The Pseudomonas aeruginosa Type III Secretion System Exoenzyme Effector ExoU Induces Mitochondrial Damage in a Murine Bone Marrow-Derived Macrophage Infection Model.

Pseudomonas aeruginosa is a Gram-negative, opportunistic pathogen that causes nosocomial pneumonia, urinary tract infections, and bacteremia. A hallmark of P. aeruginosa pathogenesis is disruption of host cell function by the type III secretion system (T3SS) and its cognate exoenzyme effectors. The T3SS effector ExoU is phospholipase A2 (PLA2) that targets the host cell plasmalemmal membrane to induce cytolysis and is an important virulence factor that mediates immune avoidance. In addition, ExoU has been shown to subvert the host inflammatory response in a noncytolytic manner. In primary bone marrow-derived macrophages (BMDMs), P. aeruginosa infection is sensed by the nucleotide-binding domain containing leucine-rich repeats-like receptor 4 (NLRC4) inflammasome, which triggers caspase-1 activation and inflammation. ExoU transiently inhibits NLRC4 inflammasome-mediated activation of caspase-1 and its downstream target, interleukin 1ß (IL-1ß), to suppress activation of inflammation. In the present study, we sought to identify additional noncytolytic virulence functions for ExoU and discovered an unexpected association between ExoU, host mitochondria, and NLRC4. We show that infection of BMDMs with P. aeruginosa strains expressing ExoU elicited mitochondrial oxidative stress. In addition, mitochondria and mitochondrion-associated membrane fractions enriched from infected cells exhibited evidence of autophagy activation, indicative of damage. The observation that ExoU elicited mitochondrial stress and damage suggested that ExoU may also associate with mitochondria during infection. Indeed, ExoU phospholipase A2 enzymatic activity was present in enriched mitochondria and mitochondrion-associated membrane fractions isolated from P. aeruginosa-infected BMDMs. Intriguingly, enriched mitochondria and mitochondrion-associated membrane fractions isolated from infected Nlrc4 homozygous knockout BMDMs displayed significantly lower levels of ExoU enzyme activity, suggesting that NLRC4 plays a role in the ExoU-mitochondrion association. These observations prompted us to assay enriched mitochondria and mitochondrion-associated membrane fractions for NLRC4, caspase-1, and IL-1ß. NLRC4 and pro-caspase-1 were detected in enriched mitochondria and mitochondrion-associated membrane fractions isolated from noninfected BMDMs, and active caspase-1 and active IL-1ß were detected in response to P. aeruginosa infection. Interestingly, ExoU inhibited mitochondrion-associated caspase-1 and IL-1ß activation. The implications of ExoU-mediated effects on mitochondria and the NLRC4 inflammasome during P. aeruginosa infection are discussed.

Analysis of pulmonary vascular injury and repair during Pseudomonas aeruginosa infection-induced pneumonia and acute respiratory distress syndrome.

Herein we describe lung vascular injury and repair using a rodent model of Pseudomonas aeruginosa pneumonia-induced acute respiratory distress syndrome (ARDS) during: 1) the exudative phase (48-hour survivors) and 2) the reparative/fibro-proliferative phase (1-week survivors). Pneumonia was induced by intratracheal instillation of P. aeruginosa strain PA103, and lung morphology and pulmonary vascular function were determined subsequently. Pulmonary vascular function was assessed in mechanically ventilated animals in vivo (air dead space, PaO2, and lung mechanics) and lung permeability was determined in isolated perfused lungs ex vivo (vascular filtration coefficient and extravascular lung water). At 48 hours post infection, histological analyses demonstrated capillary endothelial disruption, diffuse alveolar damage, perivascular cuffs, and neutrophil influx into lung parenchyma. Infected animals displayed clinical hallmarks of ARDS, including increased vascular permeability, increased dead space, impaired gas exchange, and decreased lung compliance. Overall, the animal infection model recapitulated the morphological and functional changes typically observed in lungs from patients during the exudative phase of ARDS. At 1 week post infection, there was lung histological and pulmonary vascular functional evidence of repair when compared with 48 hours post infection; however, some parameters were still impaired when compared with uninfected controls. Importantly, lungs displayed increased fibrosis and cellular hyperplasia reminiscent of lungs from patients during the fibro-proliferative phase of ARDS. Control, sham inoculated animals showed normal lung histology and function. These data represent the first comprehensive assessment of lung pathophysiology during the exudative and reparative/fibro-proliferative phases of P. aeruginosa pneumonia-induced ARDS, and position this pre-clinical model for use in interventional studies aimed at advancing clinical care.

