During infection, pathogenic bacteria selectively activate or repress genes facilitating their survival in the host. The environmental input signals are channeled through regulatory networks utilizing transcriptional and post-transcriptional mechanisms. The effectors of post-transcriptional regulation are primarily non-coding small RNAs (sRNAs). Next generation sequencing can readily identify these sRNAs, however their regulatory targets are extremely difficult to predict. The goal of this proposal is to validate a new tool developed in our laboratory, based on proximity ligation of sRNAs to their targets, catalyzed by T4 RNA ligase. In addition to providing a research tool for the community, our long-term objectives are to define complete regulatory networks controlling P. aeruginosa virulence and establishing links between transcriptional and post- transcriptional components and regulated targets. The project is based on our previous work, identifying a comprehensive set of sRNAs in P. aeruginosa and subsequent assignment a role in virulence for a subset (23) of these regulators. First, we will identify regulatory targets of these sRNAs using the RNA proximity ligation method. In the second phase of the project, we will use a number of molecular tools to identify regulators of sRNA genes, allowing us to connect virulence-associated regulatory networks containing both transcriptional as well as sRNA-based modules. This project represents an early attempt to define function for the vast amount of different sRNAs and assign them a role in bacterial physiology and virulence.
: A number of non-?coding regulatory small RNAs (sRNAs) have been implicated as regulators of bacterial virulence. Although many of the known sRNAs have been shown to play a role during infections by the opportunistic pathogen Pseudomonas aeruginosa, their regulatory targets are largely unknown. In this project, we propose a comprehensive study aimed at defining the regulatory range of virulence?associated sRNAs, and enhance our understanding of the molecular mechanisms used by this organisms to become a successful human pathogen..
| Zhang, Yi-Fan; Han, Kook; Chandler, Courtney E et al. (2017) Probing the sRNA regulatory landscape of P. aeruginosa: post-transcriptional control of determinants of pathogenicity and antibiotic susceptibility. Mol Microbiol 106:919-937 |
| Han, Kook; Tjaden, Brian; Lory, Stephen (2016) GRIL-seq provides a method for identifying direct targets of bacterial small regulatory RNA by in vivo proximity ligation. Nat Microbiol 2:16239 |
