Phosphorylation of the β2 integrin subunit CD18 contributes to Actinobacillus pleuropneumoniae ApxI-induced cytotoxicity through the downregulation of Akt activity

Actinobacillus pleuropneumoniae, the causative agent of porcine pleuropneumonia, produces various virulence factors, including the exotoxin ApxI, which contributes to disease pathogenesis. ApxI induces cytotoxicity through porcine lymphocyte function-associated antigen-1 (LFA-1; CD11a/CD18) while concurrently attenuating Akt activity. This study aimed to investigate the regulatory role of porcine CD18, focusing on the tyrosine 735 residue in its cytoplasmic tail, and to examine the involvement of Akt in ApxI-induced cytotoxicity. Through transfection experiments, we demonstrated that the expression of porcine CD18 alone in ApxI-insensitive human embryonic kidney 293 cells conferred susceptibility to ApxI, while cotransfection of porcine CD11a with porcine CD18 resulted in the highest ApxI sensitivity, accompanied by reduced Akt activity. Substituting the wild-type CD18 with a CD18 harboring a tyrosine 735-to-proline (CD18^Y735P) mutation that mimics a nonphosphomimetic state led to increased Akt activity and reduced cytotoxicity in response to ApxI. Furthermore, ApxI-induced cytotoxicity was examined in cells cotransfected with porcine CD18/CD11a together with either p110* or ectopic Akt. The expression of either p110* or ectopic Akt significantly reduced ApxI-induced cytotoxicity. This study is the first to demonstrate that the phosphorylation of CD18^Y735 underlies ApxI-induced cytotoxicity through the downregulation of Akt activity.