Oral Presentation Australian Society for Microbiology Annual Scientific Meeting 2024

Developing a Novel Tool of Pseudomonas aeruginosa Adhesion to Endothelial Cells Under Shear Stress (104183)

Sarah M Hickson 1 , Von L Torres 2 , Kate L McCarthy 3 4 , Timothy J Wells 1
  1. Frazer Institute, University of Queensland, Brisbane, QLD, Australia
  2. Institute of Molecular Bioscience, University of Queensland, Brisbane, QLD, Australia
  3. Department of Microbiology, Pathology Queensland, Brisbane, QLD, Australia
  4. Infectious Diseases Unit, Royal Brisbane and Women's Hospital, Brisbane, QLD, Australia

Introduction

Although rare, Pseudomonas aeruginosa infective endocarditis (IE) is associated with high mortality rates and need for surgical intervention. Few studies have explored P. aeruginosa IE pathogenesis, but initial adhesion is known to be vital in the establishment of infection for other bacterial species. Current models of adhesion involve incubation of bacteria with endothelial cells under static conditions. However in vivo, these cells are exposed to high blood flow rates. Under this high shear stress environment, cells undergo morphological changes, exposing epitopes that can promote bacterial adhesion. We have developed a novel tool to investigate P. aeruginosa adhesion to endothelial cells under shear stress, mimicking blood flow in the cardiac environment. Factors important for P. aeruginosa adhesion are currently being investigated, including type IV pili mutants.

Methods

Initially to characterise these novel isolates, P. aeruginosa from patients with IE (n = 6) were sequenced, genomically characterised and the proteome was analysed after growth in nutrient-rich media and human blood. In the model of adhesion under shear stress, P. aeruginosa coated in serum proteins is perfused (~2.77 dynes/cm2) through a treated flow chamber slide, seeded with endothelial cell line EA.hy926.

Results

In comparison to other clinical P. aeruginosa isolates and well-characterised laboratory strains (PAO1 and PA14), no genomic signatures were identified amongst IE clinical isolates. Proteome analysis of bacterial isolates grown in nutrient-rich media indicates a significant upregulation of phenazine synthesis in IE isolates compared to PAO1. Two laboratory strains of P. aeruginosa and five clinical IE isolates were tested for adhesion under both static and shear stress conditions. Under static conditions adhesion was low (<5%) for all strains. In contrast, under shear stress, adhesion of the strains was between 6-70%. The pilY1 gene, encoding type IV pili tip adhesin, was mutated in P. aeruginosa PA14 via CRISPR base-editing techniques. A decrease in adhesion was observed between wild-type and mutant pilY1 strains.

Conclusion

This model of adhesion under shear stress is more biologically relevant to investigate P. aeruginosa adhesion and IE establishment. In future, this model of adhesion can be translated for other cell types and bacterial species.