A genetic magnetic-extraction-proteomics (gMEP) workflow to understand the link between Shigella vacuolar rupture and autophagy – PureMagRupture
Intracellular bacterial pathogens enter host cells to form a bacterial containing vacuole (BCV). Then, the BCV matures or ruptures leading to two distinct pathogen-dependent intracellular niches; one vacuolar and one cytosolic. The specific intracellular niche is intimately linked with the bacterial capacity to grow and to evade antimicrobial pressure for bacterial persistence. Shigella is a model for BCV-rupturing pathogens and the causative agent of bacterial dysentery with a huge public health burden at the global level. Nevertheless, the molecular processes governing the intracellular niche formation of Shigella through BCV rupture have remained enigmatic. Here, we develop a genetic magnetic-extraction-proteomics workflow to obtain comprehensive insights into the molecular processes subverted for Shigella BCV rupture. To achieve this, we have already generated magnetic Shigella that can now be used to purify the bacterial containing compartments at successive invasion stages from enterocytes. The BCVs obtained through this workflow will be analyzed for the proteome, post-translational modifications and lipidome. Main regulators will be analyzed biochemically and cell biologically through approaches developed and regularly used by the PureMagRupture partners. In parallel, we have already obtained data on candidate genes that determine the intracellular niche of Shigella, such as the autophagy pathway and membrane trafficking regulators. These candidates will also be fully characterized through the novel workflows established with this project. Together, the PureMagRupture project will provide comprehensive insights into BCV rupture. Our data and newly developed methods will also be relevant for the understanding of other pathogens that access the host cytosol, as well as it will provide general insights on the accessibility of the cytosol of metazoan cells.
Madame Mariette MATONDO (IP-Plate-Forme Technologique Protéomique)
The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.
IP-Plate-Forme Technologique Protéomique IP-Plate-Forme Technologique Protéomique
PETITES GTPASES, SIGNALISATION CALCIQUE ET PATHOGENES
I2BC Institut de Biologie intégrative de la cellule
IP-Unité de Dynamique des interactions hôte-pathogène IP-Unité de Dynamique des interactions hôte-pathogène
Help of the ANR 762,949 euros
Beginning and duration of the scientific project: September 2021 - 36 Months