Gram negative bacterial pathogens, such as Shigella flexneri, which possess a Type Three Secretion System (T3SS), are able to transfer bacterial proteins, dubbed translocators and effectors, from their cytoplasm into the cytoplasm of their host cells using a syringe like needle complex. For Shigella, it has been shown that during cellular invasion, the intrabacterial pool of translocators and effectors is completely depleted upon activation of the TTS Apparatus and is then progressively replenished while bacteria remain inside host cells. Replenishment of effectors allows for cell-to-cell spreading events, which also necessitate reactivation of the T3SA, and lead to another round of depletion of intrabacterial effector stores. To understand the state of individual intracellular bacteria during infection, it is therefore of interest to be able to locate and evaluate the relative quantity of the intrabacterial and secreted pool of translocators and effectors. We recently adapted a method based on EDTA and lysozyme to permeabilize the cell wall of bacteria present within host cells in order to label the intrabacterial pool of the tip protein IpaD and the translocators IpaB and IpaC. Herein, we describe in detail the protocol to perform the successive labeling of the intrabacterial and secreted pools. This method is theoretically extendable to virulence factors secreted by other secretion systems and other bacterial pathogens.
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