Novel sRNA-mediated regulation of Rho action at a 3' untranslated region to affect mRNA stability - PROJECT SUMMARY Salmonella Typhimurium is a major foodborne pathogen that causes gastroenteritis and potentially lethal systemic infection. Colonization and invasion of the intestinal epithelium is dependent on the direct injection of effector proteins into host cells via a Type Three Secretion System (T3SS) encoded on Salmonella Pathogenicity Island 1 (SPI1). This critical system is controlled in response to numerous environmental and regulatory signals that dictate expression of the system at the proper time and place in the host. Our long-term goal is to understand the mechanisms of overall signal integration that allow this precise regulation. Our work has defined the SPI1 regulatory circuit. The three AraC-like regulators, HilD, HilC, and RtsA, act in a complex feed-forward regulatory loop to control expression of hilA, encoding the direct regulator of the SPI1 structural genes. Much of the regulatory input is integrated at the level of HilD, including at hilD mRNA translation or stability. The hilD mRNA has an unusual 300 nucleotide 3’ untranslated region (UTR) that acts as an independent module to confer instability to the mRNA. A primary hypothesis is that the hilD 3’ UTR serves as a critical node for integration of regulatory signals. Preliminary data show that mRNA stability is regulated by a novel mechanism involving interaction between Rho- mediated transcriptional termination at the 3’ UTR and RNA Degradosome-dependent degradation. Moreover, we have identified seven small RNAs (sRNAS) that bind to the hilD 3’ UTR to differentially control these factors. The first aim of this proposal is to understand the regulation of the identified sRNAS and how they affect the overall control of the SPI1 system. Expression of the sRNAs and confirmation of transcriptional regulators will be addressed using transcriptional lacZ fusions. Phenotypes conferred by deletion of the sRNA genes and/or their regulators, both in vitro and using the well-established animal model, will determine the specific roles of the sRNAs in SPI1 regulation. The second aim is to characterize the mechanism of post-transcriptional regulation via the hilD 3' UTR. Deletion analysis will identify the site of Rho action in the 3’ UTR. The roles of Rho, the RNA Degradosome, and the small RNAs in the creation and/or processing of the 3’ ends in the hilD 3’ UTR will be monitored using tagging and deep sequence analysis. In vitro transcription will more precisely define the action of Rho in creating terminated hilD transcripts. The interactions of these factors will reveal the mechanistic details of this novel regulation. The SP1 T3SS regulatory circuit serves as a paradigm for understanding the integration of host environmental signals to control a complex virulence phenotype and analysis of this system is critical to our understanding of this important pathogen.
