Pseudomonas aeruginosa injects NDK into host cells through a type III secretion system.

Pseudomonas aeruginosa is a Gram-negative opportunistic human pathogen possessing a type III secretion system (T3SS) which injects toxic effector proteins into mammalian host cells. In previous studies, P. aeruginosa strains lacking all of the known type III effectors were shown to cause cytotoxicity upon prolonged infection time. In this study, we report the identification of a new cytotoxin, nucleoside diphosphate kinase (NDK), which is injected into eukaryotic cells in a T3SS-dependent manner. Injection of NDK is inhibited by the presence of previously known effectors of the T3SS, with an effectorless strain injecting the highest amount, suggesting active competition with the known T3SS effectors. NDK is shown to cause a cytotoxic response when expressed in eukaryotic cells, and P. aeruginosa strains harbouring NDK also show a greater toxicity than strains lacking it. Interestingly, the cytotoxic effect of intracellular NDK is independent of its kinase activity. In previous studies, NDK was shown to be secreted into culture supernatants via a type I secretion system and cause cytotoxicity in a kinase-dependent manner. Therefore, the current study highlights an alternative route of NDK secretion as well as two different cytotoxic mechanisms of NDK, depending on the extra- or intra-cellular location of the protein.

JAZ mediates G1 cell cycle arrest by interacting with and inhibiting E2F1.

We discovered and reported JAZ as a unique dsRNA binding zinc finger protein that functions as a direct, positive regulator of p53 transcriptional activity to mediate G1 cell cycle arrest in a mechanism involving upregulation of the p53 target gene, p21. We now find that JAZ can also negatively regulate the cell cycle in a novel, p53-independent mechanism resulting from the direct interaction with E2F1, a key intermediate in regulating cell proliferation and tumor suppression. JAZ associates with E2F1's central DNA binding/dimerization region and its C-terminal transactivation domain. Functionally, JAZ represses E2F1 transcriptional activity in association with repression of cyclin A expression and inhibition of G1/S transition. This mechanism involves JAZ-mediated inhibition of E2F1's specific DNA binding activity. JAZ directly binds E2F1 in vitro in a dsRNA-independent manner, and JAZ's dsRNA binding ZF domains, which are necessary for localizing JAZ to the nucleus, are required for repression of transcriptional activity in vivo. Importantly for specificity, siRNA-mediated "knockdown" of endogenous JAZ increases E2F transcriptional activity and releases cells from G1 arrest, indicating a necessary role for JAZ in this transition. Although JAZ can directly inhibit E2F1 activity independently of p53, if functional p53 is expressed, JAZ may exert a more potent inhibition of cell cycle following growth factor withdrawal. Therefore, JAZ plays a dual role in cell cycle regulation by both repressing E2F1 transcriptional activity and activating p53 to facilitate efficient growth arrest in response to cellular stress, which may potentially be exploited therapeutically for tumor growth inhibition.

Expression of Pseudomonas aeruginosa toxin ExoS effectively induces apoptosis in host cells.

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 ADP-ribosylation (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 activity-dependent 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.

ExoS of Pseudomonas aeruginosa induces apoptosis through a Fas receptor/caspase 8-independent pathway in HeLa cells.

Pseudomonas aeruginosa infection is a serious complication in immunocompromised individuals and in patients with cystic fibrosis. We have previously shown that the type III secreted effector ExoS triggers apoptosis in various cultured cell lines via its ADP-ribosyltransferase (ADPRT) activity. The apoptosis process was further shown to involve intrinsic signalling pathway requiring c-Jun N-terminal kinase (JNK)-initiated mitochondrial pathway. In the present study, we investigated the role of Fas pathway activation in P. aeruginosa-induced apoptosis. P. aeruginosa infection resulted in caspase 8 cleavage in HeLa cells, which was inhibited by overexpression of a dominant negative version of Fas-associated death domain (FADD), suggesting that Fas pathway was activated. In fact, confocal laser scanning microscopy showed that P. aeruginosa induced clustering of FasR. In addition, the ADPRT activity of the ExoS was required for the induction of FasR clustering and caspase 8 cleavage. However, blocking the FasR-FasL interaction by antagonistic antibodies to FasR or to FasL had no effect on P. aeruginosa-induced caspase 8 and caspase 3 activation, neither did the silencing of FasR by small interfering RNA (siRNA), suggesting that caspase 8 activation through the FADD bypasses FasR/FasL-mediated signalling. Thus, FADD-mediated caspase 8 activation involves intracellular ExoS in an ADPRT-dependent manner. Furthermore, silencing of caspase 8 by siRNA did not interfere with P. aeruginosa-induced apoptosis, whereas it rendered HeLa cells markedly increased resistance towards FasL-induced apoptosis. In conclusion, our findings indicate that ExoS of P. aeruginosa induces apoptosis through a mechanism that is independent of Fas receptor/caspase 8 pathway.

Factors triggering type III secretion in Pseudomonas aeruginosa.

