Pseudomonas aeruginosa mediated apoptosis requires the ADP-ribosylating activity of ExoS

Abstract

Pseudomonas aeruginosa is an opportunistic bacterial pathogen that primarily infects immunocompromised individuals and patients with cystic fibrosis. Using a tissue culture system, invasive strains of P. aeruginosa were discovered to induce apoptosis at high frequency in HeLa and other epithelial and fibroblast cell lines. This apoptotic phenotype in the infected cells was determined by several criteria including (i) visual changes in cell morphology, (ii) induction of chromatin condensation and nuclear marginalization, (iii) the presence of a high percentage of cells with subG1 DNA content, and (iv) activation of caspase-3 activity. Induction of the type III secretion machinery, but not invasion of P. aeruginosa is required for induction of apoptosis. The apoptosis phenotype is independent of the cytoskeletal rearrangements that occur in the host cell early after infection. Mutants in P. aeruginosa exoS fail to induce apoptosis and complementation with wild-type exoS restored the apoptosis-inducing capacity, demonstrating that ExoS is the effector molecule. Analysis of exoS activity mutants shows that the ADP-ribosylating capacity of ExoS is essential for inducing the apoptotic pathway.

Interactions between effector proteins of the Pseudomonas aeruginosa type III secretion system do not significantly affect several measures of disease severity in mammals

“…The amino-terminal GAP activity acts on Rho family GTPases while the carboxylterminal ADPRT activity is directed towards Ras and other host-cell proteins (Fraylick et al, 2001;Goehring et al, 1999;Henriksson et al, 2000Henriksson et al, , 2002Krall et al, 2002;Olson et al, 1999;Rocha et al, 2003;Vincent et al, 1999). As a result of these enzymic activities, intoxication with ExoS is associated with several observable phenotypes, including cytotoxicity and inhibition of bacterial internalization by both phagocytic and non-phagocytic mammalian cells (Cowell et al, 2000;Fleiszig et al, 1997;Frithz-Lindsten et al, 1997;Henriksson et al, 2000;Kaufman et al, 2000;Olson et al, 1997Olson et al, , 1999Pederson & Barbieri, 1998;Vincent et al, 1999). (Throughout this discussion, the term 'cytotoxicity' will be used to refer to cytolytic cell death.)…”

Regulation of Rab5 GTPase activity during Pseudomonas aeruginosa-macrophage interaction

“…Studies have suggested that T3S effectors from P. aeruginosa are responsible for inducing rapid apoptosis is macrophage cells and epithelial cells (Hauser and Engel 1999;Kaufman, Jia et al 2000). Prior results in this study indicated down regulation of …”

Factors triggering type III secretion in Pseudomonas aeruginosa

“…It is well known that ExoS preferentially ADP-ribosylates several Ras family members (GTP-binding proteins) required for the regulation of intracellular vesicle transport, cell proliferation and differentiation (Coburn & Gill, 1991;Ganesan et al, 1998). The ADP-ribosyltransferase activity of ExoS has also been shown to be essential in triggering programmed cell death in various types of tissue-culture cells (Kaufman et al, 2000;Jia et al, 2003).…”

“…It is well known that ExoS preferentially ADP-ribosylates several Ras family members (GTP-binding proteins) required for the regulation of intracellular vesicle transport, cell proliferation and differentiation (Coburn & Gill, 1991;Ganesan et al, 1998). The ADP-ribosyltransferase activity of ExoS has also been shown to be essential in triggering programmed cell death in various types of tissue-culture cells (Kaufman et al, 2000; Jia et al, 2003).The type III secretion complex of P. aeruginosa is highly similar to that of members of the genus Yersinia and, when expressed in cells of this genus, the ExoS protein can be translocated into mammalian cells via the yersinia TTSS (Frithz-Lindsten et al, 1997, 1998. Both organisms harbour multiple regulators to tightly control the expression of the large type III secretion gene clusters in response to lowcalcium environmental stimuli.…”

“…Similarly, plant pathogens utilize TTSS to cause disease in susceptible plants and to trigger a hypersensitive response in resistant plants (Hueck, 1998;Muller et al, 2001 (Yahr et al, 1995;Frank, 1997). Upon activation, the type III secretion apparatus translocates effector molecules into the cytoplasm of host cells, resulting in cell rounding and lifting and cell death by necrosis or apoptosis (Finck-Barbancon et al, 1997;Pederson et al, 1999;Kaufman et al, 2000). There are four known effector molecules, including ExoS and ExoT, two homologous toxins with both ADP-ribosyltransferase and GTPaseactivating protein activities, an acute cytotoxin, ExoU, with lipase activity, and an adenylate cyclase, ExoY (Yahr et al, 1996(Yahr et al, , 1998Finck-Barbancon et al, 1997;Hauser et al, 1998;Sato et al, 2005).…”