Complex I inhibition by piericidin does not underlie its ability to block type III secretion.
Piericidin A1 inhibits the Y. pseudotuberculosis and Y. enterocolitica Ysc T3SS but not the Y. enterocolitica Ysa T3SS.
Piericidin A1 traps T3SS effector proteins inside bacterial cells independently of the YopN, YopD, and YopK T3SS regulatory proteins.
Piericidin A1 decreases the number of T3SS needles associated with the bacterial surface but does not alter the localization of basal body components.
Piericidin A1 blocks YscF higher-order structure formation and decreases the secretion of middle and late T3SS substrates.
Piericidin A1 inhibition of type III secretion is reversible, and this reversibility is independent of new protein synthesis.
MATERIALS AND METHODS
Bacterial strains and growth conditions.
|IP 2666pIB1||None (WT)||49|
|ΔyopK mutant||ΔyopK||This study|
|ΔyopD mutant||ΔyopD||This study|
|flhDCY. pestis mutant||ΔyopHEMOJ flhDCY. pestis||41|
|pYscF mutant||ΔyopHEMOJ ΔYscF/pTCR99-YscF||34|
|pEmpty mutant||ΔyopHEMOJ ΔYscF/pTCR99 empty vector||34|
|P. aeruginosa PA103a||None (WT)||51, 52|
|yscL mutant||yscL::mini-Tn5 KM2||53|
Measurement of membrane potential.
Type III secretion assay.
Secreted and cytosolic YopE Western blot analysis.
Detection of secreted monomeric YscF.
Immunofluorescence staining of YscF.
YscK, YscL, YscD, and YscQ analysis.
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