Pseudomonas aeruginosa ExoT Induces Atypical Anoikis Apoptosis in Target Host Cells by Transforming Crk Adaptor Protein into a Cytotoxin

Abstract

Previously, we demonstrated that Pseudomonas aeruginosa ExoT induces potent apoptosis in host epithelial cells in a manner that primarily depends on its ADP-ribosyltransferase domain (ADPRT) activity. However, the mechanism underlying ExoT/ADPRT-induced apoptosis remains undetermined. We now report that ExoT/ADPRT disrupts focal adhesion sites, activates p38β and JNK, and interferes with integrin-mediated survival signaling; causing atypical anoikis. We show that ExoT/ADPRT-induced anoikis is mediated by the Crk adaptor protein. We found that Crk-/- knockout cells are significantly more resistant to ExoT-induced apoptosis, while Crk-/- cells complemented with Crk are rendered sensitive to ExoT-induced apoptosis. Moreover, a dominant negative (DN) mutant form of Crk phenocopies ExoT-induced apoptosis both kinetically and mechanistically. Crk is generally believed to be a component of focal adhesion (FA) and its role in cellular survival remains controversial in that it has been found to be either pro-survival or pro-apoptosis. Our data demonstrate that although Crk is recruited to FA sites, its function is likely not required for FA assembly or for survival per se. However, when modified by ExoT or by mutagenesis, it can be transformed into a cytotoxin that induces anoikis by disrupting FA sites and interfering with integrin survival signaling. To our knowledge, this is the first example whereby a bacterial toxin exerts its cytotoxicity by subverting the function of an innocuous host cellular protein and turning it against the host cell.

Pseudomonas aeruginosa ExoT Induces Mitochondrial Apoptosis in Target Host Cells in a Manner That Depends on Its GTPase-activating Protein (GAP) Domain Activity

“…Under such scenarios, the GAP-induced apoptosis may be crucial in overcoming host survival mechanisms. Resistance to ADPRT-induced apoptosis could occur if the target host cell reduces the expression of Crk adaptor protein, which we have shown to be required for the ADPRT-induced apoptosis (13), or if the target host cell down-regulates the expression of FAS (a 14-3-3 cellular protein), which has been shown to be required for the ADPRT domain activity (19).…”

“…The ADPRT domain activity transforms Crk adaptor protein into a cytotoxin that induces atypical anoikis apoptosis by interfering with the integrin-survival signaling (13). However, the mechanism underlying the GAP-induced apoptosis remained unknown.…”

“…3A, infec- tion with GAP-expressing ⌬U/T(G ϩ A Ϫ ) resulted in increased expression of JNK1/2 and c-Jun and their activation (phospho-JNK1/2 and phospho-c-Jun) at 5 h after infection. To further evaluate the role of JNK in mediating GAP-induced apoptosis, we repeated the aforementioned infection studies in the presence or absence of JNK inhibitor SP600125 (added 2 h prior to infection) and evaluated GAP-induced cytotoxicity by timelapse videomicroscopy using PI uptake as a marker for cell death, as we described previously (13,22). JNK inhibition by SP600125 partially protected HeLa cells against the GAP-induced apoptosis (Fig.…”

“…Briefly, HeLa cells were grown in DMEM without phenol red with (for transfection studies) or without antibiotics (for infection studies) for 24 h. These cells were then transfected with the indicated expression vectors as described (11) or infected with the indicated strains as described (13). 1 h after transfection or at the time of infection, cells were given 7 g/ml propidium iodide (Sigma) and then placed on an Axiovert Z1 microscope (Zeiss) fitted with a live-imaging culture box (Pecon) maintaining 37°C in the presence of 5% CO 2 .…”

“…These data suggest that the GAP and the ADPRT domain activities target different apoptotic pathways to trigger cell death in infected host cells. Recently, we demonstrated that the ADPRT domain of ExoT, by ADP-ribosylating Crk adaptor protein, transforms this innocuous cellular protein into a cytotoxin that induces atypical anoikis apoptosis by disrupting focal adhesion sites and by interfering with the integrin survival signaling (13). However, the mechanism underlying the GAP-induced apoptosis remains unknown.…”