Deciphering host-pathogen interactions using an organotypic stretching device and biomechanical approaches

• Applicants: Dr.-Ing. Effie Bastounis, Ph.D.; Professorin Dr. Iordania Constantinou
• Subject Area: Biophysics ; Biomedical Systems Technology ; Medical Microbiology and Mycology, Hygiene, Molecular Infection Biology ; Cell Biology
• Term: since 2021
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 490839690

Investigating the dynamic interplay between host cells, their mechanical microenvironment, and their interaction with bacterial pathogens is critical for understanding mechanisms of virulence but also of host cell defense. In this proposal, we will investigate how biochemical and physical cues in concert orchestrate intracellular bacterial dissemination in epithelial cells in monolayer. Our model pathogen will be food-borne Listeria monocytogenes since in vivo its primary site of infection is epithelial cells lining the intestinal lumen, a critical step for achieving systemic dissemination. We will combine live-cell epifluorescence microscopy to characterize host cell and bacterial kinematics with measurements of cell mechanics via traction force microscopy, and Förster resonance energy transfer (FRET)-imaging to monitor the spatiotemporal dynamics of extracellular signal-regulated kinase (ERK). ERK has been implicated in innate immune responses to infection and can be activated when cells experience stretching, as in the intestine during peristalsis. Our preliminary data suggest that host cell ERK activation is significantly enhanced during infection, presenting distinct spatiotemporal features that culminate in the collective extrusion of infected cells out of the monolayer at late infection. Using kymographic representations and dimensionality reduction methods, we will quantify ERK dynamics and unravel its crosstalk with host cell mechanics during infection. To account for the role of extracellular physiologically relevant mechanical forces, we will develop EpiStrech, a multifunctional cell stretching platform, that will allow us to mimic peristalsis-like cellular stretching, similar to what epithelial cells are experiencing in the intestine. Our novel device will be compatible with high-resolution live-cell microscopy and conduction of biomechanical measurements and will be incrementally enhanced in complexity to account for additional relevant extracellular mechanical cues, like extracellular matrix stiffness and luminal shear flow. Using EpiStretch, we will explore for the first time in vitro how cyclic cellular stretching along different directions influences intracellular bacterial spread and collective extrusion of infected cells. Concurrently, we will determine alterations in host cell kinematics and dynamics and their crosstalk with cellular signaling, in particular, stretch-sensitive ERK activation. Through our interdisciplinary approach, we will uncover how novel virulence mechanisms work in complex biomechanical microenvironments and also use pathogens as tools to lend key insights into host epithelial cell mechanobiology. Finally, we envision that EpiStretch will find applications in the study of infection, using other host cell types and/or pathogens, and in investigating different types of cellular responses during stretching (e.g., cell differentiation, heterotypic interactions of epithelia with immune or cancer cells).

• DFG Programme: Research Grants