SUMMARY Antibiotic resistance among bacterial pathogens is spreading rapidly around the world. Therefore, it is urgent to develop strategies to discover novel antimicrobial agents. Two component system (TCSs) are ideal targets for developing novel antimicrobial treatments for at least two reasons: (i) they are often essential for bacterial growth within the host; (ii) they are common in bacteria but evidently absent in human and animals. TCSs have been studied for decades and the molecular basis of signal transduction is well known, however, important questions remain regarding regulation of these signaling systems. In this application, I will use the intracellular human pathogen Bacillus anthracis as a model organism and investigate the regulatory mechanism of the HitRS signaling system. This TCS senses the phagocyte cell environment and provides a direct fitness advantage during the interactions with the host immune cells. Furthermore, the HitRS system is activated by a variety of molecular distinct cell envelope stressors, suggesting that additional cellular factors must be required for activation of this system. Indeed, using an unbiased genetic selection strategy, we identified an RNA binding protein KreA (ComK repressor in B. anthracis) that plays a critical role in HitRS activation through modulating mRNA stability of the TCS transcripts. In addition, our preliminary data demonstrate that KreA functions as an RNA binding protein (RBP) and plays an important post-transcriptional regulatory role in HitRS signaling. The importance of bacterial post-transcriptional control has been increasingly appreciated in recent years although the mechanisms of these regulatory networks are poorly understood in bacteria. Based on our preliminary data, we propose a model that the newly identified RBP KreA binds mRNA at specific target sites, impacts expression of functionally coordinated sets of mRNAs, interacts with other RBPs dynamically to facilitate mRNA decay, and promotes bacterial survival within the mammalian hosts. In this application, we will combine a number of strategies including biochemical analysis, genetics, transcriptomics, proteomics, live cell imaging, and mouse infection models to (i) define the direct RNA targets and binding preference of KreA, (ii) elucidate the underlying mechanism of KreA in regulating HitRS signaling, (iii) determine the functional ramifications of KreA- modulated RNA stability on bacterial physiology, and (iv) dissect the contribution of HitRS signal transduction and KreA-mediated RNA regulation during phagocytosis and anthrax pathogenesis. Moreover, the results obtained from this study will provide new insights into TCS regulation, expand our knowledge of bacterial post- transcriptional regulatory networks, and lay the groundwork for developing novel antimicrobial therapeutics.