Kiyoshi Moriyama

The mechanism of action of the Pseudomonas aeruginosa-encoded type III cytotoxin, ExoU.

Pseudomonas aeruginosa delivers the toxin ExoU to eukaryotic cells via a type III secretion system. Intoxication with ExoU is associated with lung injury, bacterial dissemination and sepsis in animal model and human infections. To search for ExoU targets in a genetically tractable system, we used controlled expression of the toxin in Saccharomyces cerevisiae. ExoU was cytotoxic for yeast and caused a vacuolar fragmentation phenotype. Inhibitors of human calcium‐independent (iPLA2) and cytosolic phospholipase A2 (cPLA2) lipase activity reduce the cytotoxicity of ExoU. The catalytic domains of patatin, iPLA2 and cPLA2 align or are similar to ExoU sequences. Site‐specific mutagenesis of predicted catalytic residues (ExoUS142A or ExoUD344A) eliminated toxicity. ExoU expression in yeast resulted in an accumulation of free palmitic acid, changes in the phospholipid profiles and reduction of radiolabeled neutral lipids. ExoUS142A and ExoUD344A expressed in yeast failed to release palmitic acid. Recombinant ExoU demonstrated lipase activity in vitro, but only in the presence of a yeast extract. From these data we conclude that ExoU is a lipase that requires activation or modification by eukaryotic factors.

TLR4 signaling is essential for survival in acute lung injury induced by virulent Pseudomonas aeruginosa secreting type III secretory toxins

BackgroundThe relative contributions of the cytotoxic phenotype of P. aeruginosa expressing type III secretory toxins and an immunocompromised condition lacking normal Toll-like receptor 4 (TLR4) signaling in the pathogenesis of acute lung injury and sepsis were evaluated in a mouse model for Pseudomonas aeruginosa pneumonia. By using lipopolysaccharide-resistant C3H/HeJ mice missing normal TLR4 signaling due to a mutation on the tlr4 gene, we evaluated how TLR4 signaling modulates the pneumonia caused by cytotoxic P. aeruginosa expressing type III secretory toxins.MethodsWe infected C3H/HeJ or C3H/FeJ mice with three different doses of either a cytotoxic P. aeruginosa strain (wild type PA103) or its non-cytotoxic isogenic mutant missing the type III secretory toxins (PA103ΔUT). Survival of the infected mice was evaluated, and the severity of acute lung injury quantified by measuring alveolar epithelial permeability as an index of acute epithelial injury and the water to dry weight ratios of lung homogenates as an index of lung edema. Bacteriological analysis and cytokine assays were performed in the infected mice.ResultsDevelopment of acute lung injury and sepsis was observed in all mouse strains when the cytotoxic P. aeruginosa strain but not the non-cytotoxic strain was instilled in the airspaces of the mice. Only C3H/HeJ mice had severe bacteremia and high mortality when a low dose of the cytotoxic P. aeruginosa strain was instilled in their lungs.ConclusionThe cytotoxic phenotype of P. aeruginosa is the critical factor causing acute lung injury and sepsis in infected hosts. When the P. aeruginosa is a cytotoxic strain, the TLR4 signaling system is essential to clear the batcteria to prevent lethal lung injury and bacteremia.

Effects of monoclonal anti-PcrV antibody on Pseudomonas aeruginosa-induced acute lung injury in a rat model

BackgroundThe effects of the murine monoclonal anti-PcrV antibody Mab166 on acute lung injury induced by Pseudomonas aeruginosa were analyzed in a rat model.MethodsLung injury was induced by the instillation of P. aeruginosa strain PA103 directly into the left lungs of anesthetized rats. One hour after the bacterial instillation, rabbit polyclonal anti-PcrV IgG, murine monoclonal anti-PcrV IgG Mab166 or Mab166 Fab-fragments were administered intratracheally directly into the lungs. The degree of alveolar epithelial injury, amount of lung edema, decrease in oxygenation and extent of lung inflammation by histology were evaluated as independent parameters of acute lung injury.ResultsThese parameters improved in rats that had received intratracheal instillation of either rabbit polyclonal anti-PcrV IgG, murine monoclonal anti-PcrV IgG Mab166 or Mab166 Fab-fragments in comparison with the control group.ConclusionMab166 and its Fab fragments have potential as adjuvant therapy for acute lung injury due to P. aeruginosa pneumonia.

