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In vitro reconstitution of TTSS-mediated export across the bacterial inner membrane of Shigella flexneri


Shigella flexneri, a Gram-negative enterobacterium, causes dysentery in humans. It uses a type III secretion system (TTSS) to infect its host. The TTSS is a large protein complex that spans both bacterial membranes and the host plasma membrane. It mediates the apparent direct translocation of proteins from the bacterium through its central channel into the host cell. Therefore, unravelling the molecular mechanisms by which the TTSS functions is key to understanding how it mediates this host-pathogen interac tion. For this purpose, we aim to reconstitute in vitro its sole energy-dependent step, i.e. protein export across the bacterial inner membrane (IM). In vitro reconstitution allows us to study features of the protein export reaction that would otherwise no t be accessible, e. g. analysing the separate steps that encompasses the passage of the substrate through the IM channel.First we will analyse the in vivo properties of protein export by the IM domain, using a Shigella strain that lacks the periplasmic/ou ter membrane region of the TTSS. In this strain, the export of MxiHCdelta5, a non-polymerising mutant of the needle protein MxiH, will be analysed. Then, the components of the IM domain of the TTSS will be overproduced and inner membrane vesicles (IMVs) ha rbouring the overproduced IM domain will be isolated. Protein export assays will be conducted in vitro by the addition of the cytoplasmic TTSS components and the substrate MxiHCdelta5. These results will be compared to the data acquired in the in vivo expo rt assay. These experiments will greatly enhance our understanding of TTSS function. Many elements of TTSSs are evolutionary conserved, in particular the IM domain, thus our work should also help understand how the TTSSs in bacteria such as Chlamydia, Salm onella and Pseudomonas species, all important human, animal and/or plant pathogens, interact with the cells of their host. This should aid in the design of a new range of broad-spectrum antimicrobials.

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