Search

ABSTRACTInflammatory responses are a first line of host defense against a range of invading pathogens, consisting of the release of proinflammatory cytokines followed by attraction of polymorphonuclear neutrophils (PMNs) to the site of inflammation. Among the many virulence factors that contribute to the pathogenesis of infections, nucleoside diphosphate kinase (Ndk) mediates bacterially induced toxicity against eukaryotic cells. However, no study has examined how Ndk affects inflammatory responses. The present study examined the mechanisms by which Pseudomonas aeruginosaactivates inflammatory responses upon infection of cells. The results showed that bacterial Ndk, with the aid of an additional bacterial factor, flagellin, induced expression of the proinflammatory cytokines interleukin-1a (IL-1a) and IL-1ß. Cytokine induction appeared to be dependent on the kinase activity of Ndk and was mediated via the NF-?B signaling pathway. Notably, Ndk activated the Akt signaling pathway, which acts upstream of NF-?B, as well as caspase-1, which is a key component of inflammasome. Thus, this study demonstrated that P. aeruginosa, through the combined effects of Ndk and flagellin, upregulates the expression of proinflammatory cytokines via the Akt/NF-?B signaling pathways.

ABSTRACTPseudomonas aeruginosais an opportunistic pathogen that causes acute and chronic infections in humans. Pyocins are bacteriocins produced by P. aeruginosathat are usually released through lysis of the producer strains. Expression of pyocin genes is negatively regulated by PrtR, which gets cleaved under SOS response, leading to upregulation of pyocin synthetic genes. Previously, we demonstrated that PrtR is required for the expression of type III secretion system (T3SS), which is an important virulence component of P. aeruginosa. In this study, we demonstrate that mutation in prtRresults in reduced bacterial colonization in a mouse acute pneumonia model. Examination of bacterial and host cells in the bronchoalveolar lavage fluids from infected mice revealed that expression of PrtR is induced by reactive oxygen species (ROS) released by neutrophils. We further demonstrate that treatment with hydrogen peroxide or ciprofloxacin, known to induce the SOS response and pyocin production, resulted in an elevated PrtR mRNA level. Overexpression of PrtR by a tacpromoter repressed the endogenous prtRpromoter activity, and electrophoretic mobility shift assay revealed that PrtR binds to its own promoter, suggesting an autorepressive mechanism of regulation. A high level of PrtR expressed from a plasmid resulted in increased T3SS gene expression during infection and higher resistance against ciprofloxacin. Overall, our results suggest that the autorepression of PrtR contributes to the maintenance of a relatively stable level of PrtR, which is permissive to T3SS gene expression in the presence of ROS while increasing bacterial tolerance to stresses, such as ciprofloxacin, by limiting pyocin production.

ABSTRACTPseudomonas aeruginosais an opportunistic bacterial pathogen that primarily infects immunocompromised individuals and patients with cystic fibrosis. Invasive strains of P. aeruginosaare 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. aeruginosasecreting 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 crelease 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 melanogasterS2 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.

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.

ABSTRACTAs an opportunistic bacterial pathogen, Pseudomonas aeruginosamainly 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 NH2-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 cis 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 crelease and activation of caspases. These results highlight the modulation of host cell signaling by the type III secretion system during interaction between P. aeruginosaand host cells.

ABSTRACTDsbA is a periplasmic thiol:disulfide oxidoreductase which contributes to the process of protein folding by catalyzing the formation of disulfide bonds. In this study, we demonstrate that the dsbAgene is required for the expression of the type III secretion system under low-calcium inducing conditions, intracellular survival of P. aeruginosaupon infection of HeLa cells, and twitching motility. The diverse phenotypes of the dsbAmutant are likely due to its defect in the folding of proteins that are involved in various biological processes, such as signal sensing, protein secretion, and defense against host clearing. In light of its effect on various virulence factors, DsbA could be an important target for the control of P. aeruginosainfections.

