Poster Presentation Australian Society for Microbiology Annual Scientific Meeting 2024

Virulence plasmids of extra-intestinal pathogenic Escherichia coli (#49)

Zheng Jie Lian 1 2 3 , Minh-Duy Phan 1 2 3 , Nguyen Nhu 1 2 3 , Mark A. Schembri 1 2 3
  1. Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland, Australia
  2. School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, Queensland, Australia
  3. Australian Infectious Diseases Research Centre, The University of Queensland, Brisbane, Queensland, Australia

ColV-like virulence plasmids (ColVLPs) are large incompatibility F plasmids frequently isolated from avian and human extra-intestinal pathogenic Escherichia coli (ExPEC). ColVLPs encode virulence determinants involved in immune evasion and metal uptake and are strongly linked to increased zoonotic potential. Most ColVLP virulence genes have homologs located within genomic islands on the chromosome. Here, our bioinformatics analyses showed ColVLP-associated virulence genes and their chromosomal homologues are phylogenetically distinct, with no evidence of recombination. This striking observation suggests a divergent evolutionary pathway for the virulence genes on ColVLPs and their chromosomal counterparts. Furthermore, this also suggests that the virulence gene homologs on ColVLPs may confer gained or altered function with respect to their chromosomal homologs. To test this, we characterised OmpT, an outer membrane protease that promotes increased resistance to antimicrobial peptides (AMPs) and is encoded on both ColVLPs and the chromosome. Comparative analyses between OmpT homologs on the ColVLP (OmpTp) and the chromosome (OmpT1 and OmpT2) revealed ~73% amino acid identity between the ColVLP and chromosomal variants. All variants were confirmed to exhibit protease activity using a peptide derived from the C2 complement and the LL-37 AMP. Quantification of C2 peptide cleavage activity using a fluorescence-based assay revealed higher activity for OmpTp followed by OmpT1, but no detectable activity for OmpT2. However, all three OmpT homologs displayed activity against the LL-37 AMP based on mass spectrometry, highlighting distinct cleavage profiles for each OmpT homolog. Using a LL-37 AMP survival assay, we also demonstrated that OmpTp contributes additively (in combination with the chromosomal homologs) to LL-37 resistance, where maximal LL-37 resistance was observed with all three OmpT homologs present. These results reveal functional differences between ColVLP- and chromosomal-OmpT variants, a finding that suggests pathogenicity adapted changes may also alter the properties of other ColVLP cargo virulence genes, resulting in enhanced ExPEC virulence.