Pseudomonas aeruginosa causes acute lung injury via the catalytic activity of the patatin-like phospholipase domain of ExoU

Objective:Acute lung injury in Pseudomonas aeruginosa pneumonia depends primarily on ExoU toxin being delivered directly into the eukaryotic cell cytosol through the type III secretion system. The amino-acid sequence of ExoU has a potato patatin-like phospholipase domain, similar to the sequence of mammalian Ca2+-independent phospholipase A2. We examined whether the acute lung injury caused by cytotoxic P. aeruginosa was dependent on the patatin-like phospholipase domain of ExoU. Design:Laboratory investigation using an established mouse model for P. aeruginosa pneumonia with quantitative measurements of acute lung injury and mortality. Setting:University experimental research laboratory. Subjects:Balb/c mice. Interventions:First, a site-directional mutation was introduced in the predicted catalytically active site of the patatin-like phospholipase domain of recombinant ExoU protein. The effect of the mutation on the catalytic activity of ExoU was tested by the in vitro lysophospholipase A assay. Second, the same site-directional mutation was introduced into the exoU gene of P. aeruginosa PA103. Mice were intratracheally infected with either a wild-type P. aeruginosa strain PA103 or an isogenic mutant containing the mutation in exoU. Acute epithelial lung injury, lung edema, bacteremia, and mortality were evaluated quantitatively. Measurements and Main Results:Recombinant ExoU had lysophospholipase A activity. Site-directional mutations in the predicted catalytic site of ExoU caused a loss of the lysophospholipase A activity. Whereas the airspace instillation of PA103 caused acute lung injury and death of the infected mice, the airspace instillation of isogenic mutants secreting catalytically inactive ExoU were noncytotoxic and did not cause acute lung injury or death of the infected mice. Conclusion:Virulent P. aeruginosa causes acute lung injury and death by the cytotoxic activity derived from the patatin-like phospholipase domain of ExoU.

Performance of noninvasive partial CO2 rebreathing cardiac output and continuous thermodilution cardiac output in patients undergoing aortic reconstruction surgery

Background In the partial CO2 rebreathing method, monitored changes in CO2 elimination and end-tidal CO2 in response to a brief rebreathing period are used to estimate cardiac output. However, dynamic changes in CO2 production during ischemia and reperfusion may affect the accuracy of these estimates. This study was designed to compare measurements of cardiac output as produced by the partial CO2 rebreathing (NICO), bolus (BCO), and continuous thermodilution (CCO) methods of monitoring cardiac output. Methods Cardiac output was continuously monitored using both NICO and CCO in 28 patients undergoing aortic reconstruction. BCO measurements were taken at the following intervals when hemodynamic stability was achieved: (1) after anesthetic induction; (2) during aortic cross-clamp; (3) at reperfusion of the iliac artery; and, (4) during peritoneal closure. Results The bias and precision (1 SD) derived from all the measurements between NICO and BCO was −0.58 ± 0.9 l/min, whereas for CCO and BCO it was 0.38 ± 1.17 l/min. The bias between NICO and BCO was small after anesthetic induction and during cross-clamp, but increased following reperfusion. The bias between CCO and BCO was relatively small until reperfusion but increased significantly at peritoneal closure. Conclusions Results indicate that in aortic reconstruction surgery the performance of NICO monitoring is comparable with that of CCO; however, the direction of bias in these continuous measurement devices is the opposite.

Association between Pseudomonas aeruginosa type III secretion, antibiotic resistance, and clinical outcome: a review.

Pseudomonas aeruginosa uses a complex type III secretion system to inject the toxins ExoS, ExoT, ExoU, and ExoY into the cytosol of target eukaryotic cells. This system is regulated by the exoenzyme S regulon and includes the transcriptional activator ExsA. Of the four toxins, ExoU is characterized as the major virulence factor responsible for alveolar epithelial injury in patients with P. aeruginosa pneumonia. Virulent strains of P. aeruginosa possess the exoU gene, whereas non-virulent strains lack this particular gene. The mechanism of virulence for the exoU+ genotype relies on the presence of a pathogenic gene cluster (PAPI-2) encoding exoU and its chaperone, spcU. The ExoU toxin has a patatin-like phospholipase domain in its N-terminal, exhibits phospholipase A2 activity, and requires a eukaryotic cell factor for activation. The C-terminal of ExoU has a ubiquitinylation mechanism of activation. This probably induces a structural change in enzymatic active sites required for phospholipase A2 activity. In P. aeruginosa clinical isolates, the exoU+ genotype correlates with a fluoroquinolone resistance phenotype. Additionally, poor clinical outcomes have been observed in patients with pneumonia caused by exoU+-fluoroquinolone-resistant isolates. Therefore, the potential exists to improve clinical outcomes in patients with P. aeruginosa pneumonia by identifying virulent and antimicrobial drug-resistant strains through exoU genotyping or ExoU protein phenotyping or both.

