Regulated Secretion of YopN by the Type III Machinery of Yersinia enterocolitica

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Abstract
During infection, Yersinia enterocolitica exports Yop proteins via a type III secretion pathway. Secretion is activated when the environmental concentration of calcium ions is below 100 μM (low-calcium response). Yersiniae lacking yopN (lcrE), yscB, sycN , or tyeA do not inactivate the type III pathway even when the concentration of calcium is above 100 μM (calcium-blind phenotype). Purified YscB and SycN proteins form cytoplasmic complexes that bind a region including amino acids 16 to 100 of YopN, whereas TyeA binds YopN residues 101 to 294. Translational fusion of yopN gene sequences to the 5′ end of the npt reporter generates hybrid proteins that are transported by the type III pathway. The signal necessary and sufficient for the type III secretion of hybrid proteins is located within the first 15 codons of yopN . Expression of plasmid-borne yopN , but not of yopN 1–294 - npt , complements the calcium-blind phenotype of yopN mutants. Surprisingly, yopN mutants respond to environmental changes in calcium concentration and secrete YopN 1–294 -Npt in the absence but not in the presence of calcium. tyeA is required for the low-calcium regulation of YopN 1–294 -Npt secretion, whereas sycN and yscB mutants fail to secrete YopN 1–294 -Npt in the presence of calcium. Experiments with yopN-npt fusions identified two other signals that regulate the secretion of YopN. yopN codons 16 to 100 prevent the entry of YopN into the type III pathway, a negative regulatory effect that is overcome by expression of yscB and sycN . The portion of YopN encoded by codons 101 to 294 prevents transport of the polypeptide across the bacterial double membrane envelope in the presence of functional tyeA . These data support a model whereby YopN transport may serve as a regulatory mechanism for the activity of the type III pathway. YscB/SycN binding facilitates the initiation of YopN into the type III pathway, whereas TyeA binding prevents transport of the polypeptide across the bacterial envelope. Changes in the environmental calcium concentration relieve the TyeA-mediated regulation, triggering YopN transport and activating the type III pathway.

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