This is a MERIT extension of R37 AI075039. In the previous funding period we addressed the fundamental mechanisms by which pathogen-encoded virulence activities are detected by the innate immune system. We focused on an innate immune sensor called the NLRP1 inflammasome. This sensor was known to be activated by a protease toxin, called lethal factor (LF), that is secreted by the bacterial pathogen Bacillus anthracis. We demonstrated that LF activates NLRP1 by direct proteolytic cleavage of NLRP1. We found that this cleavage results in the N-end rule-dependent degradation of NLPR1 by the proteasome. Unexpectedly, however, we found that LF-induced degradation does not inactivate NLRP1. Instead, we found that NLRP1 underwent “functional degradation”, a unique process by which the signaling competent C-terminal CARD domain of NLRP1 was released and thereby able to undergo oligomerization and inflammasome formation. Our work identifying the molecular basis by which NLRP1 is able to sense pathogen-encoded enzymatic activity led us to ask whether NLRP1 might sense additional pathogen- encoded activities and, moreover, whether additional pathogen-encoded effectors might be detected by other innate immune sensors. Indeed, we discovered that a pathogen-encoded ubiquitin E3 ligase called IpaH7.8, secreted into host cells by the bacterial pathogen Shigella flexneri, is also sensed by NLRP1. In the extension period, we propose to continue to investigate the molecular mechanisms by which the enzymatic activities of pathogen-encoded virulence factors are detected by the innate immune system. We are conducting screens to identify novel virulence factors that stimulate host immune responses. We will then conduct mechanistic studies to identify the sensors of these virulence factors and the molecular basis of sensor activation. Together our studies will illuminate fundamental molecular mechanisms by which pathogens are sensed by the innate immune system. RELEVANCE (See instructions): Human health depends on the accurate and sensitive detection of pathogenic microbes by the innate immune system. We propose to determine fundamental mechanisms by which the virulence activities of bacterial pathogens are sensed by the innate immune system.