| Resumo: | Fish photobacteriose is a bacterial systemic and deadly infection with rapid course and very high mortalities, caused by the Photobacterium damselae subsp. piscicida (Phdp). Phdp spreads through the bloodstream and secretes the apoptogenic exotoxin AIP56 allowing the pathogen to avoid phagocytosis by inducing the apoptotic death of the host phagocytes. Although AIP56 was found to be a key virulence factor of Phdp and the AIP56-related pathology has been well characterized, the molecular targets of the toxin and the molecular pathways it affects/modulates remain to be disclosed. Recent data revealed that AIP56 is an AB-toxin, possessing an N-terminal metalloprotease domain (A domain) responsible for the catalytic activity of the toxin and a C-terminal domain (B domain) involved in the binding/internalization of the toxin into the cells. The N-terminal domain of AIP56 is homologous to the non-LEE encoded effector C (NleC), a type III secreted effector of enteric pathogenic bacteria that cleaves and inactivates the p65 of NF-kB. However, it remains to be investigated if AIP56 also targets NF-kB p65 and if NF-kB p65 inactivation by AIP56 is linked to the apoptogenic activity of the toxin. So far, AIP56 activity has been being studied using an ex vivo model consisting of freshly isolated sea bass peritoneal leukocytes because although the susceptibility of several mammalian cell lines to AIP56 has been assessed, no apoptosis was observed in any of the cell lines tested. This specificity of the toxin may result from the existence of receptors for AIP56 in phagocytes of susceptible hosts and their absence in mammalian cells and suggests that if AIP56 was able to enter mammalian cells, it would be able to induce apoptosis in those cells. The existence of a mammalian cell model for studying in detail the AIP56 activity would be very advantageous, because there is much more availability of tools to study mammalian cells than sea bass cells. In this work, we tested and optimized different protocols for the intracellular delivery of AIP56 and AIP56-related proteins into HeLa cells. We found that the most efficient methods for intracellular toxin delivery were chemical transfection with toxin-encoding expression vectors and the use of the LF/PA system of Bacillus anthracis. The chemical transfection allows not only to obtain and study transiently transfected cells, but also to develop stable transfected cell lines.
|
|---|