Life Sciences Faculties

Shigella flexneri is a bacterial entero-invasive pathogen transmitted through the fecal/oral route causing bacillary dysentery in humans. Shigella pathogenicity solely relies on a needle-like molecular syringe, the Type 3 Secretion System (T3SS) that injects more than 20 bacterial effectors to infect colonic epithelial cells. The T3SS is composed of a basal body that controls and initiates effector secretion and a needle complex that acts as a conduit for effector delivery. The needle is capped by a tip complex that regulates whether the needle is closed or whether it secretes. Sensing of host cells by the needle tip complex induces a conformational switch that remodels the tip and activates the T3SS to form a channel, the translocon pore at the distal end. Effectors are then actively secreted, promoting cell invasion and endocytosis of the bacteria in a tight vacuole derived from the host plasma membrane called Bacteria Containing Vacuole (BCV). Quickly after entry, the pathogen ruptures its BCV and establish a replicative cytosolic niche. Vacuolar rupture consists of a first step of BCV breakage followed by BCV remnants unpeeling. The team has identified bacterial effectors promoting efficient vacuole unpeeling but the direct role of the T3SS in membrane destabilization is not clear. I have overcome these limitations by investigating the T3SS/vacuole interactions at the onset of vacuolar rupture using a novel cryo-Correlative Light Electron Microscopy (CLEM) workflow applied in situ, during the host-pathogen crosstalk. Cryo-CLEM allows the combination of high-resolution information in 3D, accessed via cryo-Electron Tomography (cryo-ET) to functional information brought by light microscopy. This pipeline benefits from in-house custom-built genetically encoded reporter cell lines which are used to identify precise steps of the infection at high spatiotemporal resolution.
Using this workflow, I collected cryo-ET data on Shigella-infected epithelial cells. I have been able to visualize the Shigella T3SS at molecular resolution providing unprecedented information. Particularly, I am looking at (i) the contact sites between T3SS and BCV membrane; (ii) T3SS morphologies depending on its activation state. Together this work will allow to precisely describe the interplay between host and bacteria processes.