Oral Presentation BACPATH 2022

Deciphering the role of small RNAs in virulence regulation of hypervirulent Klebsiella pneumoniae using RIL-seq. (#13)

Kwok Jian Goh 1 2 , Liron Argaman 3 , Yael Altuvia 3 , Hanah Margalit 3 , Yunn-Hwen Gan 1 2
  1. Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, Singapore
  2. Infectious Diseases Translational Research Program, Yong Loo Lin School of Medicine, National University of Singapore, Singapore
  3. Department of Microbiology and Molecular Genetics, Institute for Medical Research Israel-Canada, Faculty of Medicine, The Hebrew University of Jerusalem, Jerusalem, Israel

Post-transcriptional regulation is mainly governed by small RNAs (sRNAs) through interactions with specific regions of bacterial mRNAs. These interactions are associated with the RNA chaperone protein Hfq, which stabilizes the sRNAs and provides the scaffold for their interactions with their targets. We apply the recently developed technique, RIL-seq (RNA interaction by ligation and sequencing), to hypervirulent Klebsiella pneumoniae str. SGH10 to interrogate the sRNA interactome. Through UV crosslinking and immunoprecipitation, RNAs on Hfq are treated and ligated to form chimeric RNA fragments, which can be identified by RNA sequencing and mapping to the genome. Chimeric fragments formed by RNAs encoded in different genomic loci represent potential functional interactions between RNAs. Our analysis has revealed over 8000 unique interacting RNA pairs. Approximately half of the interactions are found between sRNAs and the 5’-untranslated region (UTR) or coding sequence (CDS) of mRNAs. Interestingly, several interactions involve the mRNAs of virulent factors of K. pneumoniae, such as the rmp operon contributing to capsule hypermucoviscosity, the capsule locus, and siderophores. Analysis of our RIL-seq data also revealed several potential novel sRNAs. Interactions between iroN 5’-UTR and RyhB as well as ompR 5’-UTR and OmrB, have been validated in SGH10 using GFP reporter assays. In addition, we have found that sRNAs OmrB and CpxQ, which are known to regulate genes expressing outer membrane proteins, potentially interact with mRNAs of some of the virulent factors. These sRNAs induced significant phenotypic changes, such as changes to capsule and bile salt sensitivity upon overexpression in SGH10.  Ongoing work involves examining how these phenotypes are being modulated by specific sRNA-mRNA pairs to achieve fine-tuning of virulence in the bacteria.