Neutrophil depletion attenuates interleukin-8 production in mild-overstretch ventilated normal rabbit lung

ObjectiveAcute lung injury induced by lung overstretch is associated with neutrophil influx, but the pathogenic role of neutrophils in overstretch-induced lung injury remains unclear. DesignTo assess the contribution of neutrophils, we compared the effects of noninjurious large tidal volume (Vt) ventilation on lungs in normal and neutrophil-depleted animals. SettingResearch animal laboratory. SubjectsTwenty-six male Japanese white rabbits. InterventionsAnimals were mechanically ventilated for 4 hrs with one of the three following protocols: large Vt (20 mL/kg), small Vt (8 mL/kg), and large Vt (20 mL/kg) with neutrophil depletion achieved by a single dose of vinblastine injection (0.75 mg/kg) intravenously 4 days before the experiment. Measurements and Main ResultsLarge Vt ventilation produced alveolar neutrophil influx compared with low Vt (p = .002) without evidence of edema or increased epithelial permeability. The neutrophil influx was accompanied by increases in interleukin-8 in bronchoalveolar lavage fluid (p = .04). Immunohistochemistry of large Vt lungs showed increased interleukin-8 staining in bronchial epithelial cells, alveolar epithelium, alveolar macrophages, and smooth muscles of pulmonary vessels. Neutrophil depletion attenuated the interleukin-8 increase in the lung. Large Vt did not increase plasma interleukin-8 or tumor necrosis factor-&agr; in plasma and bronchoalveolar lavage fluid. No expression of p-selectin or intercellular adhesion molecule-1 was observed. ConclusionsCyclic overstretching of normal rabbit lungs with noninjurious large Vt produced neutrophil influx and interleukin-8 increase in bronchoalveolar lavage fluid. Production of pulmonary interleukin-8 by lung overstretch might require the interaction between resident lung cells and migrated neutrophils. This study suggests that large Vt ventilation potentiates the predisposed, subclinical lung injury, such as nosocomial pneumonia or aspiration of gastric contents.

Protective effects of affinity-purified antibody and truncated vaccines against Pseudomonas aeruginosa V-antigen in neutropenic mice.

Virulent P. aeruginosa strains express PcrV, one of the translocational components of the type III secretion system. PcrV has been reported to be a protective antigen against lethal P. aeruginosa infection. The PcrV region, which contributes to protective immunity against P. aeruginosa infection, was investigated by using genetically engineered, truncated PcrV proteins and affinity‐purified anti‐PcrV antibodies against the truncated PcrV proteins. The efficacy of active and passive immunization against PcrV was tested in mice with cyclophosphamide‐induced immunosuppression by intraabdominal challenge of P. aeruginosa. Active immunization with either full‐length PcrV1‐294 or PcrV139‐294 significantly improved the survival of mice infected with P. aeruginosa, while PcrV139‐258, PcrV139‐234, PcrV197‐294, and PcrV261‐294 were not protective. These results suggest that an effective PcrV vaccine needs to contain not only the Mab166 epitope (PcrV144‐257) but also the carboxyl terminal tail of PcrV. In the case of passive immunization, administration of affinity‐purified anti‐PcrV IgG against either PcrV1‐294 or PcrV139‐258 showed significantly higher efficacy against lethal P. aeruginosa infection than did original anti‐PcrV IgG and Mab166. The increased efficacy of affinity‐purified anti‐PcrV IgG implies that more potent anti‐PcrV strategies are possible. The results of this study are crucial to the development of an effective PcrV vaccine for active immunization and to an appropriate blocking anti‐PcrV antibody against P. aeruginosa infection in humans.

Proteomic Analysis of Undiluted Lung Epithelial Lining Fluid

BACKGROUND Proteomics is increasingly leading to biomarker discovery in human disease. Epithelial lining fluid (ELF), until now only recovered indirectly, diluted in BAL fluid, is an attractive sample for lung disease proteomics. The direct recovery of undiluted ELF is now possible using a bronchoscopic microsampling (BMS) probe. In this preliminary study of anesthetized ventilated rabbits, we applied this probe to recover ELF and to analyze the resulting samples with the aim of determining their potential in lung disease biomarker discovery. METHODS In order to do so, a method was devised and evaluated in preliminary experiments both for nonbronchoscopic use of the probe and for recovering undiluted ELF from probe tips. To verify the proteomic potential of the sample, the recovered ELF was separated by one-dimensional polyacrylamide gel electrophoresis, and the resulting lane was cut into multiple fractions, each of which was digested and analyzed by liquid chromatography tandem mass spectrometry. The identified proteins were then searched against Medline for association with broad categories of lung disease. RESULTS Nonbronchoscopic use of the probe allowed successful ELF sampling and the recovery of undiluted ELF from probe tips. Proteomic analysis showed that ELF contains many proteins that have already been reported as being associated with lung disease as well as proteins potentially correlated with lung disease. CONCLUSIONS This preliminary study of undiluted ELF, as recovered by the BMS probe, shows that it may be an ideal sample for lung proteomics. The potential application of this sampling technique in various lung diseases will need to be confirmed by future studies.

V-antigen genotype and phenotype analyses of clinical isolates of Pseudomonas aeruginosa.

ABSTRACT The pcrV genotype was analyzed in clinical isolates of Pseudomonas aeruginosa which showed a negative phenotype for secretion of V-antigen PcrV. The suppression of PcrV secretion in these isolates was due not to a lack of the pcrV gene but rather to suppression of PcrV expression.