Poster Presentation BACPATH 2019

Complex multi-level control of hemolysin production by uropathogenic Escherichia coli (#205)

Nhu TK Nguyen 1 2 , Minh Duy Phan 1 2 , Brian M Forde 1 2 , Ambika MV Murthy 2 3 , Kate M Peters 1 2 , Christopher J Day 4 , Jessica Poole 4 , Timothy Kidd 1 2 , Rodney A Welch 5 , Michael P Jennings 4 , Glen C Ulett 6 , Matthew J Sweet 2 3 , Scott A Beatson 1 2 , Mark A Schembri 1 2
  1. School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, QLD, Australia
  2. Australian Infectious Diseases Research Centre, The University of Queensland, Brisbane, QLD, Australia
  3. Institute for Molecular Bioscience (IMB) and IMB Centre for Inflammation and Disease Research, The University of Queensland, Brisbane, Queensland, Australia
  4. Institute for Glycomics, Griffith University Gold Coast Campus, Gold Coast, Queensland, Australia
  5. Department of Medical Microbiology and Immunology, University of Wisconsin, School of Medicine and Public Health , Madison, WI, USA
  6. School of Medical Sciences, and Menzies Health Institute Queensland, Griffith University, Southport, Queensland, Australia

Uropathogenic Escherichia coli (UPEC) are the major cause of urinary tract infections. Nearly half of all UPEC strains secrete hemolysin, a cytotoxic pore-forming toxin. Here, we show that the prevalence of the hemolysin toxin gene (hlyA) is highly variable among the most common 83 E. coli sequence types represented on the EnteroBase genome database. To explore this diversity in the context of a defined monophyletic lineage, we contextualized sequence variation of the hlyCABD operon within the genealogy of the globally-disseminated multidrug-resistant ST131 clone. We show that sequence changes in hlyCABD and its newly defined 1.616-kb long leader sequence correspond to phylogenetic designation, and that ST131 strains with the strongest hemolytic activity belong to the most extensive multidrug resistance sublineage (clade C2). To define the set of genes involved in hemolysin production, the clade C2 strain S65EC was completely sequenced and subjected to a genome-wide screen by combining saturated transposon mutagenesis and transposon-directed insertion site sequencing with the capacity to lyse red blood cells. Using this unique approach, and subsequent targeted mutagenesis and complementation, 13 genes were confirmed to be specifically required for production of active hemolysin. New hemolysin controlling elements included discrete sets of genes involved in LPS inner-core biosynthesis (waaC, waaF, waaG, rfaE) and cytoplasmic chaperone activity (dnaK, dnaJ), and we show these are required for hemolysin secretion. Overall, this work provides a unique description of hemolysin sequence diversity in a single clonal lineage and describes a complex multi-level system of regulatory control for this important toxin.