Oral Presentation The Prato Conference on the Pathogenesis of Bacterial Infections of Animals 2016

Inactivation of the Pasteurella multocida hfq results in altered expression of multiple virulence factors and reduced bacterial in vivo fitness (#2)

Marianne Mégroz 1 , Emily Gulliver 1 , Amy Wright 1 , Oded Kleifeld 2 , David Powell 3 , Paul Harrison 3 , Adele Barugahare 3 , Ben Adler 1 , Marina Harper 1 , John Boyce 1
  1. Microbiology, Monash University, Melbourne, VIC, Australia
  2. Biochemistry and Molecular Biology, Monash University, Melbourne, VIC, Australia
  3. Monash Bioinformatics Platform, Monash University, Monash, Victoria, Australia

Pasteurella multocida is a Gram-negative bacterium that causes a number of economically important animal diseases. Numerous P. multocida virulence factors have been identified, including capsule, lipopolysaccharide (LPS) and filamentous hemagglutinin, but little is known about how expression of these factors is regulated. Hfq is an RNA-binding protein that facilitates the interaction of small noncoding regulatory RNA molecules (sRNAs) with their mRNA targets. To determine the importance of sRNA regulation in P. multocida, we constructed a P. multocida hfq mutant. The hfq mutant showed reduced virulence in both mice and chickens; transcriptional and proteomic analyses identified >125 genes and >75 proteins as differentially expressed. The transcript and protein levels of genes/proteins involved in capsule biosynthesis were reduced in the hfq mutant, as were the levels of the filamentous hemagglutinin protein PfhB2 and its secretion partner LspB2. In contrast, the expression of some LPS biosynthesis genes was increased, suggesting that these are negatively regulated by Hfq-dependent mechanisms. These data provide the first evidence that Hfq and sRNAs play a crucial role in regulating P. multocida virulence. In order to identify the important regulatory sRNAs, we used two complementary approaches: transcriptomic analyses of P. multocida grown under a range of conditions and co-immunoprecipitation of Hfq and interacting sRNAs. These analyses identified 20 putative sRNAs; we have now inactivated a number of these, including one with high identity to the E. coli/Salmonella GcvB sRNA. Proteomic analyses of the gcvB mutant identified 25 proteins as differentially produced; 24 of these were predicted to be involved in amino acid biosynthesis and transport. The gene sequences of each GcvB target were compared and a consensus sequence of 5’-ACACAACA-3’ identified. Therefore, P. multocida GcvB acts to decrease production of amino acid biosynthesis and transport proteins via complementary base pairing to a highly conserved seven-nucleotide sequence on target mRNAs.