Competition between the Yops of Yersinia enterocolitica for delivery into eukaryotic cells: Role of the SycE chaperone binding domain of YopE

A type III secretion-translocation system allows Yersinia adhering at the s urface of animal cells to deliver a cocktail of effector Yops (YopH, -O, -P , -E, -M, and -T) into the cytosol of these cells. Residues or codons 1 to 77 contain all the information required for the complete delivery of YopE i nto the target cell (release from the bacterium and translocation across th e eukaryotic cell membrane). Residues or codons 1 to 15 are sufficient for release from the wild-type bacterium under Ca2+-chelating conditions but no t for delivery into target cells. Residues 15 to 50 comprise the binding do main for SycE, a chaperone specific for YopE that is necessary for release and translocation of full-length YopE, To understand the role of this chape rone, we studied the delivery of YopE-Cya reporter proteins and YopE deleta nts by polymutant Yersinia devoid of most of the Yop effecters (Delta HOPEM and Delta THE strains). We first tested YopE-Cya hybrid proteins and YopE proteins deleted of the SycE-binding site. In contrast to wild-type strains , these mutants delivered YopE(15)-Cya as efficiently as YopE(130)-Cya. The y were also able to deliver YopE(Delta 17-77). SycE was dispensable for the se deliveries. These results show that residues or codons 1 to 15 are suffi cient for delivery into eukaryotic cells and that there is no specific tran slocation signal in Yops. However, the fact that the SycE-binding site and SycE were necessary for delivery of YopE by wild-type Yersinia suggests tha t they could introduce hierarchy among the effecters to be delivered. We th en tested a YopE-Cya hybrid and YopE proteins deleted of amino acids 2 to 1 5 but containing the SycE-binding domain. These constructs were neither rel eased in vitro upon Ca2+ chelation nor delivered into cells by wild-type or polymutant bacteria, casting doubts on the hypothesis that SycE could be a secretion pilot. Finally, it appeared that residues 50 to 77 are inhibitor y to YopE release and that binding of SycE overcomes this inhibitory effect . Removal of this domain allowed in vitro release and delivery in cells in the absence as well as in the presence of SycE.