Disentangling the role of shear flow in regulating biomechanical interactions of endothelial cells with intracellular bacterial pathogens

• Applicant: Dr.-Ing. Effie Bastounis, Ph.D.
• Subject Area: Biophysics ; Cell Biology
• Term: since 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 532757564

Endothelia cells (ECs) lining the inner lumen of blood vessels are constantly exposed to shear stresses (SSs) due to blood flowing over their surfaces. While the effects of fluid SSs on regulating different aspects of EC behavior and contributing to pathologies (e.g., atherosclerosis) have been previously studied, little is known about how fluid SSs modulate the interactions of ECs with intracellular bacterial pathogens like Listeria monocytogenes (LM). The primary site of infection by food-borne LM is the intestinal epithelium. However, in vivo LM can manage to spread to distant tissues by bypassing ECs and/or infecting them, a critical step to breach important barriers, such as the blood-brain, and systemically disseminate causing lethal fatalities. In this proposal, we will investigate how fluid SSs modulate host EC biomechanics, and in turn adhesion, invasion and intercellular spread of LM using an in vitro organotypic model. Taking advantage of this model, which is compatible with time-lapse microscopy, infection assays and parallel conduction of biomechanical measurements like traction force and monolayer stress microscopy, we will first explore how varying levels of fluid SSs modulate the susceptibility of ECs to bacterial infection. We will also investigate the underlying molecular mechanisms taking advantage of a recent transcriptomic screen that revealed key differentially expressed genes between flow exposed ECs as compared to ECs under stationary conditions. Subsequently, we will determine how intracellular bacterial infection alters EC biomechanics, how those alterations are modulated by fluid SSs, and whether SS exposure ultimately acts protectively benefiting the host by obstructing bacterial dissemination, or conversely benefits the bacteria by enhancing intercellular bacterial spread. Through the unique experimental setup in conjunction with biomechanical measurements and precise spatiotemporal quantitation of the infection process, we will be able to uncover both important pathogenesis and host defense mechanisms in a context where the important role of the hemodynamic forces is considered. Moreover, by using the specific pathogen LM as a tool to subtly modulate EC biomechanics (since LM does not destroy host cell integrity because it relies on it), we anticipate revealing novel aspects of vascular EC mechanobiology.

• DFG Programme: Research Grants