A study of the YopD-LcrH interaction from Yersinia pseudotuberculosis reveals a role for hydrophobic residues within the amphipathic domain of YopD

The enteropathogen Yersinia pseudotuberculosis is a model system used to st udy the molecular mechanisms by which Gram-negative pathogens translocate e ffector proteins into target eukaryotic cells by a common type III secretio n machine. Of the numerous proteins produced by Y. pseudotuberculosis that act in concert to establish an infection, YopD (Yersinia outer protein D) i s a crucial component essential for yop regulation and Yop effector translo cation. In this study, we describe the mechanisms by which YopD functions t o control these processes. With the aid of the yeast two-hybrid system, we investigated the interaction between YopD and the cognate chaperone LcrH, W e confirmed that non-secreted LcrH is necessary for YopD stabilization befo re secretion, presumably by forming a complex with YopD in the bacterial cy toplasm. At least in yeast, this complex depends upon the N-terminal domain and a C-terminal amphipathic alpha-helical domain of YopD, Introduction of amino acid substitutions within the hydrophobic side of the amphipathic al pha-helix abolished the YopD-LcrH interaction, indicating that hydrophobic, as opposed to electrostatic, forces of attraction are important for this p rocess. Suppressor mutations isolated within LcrH could compensate for defe cts in the amphipathic domain of YopD to restore binding. Isolation of LcrH mutants unable to interact with wild-type YopD revealed no single domain r esponsible for YopD binding. The YopD and LcrH mutants generated in this st udy will be relevant tools for understanding YopD function during a Yersini a infection.