Oral Presentation Australian Society for Microbiology Annual Scientific Meeting 2018

Identification of Pasteurella multocida small RNAs: unravelling the regulatory network (#14)

Marianne N Mégroz 1 , Emily Gulliver 1 , Brandon Sy 2 , Julia Wong 2 , David Powell 3 , Adele Barugahare 3 , Jai J Tree 2 , Marina Harper 1 , John D Boyce 1
  1. Infection and Immunity Program, Monash Biomedicine Discovery Institute and Department of Microbiology, Monash University , Clayton, Victoria, Australia
  2. School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, NSW, Australia
  3. Monash Bioinformatics Platform, Monash University, Melbourne, VIC, Australia

The Gram-negative bacterium Pasteurella multocida is the causative agent of a number of economically important animal diseases, including avian fowl cholera. Numerous P. multocida virulence factors have been identified, including capsule, lipopolysaccharide (LPS) and filamentous hemagglutinin, but little is known about how the expression of these virulence factors is regulated. Small non-coding RNA molecules (sRNAs) are important regulators of bacterial gene expression and protein production, with essential roles in controlling diverse bacterial functions including virulence. Hfq is an RNA-binding protein that facilitates riboregulation via interaction with small noncoding RNA molecules (sRNAs) and their mRNA targets. A P. multocida hfq mutant produced significantly less hyaluronic acid capsule during all growth phases and displayed reduced in vivo fitness compared with the wild-type strain. The hfq mutant also displayed global changes in gene expression and protein production, including altered expression of the key P. multocida virulence factors, capsule, filamentous hemagglutinin and LPS. These data indicate that Hfq and associated sRNAs are likely to be critical regulators of P. multocida virulence. To further examine sRNA-mediated riboregulation in P. multocida, putative trans-encoded sRNAs were identified using a combination of bioinformatics, RNA-Seq, Hfq co-immunoprecipitation, Hfq UVcrosslinking and analysis of cDNA (Hfq-CRAC) and Hfq UV-crosslinking, ligation and sequencing of hybrids (Hfq-CLASH) experiments. These combined analyses identified more than 50 putative sRNAs; most were encoded in intergenic regions, highly expressed, and contained putative Rho-independent terminators. A number of these putative sRNAs were differentially expressed under specific conditions, such as growth in low-iron medium or under anaerobic conditions, suggesting that they may have roles in regulating global responses to these conditions. Finally, the Hfq co-immunoprecipitation, Hfq-CRAC and Hfq-CLASH experiments define the global set of sRNA and mRNA interaction sites. Twenty Hfq-associated hybrids were identified using Hfq-CLASH, including hfq and one sRNA. A number of putative sRNAs have been selected for experimental validation and detailed functional characterization.