Yersinia pestis, the causative agent of Plague, harbors a -71 KB plasmid that encodes many virulence-related proteins. Among the pCDl-encoded genes are genes specifying for the construction of a type III secretion system and the genes for the secreted effector proteins (collectively termed Yops). Type III secretion systems allow pathogenic bacteria to specifically translocate effectors into eukaryotic cells for the pathogen's benefit. The Yops exert their effects on targeted eukaryotic cells that results in a blockage of phagocytosis by immune cells. This effect allows Y. pestis to remain extracellular during the course of an infection. In Y. pestis the activity of the type III system is controlled by environmental stimuli, in vitro secretion is stimulated by the removal of calcium ions. In vivo Yops translocation is triggered by contact with eukaryotic cells. Appropriate control of type III secretion is required for the disease process as mutants defective in control are avirulent. Control of type III secretion relies on both positive and negative control circuits. LcrG and LcrV are two proteins whose interaction is required to control type III secretion in Y. pestis. LcrV is required to activate secretion while LcrG is required to inactivate secretion. Their interaction provides a linkage between positive and negative regulation in type III secretion control. The mechanism responsible for their controlling effects are largely unknown, although progress has been made. Published studies have shown that the interaction of LcrG and LcrV is required to control secretion. Additionally, a separate study demonstrated that a ratio of LcrV to LcrG, favoring LcrV, is required to activate secretion. Clearly, more work is needed to define the roles of LcrG and LcrV in the activation of secretion. Specifically, in this proposal we shall:
Aim 1. Define structure-function relationships within LcrG relating to LcrV interaction and secretion-blocking.
Aim 2. Screen for other proteins that may interact with LcrG.
Aim 3. Refine the subcellular localization of LcrG.
Aim 4. Examine mechanisms that alter LcrG levels relative to LcrV. Type III secretion systems have emerged recently as a common theme in the pathogenic mechanism of many gram-negative bacterial pathogens. Work in the yersiniae has been at the forefront of research on the characterization and function of type III mechanisms. An understanding of how these systems are controlled should lead to a deeper understanding of the intimate and dynamic interactions between pathogens and their hosts. ? ?
