title: A colorimetric assay for studying effector secretion through the bacterial type III secretion system.
abstract: We have devised a colorimetric method that monitors secretion of effector proteins into host cytoplasm through the bacterial type III secretion machinery. Here we used constructs of effectors fused with Bordetella adenylate cyclase as a reporter, but evaluated the effector translocation by quantifying cell viability, rather than by measuring the intracellular cAMP concentration. This is based on our findings that cells infected by a secretion-competent bacterium expressing the fusion protein lost their viability under our experimental conditions. Cell death was quantified using commercially available reagents and basic research equipment. An observation that cell death was potentiated when the infected cells were treated with 2-deoxyglucose and sodium azide suggests that the depletion of intracellular ATP is partly involved in the process. Using enteropathogenic Escherichia coli, we demonstrated that the method was applicable to at least three effectors of bacteria, Tir, EspF, and Map, and was useful for studying a secretion signal sequence for Tir. This technically simple and inexpensive method is a good alternative to the existing procedure for studying the mechanism by which effectors are secreted through the type III secretion system in a high-throughput format.
authors.affiliation: Department of Molecular Bacteriology, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan. mami@biken.osaka-u.ac.jp
authors.forename: Masami
authors.initials: M
authors.lastname: Miyake
authors.forename: Sadatsugu
authors.initials: S
authors.lastname: Sakane
authors.forename: Chiho
authors.initials: C
authors.lastname: Kobayashi
authors.forename: Miyuki
authors.initials: M
authors.lastname: Hanajima-Ozawa
authors.forename: Aya
authors.initials: A
authors.lastname: Fukui
authors.forename: Shigeki
authors.initials: S
authors.lastname: Kamitani
authors.forename: Yasuhiko
authors.initials: Y
authors.lastname: Horiguchi
mesh_terms: D037361:Adenylate Cyclase Toxin / Q000235:genetics / Q000633:toxicity; D000262:Adenylyl Cyclases / Q000235:genetics; D018829:Adhesins, Bacterial / Q000032:analysis; D023181:Antimicrobial Cationic Peptides / Q000032:analysis / Q000378:metabolism; D018938:Caco-2 Cells; D002352:Carrier Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D054804:Cathelicidins; D003124:Colorimetry / Q000191:economics / Q000379:methods*; D054308:Enteropathogenic Escherichia coli / Q000235:genetics / Q000254:growth & development / Q000472:pathogenicity*; D029968:Escherichia coli Proteins / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D021381:Protein Transport; D011956:Receptors, Cell Surface / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism
chemical_list: D037361:Adenylate Cyclase Toxin; D018829:Adhesins, Bacterial; D023181:Antimicrobial Cationic Peptides; D002352:Carrier Proteins; D054804:Cathelicidins; D029968:Escherichia coli Proteins; C489109:EspFU protein, E coli; D047908:Intracellular Signaling Peptides and Proteins; D011956:Receptors, Cell Surface; D011993:Recombinant Fusion Proteins; C109287:Tir protein, E coli; D000262:Adenylyl Cyclases