Rho GTPase activity modulates Pseudomonas aeruginosa internalization by epithelial cells

The Gram-negative pathogen Pseudomonas aeruginosa invades epithelial cells in vivo and in vitro. We have examined the pathway(s) by which epithelial c ells internalize P. aeruginosa strain PA103 using Madin-Darby canine kidney (MDCK) cells. We have recently demonstrated that P. aeruginosa internaliza tion occurs by an actin-dependent Toxin B-inhibited pathway which becomes d ownregulated as epithelial cells become polarized, suggesting that one or m ore of the Rho family GTPases is involved in bacterial internalization. Her e, we demonstrate that activation of the Rho family GTPases by cytotoxic ne crotizing factor 1 (CNF-1) stimulates P. aeruginosa internalization. Examin ation of the roles of the individual Rho family GTPases in internalization shows that expression of a constitutively active allele of RhoA (RhoAV14), but not of constitutively active Rad (Rac1V12) or Cdc42 (Cdc42V12), is suff icient to increase uptake of PA103pscJ, This relative increase persists whe n bacterial infection is established at the basolateral surface of polarize d cells, suggesting that the effect of RhoAV14 is not simply due to its kno wn ability to disrupt tight junction integrity in polarized cells. RhoAV14- mediated stimulation of bacterial uptake is actin dependent as it is abroga ted by exposure to latrunculin A. We also find that endogenous Rho GTP leve ls in epithelial cells are increased by infection with an internalized stra in of P, aeruginosa; conversely, a poorly internalized isogenic strain expr essing the bacterial anti-internalization protein ExoT causes decreased Rho GTP levels. Experimental inhibition of Rho, either by expressing dominant negative RhoAN19 or by inhibiting native Rho using a membrane permeable fus ion construct of a Rho-specific inhibitor, C3 ADP-ribosyltransferase, does not inhibit PA103pscJ internalization in MDCK or HeLa cells. Models consist ent with these data are presented.