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.
Publication types
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Evaluation Study
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Research Support, Non-U.S. Gov't
MeSH terms
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Adenylate Cyclase Toxin / genetics
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Adenylate Cyclase Toxin / toxicity
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Adenylyl Cyclases / genetics
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Adhesins, Bacterial / analysis
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Antimicrobial Cationic Peptides / analysis
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Antimicrobial Cationic Peptides / metabolism
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Caco-2 Cells
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Carrier Proteins / analysis
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Carrier Proteins / genetics
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Carrier Proteins / metabolism
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Cathelicidins
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Colorimetry / economics
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Colorimetry / methods*
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Enteropathogenic Escherichia coli / genetics
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Enteropathogenic Escherichia coli / growth & development
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Enteropathogenic Escherichia coli / pathogenicity*
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Escherichia coli Proteins / analysis*
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Escherichia coli Proteins / genetics
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Escherichia coli Proteins / metabolism
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Humans
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Intracellular Signaling Peptides and Proteins
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Protein Transport
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Receptors, Cell Surface / analysis*
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Receptors, Cell Surface / genetics
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Receptors, Cell Surface / metabolism
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Recombinant Fusion Proteins / analysis
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Recombinant Fusion Proteins / genetics
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Recombinant Fusion Proteins / metabolism
Substances
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Adenylate Cyclase Toxin
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Adhesins, Bacterial
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Antimicrobial Cationic Peptides
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Carrier Proteins
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Cathelicidins
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Escherichia coli Proteins
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EspFU protein, E coli
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Intracellular Signaling Peptides and Proteins
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Receptors, Cell Surface
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Recombinant Fusion Proteins
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Tir protein, E coli
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Adenylyl Cyclases