The gram-negative bacterium Yersinia pestis is the causative agent of plague. Y. pestis and some other pathogens have the ability to modify the acyl chains in their lipopolysaccharide (LPS) /lipid A in order to minimize Toll-like receptor 4 (TLR4) signaling. Consequently, Y. pestis produces a tetra-acyl LPS with poor TLR4-activating ability at 37C, while synthesizing a potent hexa-acyl lipid at 26C. We have shown that the resulting evasion of innate immune responses at 37C is necessary for Y. pestis virulence via the peripheral route, by generating a modified bacterial strain synthesizing a hexa-acylated TLR4-activating LPS also at 37C. The modified strain contained LpxL, a lipid A biosynthesis enzyme from E. coli, that is absent in Y. pestis, and has more than a million-fold reduced virulence. Our main hypothesis is that evasion of LPS-TLR4 signaling is essential for the virulence of Y. pestis, and that a tight regulation of lipid A structure is necessary for this evasion to occur. We propose to use Y. pestis strains that generate modified LPS to study evasion and activation of innate immunity by the plague bacillus. Our model system appears well suited to describe efficient innate immune mechanisms against Y. pestis, and our long-term goal is to define such mechanisms and bacterial countermeasures. 1) Y. pestis expresses LpxP, a lipid A biosynthesis gene that likely is necessary for the production of a hexa-acyl TLR4-activating LPS at lower temperatures. We propose to study regulation of LpxP expression at 37C, as regulation appears necessary for virulence. 2) We also wish to study the role of evasion of LPS-TLR4 signaling in the evolution of Y. pestis to a highly virulent pathogen from its closest ancestor, Y. pseudotuberculosis (Y. ptb), which only may cause a mild gastroenteritis. Interestingly, Y. ptb harbors an LpxL gene. Our proposal suggests studies of Y. ptb LpxL function, this will include expression of Y. ptb LpxL in Y. pestis, and study LPS activity and structures. 3) Preliminary results indicate that interleukin-1 (IL-1) release and signaling is effective in clearing infection with Y. pestis-LpxL, more so than TNF and type I IFN. We will analyze mechanisms by which Y. pestis induces and is controlled by IL-1, in vitro and in vivo.