The type III secretion chaperone LcrH co-operates with YopD to establish anegative, regulatory loop for control of Yop synthesis in Yersinia pseudotuberculosis

The enteropathogen Yersinia pseudotuberculosis is a model system used to st udy the molecular mechanisms by which Gram-negative pathogens secrete and s ubsequently translocate antihost effector proteins into target eukaryotic c ells by a common type III secretion system (TTSS). In this process, YopD (Y ersinia outer protein D) is essential to establish regulatory control of Yo p synthesis and the ensuing translocation process. YopD function depends up on the non-secreted TTSS chaperone LcrH low-calcium response H), which is r equired for presecretory stabilization of YopD. However, as a new role for TTSS chaperones in virulence gene regulation has been proposed recently, we undertook a detailed analysis of LcrH. A IcrH null mutant constitutively p roduced Yops, even when this strain was engineered to produce wild-type lev els of YopD. Furthermore, the YopD-LcrH interaction was necessary to regain the negative regulation of virulence associated genes yops). This finding was used to investigate the biological significance of several LcrH mutants with varied YopD binding potential. Mutated LcrH alleles were introduced i n trans into a IcrH null mutant to assess their impact on yop regulation an d the subsequent translocation of YopE, a Rho-GTPase activating protein, ac ross the plasma membrane of eukaryotic cells. Two mutants, LcrH(K20E), (E30 G), (131V), (M99V), (D135G) and LcrH(E30G) lost all regulatory control, eve n though YopD binding and secretion and the subsequent translocation of Yop E was indistinguishable from wild type. Moreover, these regulatory deficien t mutants showed a reduced ability to bind YscY in the two-hybrid assay. Co llectively, these findings confirm that LcrH plays an active role in yop re gulation that might be mediated via an interaction with the Ysc secretion a pparatus. This chaperone-substrate interaction presents an innovative means to establish a regulatory hierarchy in Yersinia infections. It also raises the question as to whether or not LcrH is a true chaperone involved in sta bilization and secretion of YopD or a regulatory protein responsible for co -ordinating synthesis of Yersinia virulence determinants. We suggest that L crH can exhibit both of these activities.