Multi-reporter: fluorescent reporter for temporal detection of cell death in infection

Most microbial pathogens, including bacteria, fungi, and viruses, can lead to the death of their host cells. Host cells, however, die in a multitude of different ways. Some are inflammatory, such as pyroptosis and necroptosis, and some are not, such as apoptosis. The currently available tools for cell stress and death detection are limited and cannot easily distinguish between the diverse cellular events leading to cell death.

Therefore, we are creating fluorescent reporters for cellular stress and different cell death pathways such as apoptosis, pyroptosis or necroptosis, which allow real-time detection by fluorescent microscopy. Cell lines are created via lentiviral transduction, which also allows multiple transductions into a single cell line to create multi-pathway reporters. Cell lines include THP-1 macrophages and C2BBe1 intestinal epithelial cells. We have created fluorescent reporters for redox status (stress), using a roGFP2 fluorophore, and apoptosis (cell death), using a bimolecular fluorescence complementation (BiFC) reporter for live detection of caspase 3/7 activation. Another fluorescent reporter for live detection of pyroptosis, via visualization of Gasdermin-D oligomerization, is being validated. As all reporters work with non-overlapping spectra, we aim to create a single "multi-reporter" cell line, which will enable us to detect simultaneously different types of cell stress and death pathways.

This reporter system will allow to monitor cellular stress and to differentiate the different host cell death pathways that are active during infection. Importantly, it will be possible to follow the state of the host cells in a temporally and spatially resolved manner. Our new reporter will thereby help to answer many of the open questions on host-pathogen interactions during infection.