ABSTRACTClinical isolates of Pseudomonas aeruginosaare classified into invasive and noninvasive (cytolytic) strains. In a noninvasive PA103 background, ExoS and ExoT have recently been shown to function as anti-internalization factors. However, these two factors seemed not to have such a function in an invasive strain PAK background. In this study, using HeLa tissue culture cells, we observed that the internalization of invasive strain PAK is dependent on its growth phases, with the stationary-phase cells internalized about 100-fold more efficiently than the exponential-phase cells. This growth phase-dependent internalization was not observed in the noninvasive PA103 strain. Further analysis of various mutant derivatives of the invasive PAK and the noninvasive PA103 strains demonstrated that ExoS or ExoT that is injected into host cells by a type III secretion machinery functions as an anti-internalization factor in both types of strains. In correlation with the growth phase-dependent internalization, the invasive strain PAK translocates much higher amount of ExoS and ExoT into HeLa cells when it is in an exponential-growth phase than when it is in a stationary-growth phase, whereas the translocation of ExoT by the noninvasive strain PA103 is consistently high regardless of the growth phases, suggesting a difference in the regulatory mechanism of type III secretion between the two types of strains. Consistent with the invasive phenotype of the parent strain, an internalized PAK derivative survived well within the HeLa cells, whereas the viability of internalized PA103 derivative was dramatically decreased and completely cleared within 48 h. These results indicate that the invasive strains of P. aeruginosahave evolved the mechanism of intracellular survival, whereas the noninvasive P. aeruginosastrains have lost or not acquired the ability to survive within the epithelial cells.

ABSTRACTBurn wounds are prone to infection by Pseudomonas aeruginosa, which is an opportunistic pathogen causing various human diseases. During infection, the bacterium senses environmental changes and regulates the expression of genes appropriate for survival. A purine-auxotrophic mutant of P. aeruginosawas unable to replicate efficiently on burn wounds, suggesting that burn wounds are purine-deficient environments. An in vivo expression technology based on purEKgene expression was applied to the burned mouse infection model to isolate P. aeruginosagenes that are specifically induced during infection. Four such in vivo-inducible (ivi) genetic loci were identified, including the gene for a superoxide response regulator (soxR), the gene for a malate synthase G homologue (glcG), an antisense transcript of a putative regulator responding to copper (copR), and an uncharacterized genetic locus. SoxR of Escherichia coliis known to regulate genes involved in protecting the bacterium against oxidative stress. The expression of soxRwas proven to be highly inducible during the infection of burned mice and also inducible by treatment with paraquat, which is a redox-cycling reagent generating intracellular superoxide. The SoxR protein functions as an autorepressor in the absence of paraquat, whereas in the presence of paraquat, this autorepression is diminished. Furthermore, asoxRnull mutant was shown to be much more sensitive than wild-type P. aeruginosato macrophage-mediated killing. In support of this observation, a soxRnull mutant exhibited a significant delay in causing systemic infections in the burned mice. Since most mortality in burn patients is caused by systemic infection, the defect in the ability to cause efficient bacteremia in burned mice suggests an important role of the soxRgene in the infection of burn wounds.

Pseudomonas aeruginosais an opportunistic pathogenic bacterium whose type III secretion system (T3SS) plays a critical role in acute infections. Translocation of the T3SS effectors into host cells induces cytotoxicity. In addition, the T3SS promotes the intracellular growth of P. aeruginosaduring host infections. The T3SS regulon genes are regulated by an AraC-type regulator, ExsA. In this study, we found that an extracellular metalloprotease encoded by impA(PA0572) is under the regulation of ExsA.

ABSTRACTAs one of the first lines of host defense, monocytes play important roles in clearing infected microbes. The defensive response is triggered by recognition of diverse microbial moieties, including released factors, which modulate host immune responses to establish a harsh environment for clinically important bacterial pathogens. In this study, we found that the expression of PTX3, a soluble form of pattern recognition receptor, was induced by infection with live Pseudomonas aeruginosaor treatment of cells with its supernatant. P. aeruginosaGroEL, a homolog of heat shock protein 60, was identified as one of the factors responsible for inducing the expression of PTX3in host cells. GroEL induced PTX3expression by activating the Toll-like receptor 4 (TLR4)-dependent pathway via nuclear factor-kappa B (NF-κB), while simultaneously inhibiting expression of microRNA-9, which targets the PTX3transcript. Finally, by acting as an opsonin, GroEL-induced PTX3 promoted the association and phagocytosis of Staphylococcus aureusinto macrophages. These data suggest that the host defensive environment is supported by the production of PTX3 in response to GroEL, which thus has therapeutic potential for clearance of bacterial infections.