Project summary
The E. coli strain LF82 is a clinical isolate representative of adherent-invasive E. coli (AIEC), which are found much more frequently in individuals suffering from Crohn’s disease than in healthy individuals. Unlike other pathogens, AIEC do not possess any specific genetic markers, but they are distinguished by their ability to survive and replicate within macrophage phagolysosomes after invading host tissues. During phagocytosis, bacteria are exposed to various stresses, including genotoxic stress induced by the production of reactive oxygen species (ROS) by macrophages. These stresses activate the SOS response, a mechanism known to trigger prophage induction (the activation of latent viruses integrated into the bacterial genome). The LF82 strain contains five prophages, including Gally, which can be induced by genotoxic stress in vitro¹.
Our previous work revealed that Gally is induced in macrophages, but that its viral cycle is prematurely interrupted, suggesting that its activity is actively controlled by the bacterium¹. This phenomenon resembles that observed in Listeria monocytogenes, although the underlying mechanisms differ²˒³. Transcriptomic data suggest that the Gally prophage is blocked at the stage of Q antiterminator synthesis.
The aim of this internship will be to confirm, through in vitro genetic experiments and macrophage infection experiments, that the induction blockade is indeed due to inhibition of Q synthesis, and to investigate which phage transcription factors are involved in this regulation. The phenotypes observed in macrophages will be directly analyzed using fluorescent reporter constructs monitoring Gally prophage activity.
The intern will learn microbiology and molecular biology techniques for the cloning and genetic deletions required for the project, as well as for the genetic manipulation of the Gally phage and the E. coli LF82 strain. They will also receive training in cell culture and fluorescence microscopy using the THP-1 macrophage model.
Informations
Micalis contact: Jeffrey Cornuault :
Team: Dynphages
Starting period: January 2027
References
- Misson, P., Bruder, E., Cornuault, J.K., Paepe, M.D., Nicolas, P., Demarre, G., Lakisic, G., Petit, M.-A., Espeli, O., and Lecointe, F. (2023). Phage production is blocked in the adherent-invasive Escherichia coli LF82 upon macrophage infection. PLOS Pathogens 19, e1011127. https://doi.org/10.1371/journal.ppat.1011127.
- Pasechnek, A., Rabinovich, L., Stadnyuk, O., Azulay, G., Mioduser, J., Argov, T., Borovok, I., Sigal, N., and Herskovits, A.A. (2020). Active Lysogeny in Listeria Monocytogenes Is a Bacteria-Phage Adaptive Response in the Mammalian Environment. Cell Reports 32. https://doi.org/10.1016/j.celrep.2020.107956.
- Azulay, G., Pasechnek, A., Stadnyuk, O., Ran-Sapir, S., Fleisacher, A.M., Borovok, I., Sigal, N., and Herskovits, A.A. (2022). A dual-function phage regulator controls the response of cohabiting phage elements via regulation of the bacterial SOS response. Cell Reports 39. https://doi.org/10.1016/j.celrep.2022.110723.
