (FRET) provides a powerful tool for monitoring intermolecular interactions and a sensitive technique for studying \u00c5-level protein conformational changes. One system that has particularly benefited from the sensitivity and diversity of FRET measurements is the maturation of the Shigella type III secretion apparatus (T3SA) needle tip complex. The Shigella T3SA delivers effector proteins into intestinal cells to promote bacterial invasion and spread. The T3SA is comprised of a basal body that spans the bacterial envelope and a needle with an exposed tip complex that matures in response to environmental stimuli. FRET measurements demonstrated bile salt binding by the nascent needle tip protein IpaD and also mapped resulting structural changes which led to the recruitment of the translocator IpaB. At the needle tip IpaB acts as a sensor for host cell contact but prior to secretion, it is stored as a heterodimeric complex with the chaperone IpgC. FRET analyses showed that chaperone binding to IpaB\u2019s N-terminal domain causes a conformational change in the latter. These FRET analyses, with other biophysical methods, have been central to understanding T3SA maturation and will be highlighted, focusing on the details of the FRET measurements and the relevance to this particular system.<\/jats:p>","DOI":"10.3390\/ijms131115137","type":"journal-article","created":{"date-parts":[[2012,11,16]],"date-time":"2012-11-16T11:03:15Z","timestamp":1353063795000},"page":"15137-15161","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":15,"title":["F\u00f6rster Resonance Energy Transfer (FRET) as a Tool for Dissecting the Molecular Mechanisms for Maturation of the Shigella Type III Secretion Needle Tip Complex"],"prefix":"10.3390","volume":"13","author":[{"given":"Nicholas","family":"Dickenson","sequence":"first","affiliation":[{"name":"Department of Microbiology and Molecular Genetics, Oklahoma State University, Stillwater, OK 74078, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"William","family":"Picking","sequence":"additional","affiliation":[{"name":"Department of Microbiology and Molecular Genetics, Oklahoma State University, Stillwater, OK 74078, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2012,11,16]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"448","DOI":"10.1016\/j.coi.2011.06.001","article-title":"Shigella are versatile mucosal pathogens that circumvent the host innate immune system","volume":"23","author":"Ashida","year":"2011","journal-title":"Curr. Opin. Immunol"},{"key":"ref_2","first-page":"554","article-title":"Early Steps in Inducing Type III Secretion in Shigella","volume":"4","author":"Picking","year":"2009","journal-title":"Microbes Infect"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"18646","DOI":"10.1074\/jbc.M802799200","article-title":"Deoxycholate interacts with IpaD of Shigella flexneri in inducing the recruitment of IpaB to the type III secretion apparatus needle tip","volume":"283","author":"Stensrud","year":"2008","journal-title":"J. Biol. Chem"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"172","DOI":"10.1021\/bi101365f","article-title":"Conformational changes in IpaD from Shigella flexneri upon binding bile salts provide insight into the second step of type III secretion","volume":"50","author":"Dickenson","year":"2011","journal-title":"Biochemistry"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"2626","DOI":"10.1128\/IAI.01599-06","article-title":"Bile salts stimulate recruitment of IpaB to the Shigella flexneri surface, where it colocalizes with IpaD at the tip of the type III secretion needle","volume":"75","author":"Olive","year":"2007","journal-title":"Infect. Immun"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"4062","DOI":"10.1021\/bi300243z","article-title":"Binding affects the tertiary and quaternary structures of the Shigella translocator protein IpaB and its chaperone IpgC","volume":"51","author":"Adam","year":"2012","journal-title":"Biochemistry"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"427","DOI":"10.1002\/bies.201100148","article-title":"Image analysis in fluorescence microscopy: Bacterial dynamics as a case study","volume":"34","author":"Shaevitz","year":"2012","journal-title":"Bioessays"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"62","DOI":"10.1006\/meth.1999.0906","article-title":"Green fluorescent protein as a reporter for macromolecular localization in bacterial cells","volume":"20","author":"Margolin","year":"2000","journal-title":"Methods"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"527","DOI":"10.1083\/jcb.150.3.527","article-title":"Three regions within ActA promote Arp2\/3 complex-mediated actin nucleation and Listeria monocytogenes motility","volume":"150","author":"Skoble","year":"2000","journal-title":"J. Cell Biol"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1159\/000329667","article-title":"Subgingival