Expression ofPseudomonas aeruginosaToxin ExoS Effectively Induces Apoptosis in Host Cells

Abstract

Pseudomonas aeruginosa is an opportunistic bacterial pathogen that primarily infects immunocompromised individuals and patients with cystic fibrosis. Invasive strains of P. aeruginosa are known to induce apoptosis at a high frequency in HeLa cells and in many other cell lines, a process that is dependent on the ADPribosylation (ADPRT) activity of a type III secreted protein ExoS. In our previous report, it was proposed that P. aeruginosa secreting ExoS, upon infection, shuts down host cell survival signal pathways by inhibiting ERK1/2 and p38 activation, and it activates proapoptotic pathways through activation of JNK1/2, leading ultimately to cytochrome c release and activation of caspases. In this study, we demonstrate that the expression of ExoS in HeLa cells by eukaryotic expression vector effectively caused apoptosis in an ADPRT activitydependent manner, indicating that ExoS alone is sufficient to trigger apoptotic death of host cells independent of any other bacterial factors. By expressing an EGFP-ExoS fusion protein, we were able to directly correlate the death of HeLa cells with the presence of intracellular ExoS and further proved the dependence of this process on both JNK activation and mitochondrial proapoptotic event. The cellular pathway responsible for the ExoS-induced cytotoxicity appears to be well conserved, since the expression of the ADPRT-competent ExoS also induced rapid cell death in the Drosophila melanogaster S2 cell lines. The presented study not only highlights the ability of ExoS ADPRT to modulate host cell signaling, eventually leading to apoptosis, but also establishes ExoS as a valuable tool, in principle, for the elucidation of apoptosis mechanisms.

The ADP-Ribosylation Domain of Pseudomonas aeruginosa ExoS Is Required for Membrane Bleb Niche Formation and Bacterial Survival within Epithelial Cells

“…The data show that the ADP-r activity of ExoS is involved, which could relate to known cellular effects of ExoS in disrupting epithelial cytoskeleton function, endosome trafficking, signal transduction, and the induction of apoptosis, each of which involves ADP-ribosylation of various host cell targets (3,9,20,29,36,39). Future studies will advance our understanding of the relationship between T3SS P. aeruginosa invasion bleb niche formation and intracellular survival within epithelial cells, the role of ExoS (and its ADP-r activity) in promoting P. aeruginosa survival within, and traversal of, epithelial barriers (48), and the contributions of ExoS to virulence in vivo (32,47).…”

“…The uncoupling of Rab signaling may enable P. aeruginosa to evade trafficking to perinuclear vacuoles, which we previously showed display late endocytic markers when T3SS mutants traffic, while endowing P. aeruginosa with the capacity for intracellular replication. Finally, ExoS ADP-r activity has been shown to mediate P. aeruginosa-induced apoptosis in epithelial cells (29,30). Cells with bleb niches not only tend to be rounded, which is characteristic of apoptosis, but also tend to be labeled with annexin V-EGFP, an early apoptotic marker (51).…”

Regulatory Role of PopN and Its Interacting Partners in Type III Secretion of Pseudomonas aeruginosa

“…ExoS preferentially ADP ribosylates several Ras families of GTP-binding proteins required for the regulation of intracellular vesicle transport, cell proliferation, and differentiation (7,18). The ADP ribosyltransferase activity of ExoS causes programmed cell death in various types of host cells (26,27,29). ExoT preferentially ADP ribosylates Crk-I and Crk-II proteins in vivo (48) and inhibits host cell division by targeting cytokinesis (46).…”

Pseudomonas aeruginosa Induces Membrane Blebs in Epithelial Cells, Which Are Utilized as a Niche for Intracellular Replication and Motility

“…While in vitro research has focused mostly on deciphering the active domains of T3SS effectors and their host cell molecular targets (1,5,12,30,31,33,43,57,60), in vivo studies have concentrated largely on proving that effectors and their in vitro-defined active domains contribute to disease (42,53,61,71). Molecular and cellular relationships between in vitrodefined activities and the in vivo roles of the T3SS are yet to be established, in particular as they relate to the sequence of events enabling bacterial survival and to the numerous cell types and conditions under which bacteria interact with the infected host.…”