Recombinant AexU effector protein of Aeromonas veronii bv. sobria disrupts the actin cytoskeleton by downregulation of Rac1 and induces direct cytotoxicity to β4-integrin expressing cell lines

Abstract

AexU is a type three secretion system (TTSS) effector of Aeromonas hydrophila which has an in vitro ADP-ribosyltransferase (ART) and GTPase-activating protein (GAP) activities on Rac1, RhoA and Cdc42. Here we show that, AexU of Aeromonas veronii bv. sobria AeG1 strain disrupts actin cytoskeleton of HeLa cells during AeG1 infection, aexU transfection or direct application of AexU protein. Such cellular disruption was rescued by either inactivation of AexU-GAP activity by substitution of arginine residue 143 to alanine or expression of a constitutively active (CA) Rac1 but not CA RhoA or CA Cdc42. On the other hand, AexU was found co-localized with β4-integrin probably through its Arg-Gly-Asp (RGD) integrin binding motif (319-321) residues. Interestingly, direct application of GST-AexU-HA fusion protein caused significant cytotoxic effect on β4-integrin expressing HT-29 cells. In contrast, β4-integrin blockade with a specific antibody reduced such cytotoxicity. Consequently, AexU cytotoxic effect was exaggerated with a greater expression of β4-integrin in Caco-2 and HeLa cells, while it was incompetent on β4-integrin non-expressing CHO cells. As far as we know, this is a novel TTSS effector which specifically inactivates Rac1 to disrupt actin cytoskeleton and has an alternative cytotoxic pathway through β4-integrin mediation.