biofilm structure","volume":"15","author":"Zijnge","year":"2012","journal-title":"Front. Oral. Biol"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"373","DOI":"10.1146\/annurev.micro.112408.134205","article-title":"Use of fluorescence microscopy to study intracellular signaling in bacteria","volume":"64","author":"Kentner","year":"2010","journal-title":"Annu. Rev. Microbiol"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"312","DOI":"10.1002\/bip.21585","article-title":"How to quantify protein diffusion in the bacterial membrane","volume":"95","author":"Bollen","year":"2011","journal-title":"Biopolymers"},{"key":"ref_13","doi-asserted-by":"crossref","unstructured":"Lakowicz, J.R. (1983). Principles of Fluorescence Spectroscopy, Plenum Press.","DOI":"10.1007\/978-1-4615-7658-7"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"508","DOI":"10.1016\/j.cell.2012.05.046","article-title":"Plasma membrane reshaping during endocytosis is revealed by time-resolved electron tomography","volume":"150","author":"Kukulski","year":"2012","journal-title":"Cell"},{"key":"ref_15","first-page":"319","article-title":"The three dimensionality of cell membranes: Lamellar to cubic membrane transition as investigated by electron microscopy","volume":"108","author":"Chong","year":"2012","journal-title":"Methods Cell Biol"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"29","DOI":"10.1016\/j.jmb.2012.03.025","article-title":"Ultrastructural analysis of IpaD at the tip of the nascent MxiH type III secretion apparatus of Shigella flexneri","volume":"420","author":"Epler","year":"2012","journal-title":"J. Mol. Biol"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"235","DOI":"10.1016\/j.jsb.2008.05.008","article-title":"What transmission electron microscopes can visualize now and in the future","volume":"163","author":"Muller","year":"2008","journal-title":"J. Struct. Biol"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"e36749","DOI":"10.1371\/journal.pone.0036749","article-title":"Transmission electron microscopy reveals distinct macrophage- and tick cell-specific morphological stages of Ehrlichia chaffeensis","volume":"7","author":"Dedonder","year":"2012","journal-title":"PLoS One"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"993","DOI":"10.1146\/annurev.biochem.77.061906.092014","article-title":"Super-resolution fluorescence microscopy","volume":"78","author":"Huang","year":"2009","journal-title":"Annu. Rev. Biochem"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"967","DOI":"10.1366\/11-06398","article-title":"Review of super-resolution fluorescence microscopy for biology","volume":"65","author":"Leung","year":"2011","journal-title":"Appl. Spectrosc"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"011003","DOI":"10.1117\/1.JBO.17.1.011003","article-title":"Forster\u2019s resonance excitation transfer theory: Not just a formula","volume":"17","author":"Knox","year":"2012","journal-title":"J. Biomed. Opt"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"12","DOI":"10.1186\/jbiol225","article-title":"No better time to FRET: Shedding light on host pathogen interactions","volume":"9","author":"Hayward","year":"2010","journal-title":"J. Biol"},{"key":"ref_23","first-page":"55","article-title":"Intermolecular energy migration and fluorescence","volume":"2","year":"1948","journal-title":"Ann. Phys"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"379","DOI":"10.1128\/MMBR.62.2.379-433.1998","article-title":"Type III protein secretion systems in bacterial pathogens of animals and plants","volume":"62","author":"Hueck","year":"1998","journal-title":"Microbiol. Mol. Biol. Rev"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"50","DOI":"10.1128\/mr.57.1.50-108.1993","article-title":"The complete general secretory pathway in gram-negative bacteria","volume":"57","author":"Pugsley","year":"1993","journal-title":"Microbiol. Rev"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"53","DOI":"10.1016\/j.resmic.2003.10.002","article-title":"The autotransporter secretion system","volume":"155","author":"Desvaux","year":"2004","journal-title":"Res. Microbiol"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"a000299","DOI":"10.1101\/cshperspect.a000299","article-title":"Bacterial nanomachines: The flagellum and type III injectisome","volume":"2","author":"Erhardt","year":"2010","journal-title":"Cold Spring Harb. Perspect. Biol"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"47","DOI":"10.1016\/j.mib.2009.11.001","article-title":"Type III secretion systems shape up as they ship out","volume":"13","author":"Marlovits","year":"2010","journal-title":"Curr. Opin. Microbiol"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"277","DOI":"10.1038\/nsmb.1977","article-title":"Common architecture of the flagellar type III protein export apparatus and F- and V-type