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Intracellular pathogens include all viruses, many bacteria and parasites capable of invading and surviving within host cells. Key to survival is the subversion of host cell pathways by the pathogen for the purpose of propagation and evading the immune system. The intracellular bacterium Shigella flexneri, the causative agent of bacillary dysentery, invades host cells in a vacuole that is subsequently ruptured to allow growth of the pathogen within the host cytoplasm. S. flexneri invasion has been classically described as a macropinocytosis-like process, however the underlying details and the role of macropinosomes in the intracellular bacterial lifestyle have remained elusive. We applied dynamic imaging and advanced large volume correlative light electron microscopy (CLEM) to study the highly transient events of S. flexneri’s early invasion into host epithelial cells and elucidate some of its fundamental features. First, we demonstrate a clear distinction between two compartments formed during the first step of invasion: the bacterial containing vacuole and surrounding macropinosomes, often considered identical. Next, we report a functional link between macropinosomes and the process of vacuolar rupture, demonstrating that rupture timing is dependent on the availability of macropinosomes as well as the activity of the small GTPase Rab11 recruited directly to macropinosomes. We go on to reveal that the bacterial containing vacuole and macropinosomes come into direct contact at the onset of vacuolar rupture. Finally, we demonstrate that S. flexneri does not subvert pre-existing host endocytic vesicles during the invasion steps leading to vacuolar rupture, and propose that macropinosomes are the major compartment involved in these events. These results provide the basis for a new model of the early steps of S. flexneri epithelial cell invasion, establishing a different view of the enigmatic process of cytoplasmic access by invasive bacterial pathogens. [ABSTRACT FROM AUTHOR]

Intracellular bacterial pathogens have evolved a plethora of strategies to invade eukaryotic cells. By manipulating host signaling pathways, in particular vesicular trafficking, these microbes subvert host functions to promote their internalization and to establish an intracellular niche. During these events, host endomembrane compartments are dynamically reorganized. Shigella flexneri, the causative agent of bacillary dysentery, recruits components of the host recycling pathway and the exocyst of non-phagocytic enterocytes in the vicinity of its entry site to facilitate its access to the host cytosol. These factors are either dynamically tethered to in situ formed macropinosomes or to the bacteria-containing vacuole itself. The underlying interactions cannot readily be monitored as individual bacterial infection events take place without synchronicity using cellular infection models. Therefore, time-resolved screens by fluorescence microscopy represent a powerful tool for the study of host subversion. Such screens can be performed with libraries of fluorescently tagged host factors. Using the cytosolic pathogenic agent Shigella flexneri as a model, we provide detailed protocols for such medium-to-high throughput multidimensional imaging screening of the dynamic host-pathogen cross talk. Our workflow is designed to be easily adapted for the study of different host factor libraries and different pathogen models., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Small molecule-based fluorescent probes offer great opportunities for specifically tracking proteins in living systems with minimal perturbation on the protein function and localization. Herein, we report a small green fluorescent probe (Ni 2+ - NTA-AF) consisting of a Ni 2+ -NTA moiety, a fluorescein, and an arylazide group, that binds specifically to His 6 -tagged proteins with fluorescence enhancement in vitro upon photoactivation of the arylazide group. Importantly, the probe can cross the cell membranes and stoichiometrically label His 6 -tagged proteins rapidly (∼15 min) in living prokaryotic and eukaryotic cells exemplified by a DNA repair protein Xeroderma pigmentosum group A (XPA). Using the probe, we successfully visualized Sirtuin 5, which is localized to the mitochondria. This probe exhibits high quantum yields and improved solubility, offering a new opportunity for imaging intracellular His 6 -tagged proteins inside living cells with better contrast.