Unravelling host-bacterial interactions using tissue microbiology approaches

To fully grasp the complexity of host-pathogen interactions, there is a pressing need for more advanced and comprehensive infection models. Infections can occur at various sites within the body, each with its own unique microenvironment. Dynamic factors, such as shear stress and desiccation, are increasingly recognised as critical in host-pathogen interactions, particularly in the context of adhesion. Understanding the tissue structure and the host's antimicrobial response within the infection microenvironment is essential for comprehending how pathogens colonise and adapt to infectious niches.

While in vivo models can somewhat replicate this complexity, they are often technically demanding and time-consuming. Conversely, simple in vitro models, do not always recapitulate the complex conditions found in host tissues. In this thesis, we utilise Tissue Microbiology approaches to strike a balance between complexity and throughput, allowing us to explore the infectious mechanisms of two distinct bacterial infections: urinary tract infections caused by Uropathogenic Escherichia coli (UPEC), and skin infections caused by Staphylococcus.

In Paper I, we investigated the initial attachment and development of microcolonies of UPEC using a proximal-tubule-on-chip (PToC) model. This model replicated a key microenvironmental factor, the shear stress of urine flow. We examined the potential role of biofilm in microcolony formation using a novel, non- toxic biofilm extracellular matrix (ECM) tracer, Optotracer EbbaBiolight 680 (Ebba680), in a real-time imaging system. Our findings revealed a synergistic effect between UPEC adhesion factors, P fimbriae facilitated the initial attachment to the renal epithelium, while type 1 fimbriae promoted intercellular binding and the development of the three-dimensional microcolonies. Notably, we observed no biofilm ECM in the early stages of microcolony development.

The need to study live biofilm development in real-time over extended periods led us to establish a novel agar model, the Ebba680-biofilm assay, in Papers II, III. This versatile model allowed us to observe UPEC biofilm development with temporal and spatial resolution in detail, and to quantify ECM expression. We identified UPEC strain No. 12 (UPEC12) as a capable ECM producer at both ambient and physiological temperatures. We see that UPEC12 began producing ECM curli after approximately 14 h in our model. Additionally, we employed two-photon imaging to visualise live macrocolony biofilms on agar in 3D. This approach revealed structural heterogeneity and varying ECM expression within the biofilm across the x, y, and z axis. To explore the potential roles of ECM production, we applied a wettability test, a technique commonly used in materials science. This revealed that it was the nanoscale interactions between ECM components, rather than the macrostructure of the biofilm, that contributed to the increased hydrophobicity of the macrocolony surface.

In Paper IV, we examined the attachment and colonisation of Staphylococcus species on human skin using both ex vivo and in vitro skin models. Our findings indicated that the attachment capability of different staphylococci does not necessarily correlate with their ability to colonise and grow on human skin. We demonstrated that S. aureus and S. epidermidis have a significantly higher affinity for binding to the stratum corneum, the topmost layer of human skin, compared to S. lugdunensis, a strain that shows potential in inhibiting S. aureus growth. Using confocal imaging, we identified that the primary binding sites of staphylococci are located at the edges of corneocytes, the predominant cell type in the stratum corneum. In our search for the adherence mechanisms of staphylococci, we began screening for potential binding targets expressed in this biological niche.

List of scientific papers

I. Antypas, H., Zhang, T., Choong, F. X., Melican, K .* & Richter-Dahlfors, A .* Dynamic single cell analysis in a proximal-tubule-on-chip reveals heterogeneous epithelial colonisation strategies of uropathogenic Escherichia coli under shear stress. FEMS Microbes. 4, xtad007 (2023). *Corresponding author
https://doi.org/10.1093/femsmc/xtad007

II. Choong, F. X., Huzell, S., Rosenberg, M., Eckert, J. A., Nagaraj, M., Zhang, T., Melican, K., Otzen, D. E. & Richter-Dahlfors, A .* A semi high- throughput method for real-time monitoring of curli producing Salmonella biofilms on air-solid interfaces. Biofilm. 3, 100060 (2021). *Corresponding author
https://doi.org/10.1016/j.bioflm.2021.100060

III. Zhang, T., Ray, S., Melican, K .* & Richter-Dahlfors, A .* The maturation of native uropathogenic Escherichia coli biofilms seen through a non-interventional lens. Biofilm. 8, 100212 (2024). *Corresponding author
https://doi.org/10.1016/j.bioflm.2024.100212

IV. Zhang, T., Luo, R. & Melican, K .* Staphylococcal adhesion dynamics on human skin stratum corneum. *Corresponding author [Manuscript]