ATPases","volume":"18","author":"Ibuki","year":"2011","journal-title":"Nat. Struct. Mol. biol"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"1085","DOI":"10.1111\/j.1365-2958.2008.06237.x","article-title":"The type III secretion system tip complex and translocon","volume":"68","author":"Mueller","year":"2008","journal-title":"Mol. Microbiol"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"4391","DOI":"10.1128\/IAI.00440-06","article-title":"IpaD Localizes to the tip of the type III secretion system needle of Shigella flexneri","volume":"74","author":"Espina","year":"2006","journal-title":"Infect. Immun"},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"674","DOI":"10.1126\/science.1118476","article-title":"The V-antigen of Yersinia forms a distinct structure at the tip of injectisome needles","volume":"310","author":"Mueller","year":"2005","journal-title":"Science"},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"2754","DOI":"10.1128\/IAI.00190-09","article-title":"Liposomes recruit IpaC to the Shigella flexneri type III secretion apparatus needle as a final step in secretion induction","volume":"77","author":"Epler","year":"2009","journal-title":"Infect. Immun"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"387","DOI":"10.1046\/j.1462-5822.2000.00068.x","article-title":"Type III secretion systems: What is the translocator and what is translocated?","volume":"2","author":"Blocker","year":"2000","journal-title":"Cell. Microbiol"},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"683","DOI":"10.1083\/jcb.147.3.683","article-title":"The tripartite type III secreton of Shigella flexneri inserts IpaB and IpaC into host membranes","volume":"147","author":"Blocker","year":"1999","journal-title":"J. Cell Biol"},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"187","DOI":"10.1046\/j.1462-5822.2000.00046.x","article-title":"Bacterial signals and cell responses during Shigella entry into epithelial cells","volume":"2","author":"Dumenil","year":"2000","journal-title":"Cell. Microbiol"},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"4461","DOI":"10.1073\/pnas.1116126109","article-title":"Structure of a type III secretion needle at 7-A resolution provides insights into its assembly and signaling mechanisms","volume":"109","author":"Fujii","year":"2012","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"42929","DOI":"10.1074\/jbc.M508377200","article-title":"The needle component of the type III secreton of Shigella regulates the activity of the secretion apparatus","volume":"280","author":"Kenjale","year":"2005","journal-title":"J. Biol. Chem"},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"3027","DOI":"10.1073\/pnas.0535335100","article-title":"Type III secretion systems and bacterial flagella: Insights into their function from structural similarities","volume":"100","author":"Blocker","year":"2003","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"4488","DOI":"10.1002\/jps.22180","article-title":"Formulation and immunogenicity studies of type III secretion system needle antigens as vaccine candidates","volume":"99","author":"Barrett","year":"2010","journal-title":"J Pharm. Sci"},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"12529","DOI":"10.1073\/pnas.0602689103","article-title":"Molecular model of a type III secretion system needle: Implications for host-cell sensing","volume":"103","author":"Deane","year":"2006","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"322","DOI":"10.1016\/j.jmb.2006.03.028","article-title":"Solution structure of monomeric BsaL, the type III secretion needle protein of Burkholderia pseudomallei","volume":"359","author":"Zhang","year":"2006","journal-title":"J. Mol. Biol"},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"1304","DOI":"10.1016\/j.jmb.2007.06.034","article-title":"Differences in the electrostatic surfaces of the type III secretion needle proteins PrgI, BsaL, and MxiH","volume":"371","author":"Wang","year":"2007","journal-title":"J. Mol. Biol"},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"17103","DOI":"10.1074\/jbc.M300091200","article-title":"Helical structure of the needle of the type III secretion system of Shigella flexneri","volume":"278","author":"Cordes","year":"2003","journal-title":"J. Biol. Chem"},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"1719","DOI":"10.1111\/j.1365-2958.2005.04790.x","article-title":"The Yersinia pestis type III secretion needle plays a role in the regulation of Yop secretion","volume":"57","author":"Torruellas","year":"2005","journal-title":"Mol. Microbiol"},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"935","DOI":"10.1002\/prot.23251","article-title":"Identification of the bile salt binding site on IpaD from Shigella flexneri and the influence of ligand binding on IpaD structure","volume":"80","author":"Barta","year":"2012","journal-title":"Proteins"},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"6923","DOI":"10.1128\/JB.05555-11","article-title":"Translocators