Sunday, October 6, 2024
10/6/2024
Project Summary Streptococcus pneumoniae is a leading cause of pneumonia, acute otitis media (ear infection), and meningitis in the United States. S. pneumoniae remains an important pathogen despite a vaccine and effective antibiotic treatments due to increasing levels of antibiotic resistance and vaccine evasion through serotype switching. Virulence in S. pneumoniae is mediated by a complex gene regulatory program that is only partially understood. In many bacteria, trans-acting small non-coding RNAs (sRNAs) play key roles in regulating stress and virulence responses. While many sRNAs have been identified in S. pneumoniae via high-throughput sequencing studies, biological function has been assigned to only a few. The goal of this exploratory proposal is to identify the mRNA targets for sRNA candidates in S. pneumoniae, focusing specifically on those implicated in mediating virulence. We have two aims to reach this goal: First, we will determine the mRNA targets for a set of prioritized sRNA candidates implicated in S. pneumoniae virulence during mouse infections. Second, we will characterize the entire S. pneumoniae sRNA:mRNA interaction network. To achieve the first aim, we will use MS2-afinity purification coupled with RNA sequencing (MAPS) to identify mRNA targets for a set of prioritized sRNAs associated with fitness defects in mouse infections. MAPS tags each sRNA individually to allow specific capture of sRNA targets, but allows unbiased identification of potential mRNA partners via high-throughput sequencing of the captured RNAs. This approach will be combined with computational tools that predict the targets of sRNAs using thermodynamic properties. A set of 5- 10 of the most promising mRNA targets identified from the combined approaches will be assessed using reporter gene assays to validate the sRNA impact on gene expression. To achieve the second aim, we will use RIL-seq, a technique which captures mRNA:sRNA pairing through crosslinking followed by ligation. Subsequent sequencing identifies chimeric reads corresponding two interacting RNAs. To enrich for sRNA:mRNA pairs, a FLAG-tagged RNA-binding protein partner to immunoprecipitated the sRNAs. In S. pneumoniae, which lacks an RNA chaperone, we will use Cbf1, an exonuclease shown to process sRNAs, to enrich our population. Taken together, our two aims will identify mRNA targets for sRNA candidates associated with virulence, and move the field from cataloging sRNA presence toward identification of sRNA biological function, and thus therapeutic intervention.
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