Abstract
Pathogenic bacteria induce eukaryotic cell damage which range from discrete modifications of signalling pathways, to morphological alterations and even to cell death. Accurate quantitative detection of these events is necessary for studying host-pathogen interactions and for developing strategies to protect host organisms from bacterial infections. Investigation of morphological changes is cumbersome and not adapted to high-throughput and kinetics measurements. Here, we describe a simple and cost-effective method based on automated analysis of live cells with stained nuclei, which allows real-time quantification of bacteria-induced eukaryotic cell damage at single-cell resolution. We demonstrate that this automated high-throughput microscopy approach permits screening of libraries composed of interference-RNA, bacterial strains, antibodies and chemical compounds in ex vivo infection settings. The use of fluorescently-labelled bacteria enables the concomitant detection of changes in bacterial growth. Using this method named CLIQ-BID (Cell Live Imaging Quantification of Bacteria Induced Damage), we were able to distinguish the virulence profiles of different pathogenic bacterial species and clinical strains.
Highlights
Bacterial toxins targeting eukaryotic cells can either directly affect plasma membrane integrity or alternatively they may be internalized, translocated or injected inside the cells
The injection of the exotoxins ExoS, T, Y and ExoU by the T3SS machinery is one of the main virulence determinants of P. aeruginosa clinical strains[7]. Those toxins have profound effects on eukaryotic cell biology, provoking plasma membrane disruption or inhibition of phagocytosis followed by a delayed apoptosis[8]
In the search for robust descriptors of this phenomenon, we observed that the Hoechst-stained nuclei of Human Umbilical Vascular Endothelial Cells (HUVECs) become gradually smaller and brighter during incubation with the wild-type P. aeruginosa strain PAO1 harbouring ExoS and ExoT
Summary
Bacterial toxins targeting eukaryotic cells can either directly affect plasma membrane integrity or alternatively they may be internalized, translocated or injected inside the cells. The analysis of early events such as the morphological changes induced by cytoskeleton rearrangements are usually based on fixed and stained cells, rendering fine kinetics studies laborious, or on expression of fluorescent chimeric markers, a time-consuming procedure to which some cells are refractory[6]. These approaches are not accessible to non-expert scientists. We present the CLIQ-BID method, based on automated high-throughput monitoring of the fluorescence intensity of eukaryotic cell nuclei stained with vital-Hoechst This live-imaging method permits real-time quantification of bacteria-induced cell damage at single-cell resolution. The CLIQ-BID method could be used in other cytotoxicity and cell-stress studies
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