YopB and YopD from Yersinia enterocolitica form a multimeric integral membrane complex in eukaryotic cell membranes","volume":"193","author":"Montagner","year":"2011","journal-title":"J. Bacteriol"},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"462","DOI":"10.1002\/cphc.201000664","article-title":"FRET microscopy in 2010: The legacy of Theodor Forster on the 100th anniversary of his birth","volume":"12","author":"Sun","year":"2011","journal-title":"Chemphyschem"},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"39158","DOI":"10.1074\/jbc.M112.395228","article-title":"GRB14 is a negative regulator of ceacam3-mediated phagocytosis of pathogenic bacteria","volume":"287","author":"Kopp","year":"2012","journal-title":"J. Biol. Chem"},{"key":"ref_50","doi-asserted-by":"crossref","first-page":"e16","DOI":"10.1371\/journal.ppat.0010016","article-title":"Yersinia pseudotuberculosis spatially controls activation and misregulation of host cell Rac1","volume":"1","author":"Wong","year":"2005","journal-title":"PLoS Pathog"},{"key":"ref_51","doi-asserted-by":"crossref","first-page":"269","DOI":"10.1126\/science.281.5374.269","article-title":"Specific covalent labeling of recombinant protein molecules inside live cells","volume":"281","author":"Griffin","year":"1998","journal-title":"Science"},{"key":"ref_52","doi-asserted-by":"crossref","first-page":"515","DOI":"10.1016\/0092-8674(94)90260-7","article-title":"Extracellular association and cytoplasmic partitioning of the IpaB and IpaC invasins of S. flexneri","volume":"79","author":"Menard","year":"1994","journal-title":"Cell"},{"key":"ref_53","doi-asserted-by":"crossref","first-page":"8128","DOI":"10.1021\/bi700099c","article-title":"Preparation and characterization of translocator\/chaperone complexes and their component proteins from Shigella flexneri","volume":"46","author":"Birket","year":"2007","journal-title":"Biochemistry"},{"key":"ref_54","doi-asserted-by":"crossref","first-page":"395","DOI":"10.1016\/j.jmb.2012.01.026","article-title":"The structures of coiled-coil domains from type III secretion system translocators reveal homology to pore-forming toxins","volume":"417","author":"Barta","year":"2012","journal-title":"J. Mol. Biol"},{"key":"ref_55","doi-asserted-by":"crossref","first-page":"39965","DOI":"10.1074\/jbc.M110.135616","article-title":"Combination of two separate binding domains defines stoichiometry between type III secretion system chaperone IpgC and translocator protein IpaB","volume":"285","author":"Lokareddy","year":"2010","journal-title":"J. Biol. Chem"},{"key":"ref_56","doi-asserted-by":"crossref","first-page":"6950","DOI":"10.1128\/JB.05163-11","article-title":"Disulfide bonding within components of the Chlamydia type III secretion apparatus correlates with development","volume":"193","author":"Fields","year":"2011","journal-title":"J. Bacteriol"},{"key":"ref_57","doi-asserted-by":"crossref","first-page":"10353","DOI":"10.1021\/bi901200y","article-title":"Biophysical characterization of Chlamydia trachomatis CT584 supports its potential role as a type III secretion needle tip protein","volume":"48","author":"Markham","year":"2009","journal-title":"Biochemistry"},{"key":"ref_58","doi-asserted-by":"crossref","first-page":"1150","DOI":"10.1128\/IAI.72.2.1150-1154.2004","article-title":"Type III secretion: A virulence factor delivery system essential for the pathogenicity of Burkholderia mallei","volume":"72","author":"Ulrich","year":"2004","journal-title":"Infect. Immun"},{"key":"ref_59","doi-asserted-by":"crossref","first-page":"41","DOI":"10.1080\/09687860500041858","article-title":"Type III secretion: A secretory pathway serving both motility and virulence (review)","volume":"22","author":"Journet","year":"2005","journal-title":"Mol. Membr. Biol"},{"key":"ref_60","doi-asserted-by":"crossref","first-page":"54","DOI":"10.4161\/viru.2.1.14666","article-title":"Monitoring Shigella flexneri vacuolar escape by flow cytometry","volume":"2","author":"Nothelfer","year":"2011","journal-title":"Virulence"},{"key":"ref_61","doi-asserted-by":"crossref","first-page":"e1002507","DOI":"10.1371\/journal.ppat.1002507","article-title":"Phagosomal rupture by Mycobacterium tuberculosis results in toxicity and host cell death","volume":"8","author":"Simeone","year":"2012","journal-title":"PLoS Pathog"}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/13\/11\/15137\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T21:53:38Z","timestamp":1760219618000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/13\/11\/15137"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2012,11,16]]},"references-count":61,"journal-issue":{"issue":"11","published-online":{"date-parts":[[2012,11]]}},"alternative-id":["ijms131115137"],"URL":"https:\/\/doi.org\/10.3390\/ijms131115137","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2012,11,16]]}}}