Control of type III secretion in Yersinia pestis by the negative regulator, LcrG

Yersinia pestis, the causative agent of bubonic plague, downregulates the host immune response using a type III secretion system. The activity of the type III secretion system of Y. pestis is regulated by several proteins, including the negative secretion regulator, LcrG. LcrG has been proposed to block the secretion apparatus from the cytoplasmic face of the inner membrane under conditions that do not favor Yops secretion. LcrG has been demonstrated to interact and form a stable complex with a positive regulator of secretion, LcrV. Under secretion-permissive conditions, it is thought that increased levels of LcrV titrate LcrG away from the secretion apparatus, allowing secretion of Yops to occur. The goal of this project was to determine the role of the LcrG-LcrV interaction in Yops secretion control. First, the region of LcrG required for interaction with LcrV was identified. This information was used to construct an LcrG mutant that could not interact with LcrV. Expression of this mutant blocked Yops secretion even under secretion-permissive conditions. These results demonstrate that the LcrG-LcrV interaction is necessary for control of Yops secretion. Structure function relationships within LcrG were also examined to gain information about the domains responsible for LcrG's two currently described activities: interaction with LcrV and secretion blocking. The N-terminus of LcrG (amino acids 7--40) is required for interaction with LcrV and is predicted to form a coiled-coil motif. Three residues within that region were identified as important for interaction with LcrV. These residues are conserved in the LcrG homolog, PcrG, which was found to complement both the LcrV-interaction and secretion-blocking activities of LcrG. The domain of LcrG responsible for secretion blocking was preliminarily characterized as well. Our initial evidence suggests that the secretion-blocking domain resides in a central region of LcrG and may overlap with the LcrV-interaction domain. In conclusion, this study has identified structural domains within LcrG and attempted to characterize their function in terms of the Low Calcium Response. This information has advanced our knowledge of the complex regulation of type III secretion in Yersinia pestis.