Analysis of the molecular mechanisms involved in a cytoplasmic purge of enterocytes
As the intestine represents a wide surface of exchange for nutrient uptake, it constantly faces challenges from the environment, and thus needs to efficiently deal with exposure to noxious chemical and biological agents that may contaminate the ingested food.
We have recently discovered that a cytoplasmic purge occurs in Drosophila enterocytes to alleviate the damage upon Serratia marcescens ingestion via the limited extrusion of their apical cytoplasm containing damaged organelles into the intestinal lumen through a large aperture (Lee KZ et al. Cell Host Microbe. 2016 Dec 14;20(6):716-730). This process includes a transient cell thinning (3h post infection) followed by a subsequent recovery (from 6h to 12h post infection). The bacterial pore-forming hemolysin is necessary and sufficient to induce the purge as: i) a Serratia marcescens hemolysin mutant fails to induce it; ii) an E. coli strain expressing the S. marcescens hemolysin does trigger it. The hemolysin-dependent cytoplasmic purge is evolutionarily conserved in the mouse intestine and in human intestinal cell lines (Caco-2 cells). It appears to be a protective response, since flies are more susceptible to the S. marcescens hemolysin mutant than to wild-type bacteria after oral infection.
This process can be divided in two phases: a thinning phase, followed by a recovery phase. We started to identify some factors involved in the recovery phase, such as cyclinJ that participates in a transcriptional response. However, the molecular mechanisms underlying the thinning phase remain uncharacterized to date. A candidate-based RNAi screen has been started in order to better understand this process, by targeting genes associated with cell death, cytoskeleton dynamics, vesicular trafficking.
The PhD student will first finish to test all the candidate genes selected for this screen. In parallel, the hits already identified will be confirmed and their relationships with the cytoskeleton dynamics will be studied thanks to fluorescent reporter lines. Various approaches will be used to better understand the function of the candidate genes in the gut, such as RT-qPCR, immunocytochemistry, western-blots, CrispR-Cas9 mutants.
Then, when the hits will be confirmed in Drosophila, the vertebrate homologs will be studied in human intestinal Caco2 cell cultures, to study their level of expression, their location and the inhibition of their function by RNAi and CrispR-Cas9.
This subject will allow understanding a fascinating cell biology process whereby a large amount of cytoplasm is expelled through a large aperture in a controlled manner without killing the enterocyte. The major asset of this study is the large palette of reporter and genetic tools available in the Drosophila model.
Contact: Dominique Ferrandon