The type III secretion system of Pseudomonas aeruginosa is tightly regulated by various environmental signals, such as low calcium and contact with the host cell. However, the exact signals triggering type III secretion are unknown. The present study describes the finding that secretion of P. aeruginosa type III effector molecules requires protein factors from serum and L broth, designated type III secretion factors (TSFs), in addition to the low-calcium environment. In the absence of TSF or calcium chelator EGTA, basal levels of type III effector molecules are accumulated intracellularly. Addition of TSF and EGTA together effectively triggers the secretion of pre-existing effector molecules in a short time, even before the active expression of type III genes; thus, active type III gene expression does not seem to be a prerequisite for type III secretion. A search for TSF molecules in serum and L broth resulted in the identification of albumin and casein as the functional TSF molecules. Although there is no clear sequence similarity between albumin and casein, both proteins are known to have a low-affinity, high-capacity calcium-binding property. Tests of well-studied calcium-binding proteins seemed to indicate that low-affinity calcium-binding proteins have TSF activity, although the requirement of low-affinity calcium-binding ability for the TSF activity is not clear. P. aeruginosa seems to have evolved a sensing mechanism to detect target cells for type III injection through host-derived proteins in combination with a low-calcium signal. Disruption of the bacterial ability to sense low calcium or TSF might be a valid avenue to the effective control of this bacterial pathogen.

The truA gene of Pseudomonas aeruginosa is required for the expression of type III secretory genes.

Invasive strains of Pseudomonas aeruginosa can cause rapid host cell apoptosis by injecting the type III effector molecule ExoS. A transposon insertional mutant bank of P. aeruginosa was screened to identify P. aeruginosa genes that contribute to the ability of the bacteria to trigger host cell apoptosis. Several isolated mutants had disruptions in the fimV gene. A fimV mutant was unable to induce the expression of exoS, exoT and exsA genes under type III inducing conditions, thus exhibiting a defect in type III protein secretion. Furthermore, this mutant was defective in twitching motility, although type IV pili were present on the bacterial surface. Complementation by a fimV-containing cosmid clone restored both phenotypes to the wild-type levels. However, expression of the type III genes in the fimV mutant was not restored by the introduction of a fimV gene alone, although it restored the twitching motility. A gene downstream of fimV, encoding a tRNA pseudouridine synthase (truA) homologue, was able to complement the type III gene expression defect of the fimV mutant. Thus fimV and truA form an operon and fimV mutation has a polar effect on truA. Indeed, a truA mutant is defective in type III gene expression while its twitching motility is unaffected, and a truA clone is able to complement the type III secretion defect. Pseudouridination of tRNAs is important for tRNA structure, thereby improving the fidelity of protein synthesis and helping to maintain the proper reading frame; thus the results imply that truA controls tRNAs that are critical for the translation of type III genes or their regulators.

Pseudomonas aeruginosa mediated apoptosis requires the ADP-ribosylating activity of exoS.

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.

c-Jun NH2-terminal kinase-mediated signaling is essential for Pseudomonas aeruginosa ExoS-induced apoptosis.

As an opportunistic bacterial pathogen, Pseudomonas aeruginosa mainly affects immunocompromised individuals as well as patients with cystic fibrosis. In a previous study, we showed that ExoS of P. aeruginosa, when injected into host cells through a type III secretion apparatus, functions as an effector molecule to trigger apoptosis in various tissue culture cells. Here, we show that injection of the ExoS into HeLa cells activates c-Jun NH(2)-terminal kinase (JNK) phosphorylation while shutting down ERK1/2 and p38 phosphorylation. Inhibiting JNK activation by expression of a dominant negative JNK1 or with a specific JNK inhibitor abolishes ExoS-triggered apoptosis, demonstrating the requirement for JNK-mediated signaling. Following JNK phosphorylation, cytochrome c is released into the cytosol, leading to the activation of caspase 9 and eventually caspase 3. Although c-Jun phosphorylation is also observed as a result of JNK activation, ongoing host protein synthesis is not essential for the apoptotic induction, suggesting that c-Jun- or other AP-1-driven activation of gene expression is dispensable in this process. Therefore, ExoS has opposing effects on different cellular pathways that regulate apoptosis: it 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. These results highlight the modulation of host cell signaling by the type III secretion system during interaction between P. aeruginosa and host cells.

An in vivo inducible gene of Pseudomonas aeruginosa encodes an anti-ExsA to suppress the type III secretion system.

We have previously reported on the isolation of in vivo inducible genes of Pseudomonas aeruginosa using IVET system. One of such genes isolated from burn mouse infection model encodes a short open reading frame with unknown function. In this study, we demonstrate that this gene product specifically suppresses the expression of type III secretion genes in P. aeruginosa, thus named PtrA (Pseudomonas type III repressor A). A direct interaction between the PtrA and type III transcriptional activator ExsA was demonstrated, suggesting that its repressor function is probably realized through inhibition of the ExsA protein function. Indeed, an elevated expression of the exsA compensates the repressor effect of the PtrA. Interestingly, expression of the ptrA is highly and specifically induced by copper cation. A copper- responsive two-component regulatory system, copR-copS, has also been identified and shown to be essential for the copper resistance in P. aeruginosa as well as the activation of ptrA in response to the copper signal. Elevated expression of the ptrA during the infection of mouse burn wound suggests that P. aeruginosa has evolved tight regulatory systems to shut down energy-expensive type III secretion apparatus in response to specific environmental signals, such as copper stress.