EVs from C. jejuni CDT intoxicated-Caco-2 cells differently inhibit proliferation in tumour intestinal epithelial and myeloid cells: potential utility for antitumor strategies

Campylobacter jejuni is a Gram-negative spiral-shaped bacterium that is the most prevalent cause of human’s gastroenteritis, responsible for over 500 million cases of gastroenteritis in the world over each year. The Cytolethal Distending Toxin (CDT) represents an important virulence factor, it is a heterotrimeric complex composed of CdtA, CdtB, and CdtC. CdtA and CdtC constitute the regulatory subunits and CdtB the catalytic subunit exhibiting phosphatase and DNase activities, resulting in cell cycle arrest and cell death. Moreover, an important complication of C. jejuni enteritis is Guillain Barré syndrome (GBS), an acute autoimmune inflammatory demyelinating polyneuropathy. Macrophages have been implicated in both initiation and resolution of Experimental Autoimmune Neuritis (EAN), the animal model of GBS. Effective functioning of these cells requires intercellular communication between myeloid cells, primary epithelial infected cells, and other types of immune cells. Mediators of intercellular communication are extracellular vesicles (EVs). They play important roles in normal cell-to-cell communications but can also spread pathogen- and host-derived molecules during infections to alter immune responses. The thesis focuses on multiple aspects: 1. The effects of CDT on U937 myeloid cells, drawing the involvement of lysosomal and endosomal compartment, ER-remodelling and mitochondria network. Rapamycin, an efficient inhibitor of the mammalian target of rapamycin (mTOR), acts as a mitigating factor of the intoxication. 2. Mitochondrial modifications, deep changes in lysosomal exocytosis, secretory autophagy and EV release were registered in intestinal epithelial Caco-2 cells, particularly induced by the wild-type strain, compared to the mutant strain. Caco-2 cells respond to stimuli by activating the crosstalk between the autophagic and the endosomal system, largely involved in bacterial infections. Through the co-culture model between Caco-2 and U937, we deduce that CDT-like effects are transferred by Caco-2 cells to uninfected U937 cells. 3. The role of EVs from C. jejuni Caco-2 infected cells in modulating the myeloid response during CDT treatment (mimicking the in vivo cellular interactions, possibly leading to GBS). Indeed, EVs represent a mechanism to spread the active CDT on both myeloid and intestinal cells revealing that in their cargo the active toxin is still present. Since CDT is an antiproliferative factor, these EVs act as antitumor agents, in our in vitro model. In summary, CDT targets endo-lysosomal compartment, partially eluding lysosomal degradation and exploiting unconventional secretion (EV release). This scenario is observed in both intestinal epithelial and myeloid cells. EVs are the primary routes to spread the efficient CDT among homologous and heterologous cells, and, due to the typical arrest of proliferation, their possible use is envisaged in anticancer strategies, on which engineered CDTs are recently considered for targeted therapies.