Examining the role of actin-plasma membrane association in Pseudomonas aeruginosa infection and type III secretion translocation in migratory T24 epithelial cells.

The opportunistic pathogen Pseudomonas aeruginosa targets wounded epithelial barriers, but the cellular alteration that increases susceptibility to P. aeruginosa infection remains unclear. This study examined how cell migration contributes to the establishment of P. aeruginosa infections using (i) highly migratory T24 epithelial cells as a cell culture model, (ii) mutations in the type III secretion (T3S) effector ExoS to manipulate P. aeruginosa infection, and (iii) high-resolution immunofluorescent microscopy to monitor ExoS translocation. ExoS includes both GTPase-activating (GAP) and ADP-ribosyltransferase (ADPRT) activities, and P. aeruginosa cells expressing wild-type ExoS preferentially bound to the leading edge of T24 cells, where ExoS altered leading-edge architecture and actin anchoring in conjunction with interrupting T3S translocation. Inactivation of ExoS GAP activity allowed P. aeruginosa to be internalized and secrete ExoS within T24 cells, but as with wild-type ExoS, translocation was limited in association with disruption of actin anchoring. Inactivation of ExoS ADPRT activity resulted in significantly enhanced T3S translocation by P. aeruginosa cells that remained extracellular and in conjunction with maintenance of actin-plasma membrane association. Infection with P. aeruginosa expressing ExoS lacking both GAP and ADPRT activities resulted in the highest level of T3S translocation, and this occurred in conjunction with the entry and alignment of P. aeruginosa and ExoS along actin filaments. Collectively, in using ExoS mutants to modulate and visualize T3S translocation, we were able to (i) confirm effector secretion by internalized P. aeruginosa, (ii) differentiate the mechanisms underlying the effects of ExoS GAP and ADPRT activities on P. aeruginosa internalization and T3S translocation, (iii) confirm that ExoS ADPRT activity targeted a cellular substrate that interrupted T3S translocation, (iv) visualize the ability of P. aeruginosa and ExoS to align with actin filaments, and (v) demonstrate an association between actin anchoring at the leading edge of T24 cells and the establishment of P. aeruginosa infection. Our studies also highlight the contribution of ExoS to the opportunistic nature of P. aeruginosa infection through its ability to exert cytotoxic effects that interrupt T3S translocation and P. aeruginosa internalization, which in turn limit the P. aeruginosa infectious process.