The Highly Selective Caspase-1 Inhibitor VX-765 Provides Additive Protection Against Myocardial Infarction in Rat Hearts When Combined With a Platelet Inhibitor.

Use of ischemic postconditioning and other related cardioprotective interventions to treat patients with acute myocardial infarction (AMI) has failed to improve outcomes in clinical trials. Because P2Y12 inhibitors are themselves postconditioning mimetics, it has been postulated that the loading dose of platelet inhibitors routinely given to patients treated for AMI masks the anti-infarct effect of other intended cardioprotective interventions. To further improve outcomes of patients with AMI, an intervention must be able to provide additive protection in the presence of a P2Y12 platelet inhibitor. Previous studies reported an anti-infarct effect using a peptide inhibitor of the pro-inflammatory caspase-1 in animal models of AMI. Herein we tested whether a pharmacologic caspase-1 inhibitor can further limit infarct size in open-chest, anesthetized rats treated with a P2Y12 inhibitor. One hour occlusion of a coronary branch followed by 2 hours of reperfusion was used to simulate clinical AMI and reflow. One group of rats received an intravenous bolus of 16 mg/kg of the highly selective caspase-1 inhibitor VX-765 30 minutes prior to onset of ischemia. A second group received a 60 µg/kg intravenous bolus of the P2Y12 inhibitor cangrelor 10 minutes prior to reperfusion followed by 6 µg/kg/min continuous infusion. A third group received treatment with both inhibitors as above. Control animals received no treatment. Infarct size was measured by tetrazolium stain and volume of muscle at risk by fluorescent microspheres. In untreated hearts, 73.7% ± 4.1% of the ischemic zone infarcted. Treatment with either cangrelor or VX-765 alone reduced infarct size to 43.8% ± 2.4% and 39.6% ± 3.6% of the ischemic zone, respectively. Combining cangrelor and VX-765 was highly protective, resulting in only 14.0% ± 2.9% infarction. The ability of VX-765 to provide protection beyond that of a platelet inhibitor alone positions it as an attractive candidate therapy to further improve outcomes in today's patients with AMI.

Rickettsia prowazekii methionine aminopeptidase as a promising target for the development of antibacterial agents.

Methionine aminopeptidase (MetAP) is a class of ubiquitous enzymes essential for the survival of numerous bacterial species. These enzymes are responsible for the cleavage of N-terminal formyl-methionine initiators from nascent proteins to initiate post-translational modifications that are often essential to proper protein function. Thus, inhibition of MetAP activity has been implicated as a novel antibacterial target. We tested this idea in the present study by targeting the MetAP enzyme in the obligate intracellular pathogen Rickettsia prowazekii. We first identified potent RpMetAP inhibitory species by employing an in vitro enzymatic activity assay. The molecular docking program AutoDock was then utilized to compare published crystal structures of inhibited MetAP species to docked poses of RpMetAP. Based on these in silico and in vitro screens, a subset of 17 compounds was tested for inhibition of R. prowazekii growth in a pulmonary vascular endothelial cell (EC) culture infection model system. All compounds were tested over concentration ranges that were determined to be non-toxic to the ECs and 8 of the 17 compounds displayed substantial inhibition of R. prowazekii growth. These data highlight the therapeutic potential for inhibiting RpMetAP as a novel antimicrobial strategy and set the stage for future studies in pre-clinical animal models of infection.

In the absence of effector proteins, the Pseudomonas aeruginosa type three secretion system needle tip complex contributes to lung injury and systemic inflammatory responses.

Herein we describe a pathogenic role for the Pseudomonas aeruginosa type three secretion system (T3SS) needle tip complex protein, PcrV, in causing lung endothelial injury. We first established a model in which P. aeruginosa wild type strain PA103 caused pneumonia-induced sepsis and distal organ dysfunction. Interestingly, a PA103 derivative strain lacking its two known secreted effectors, ExoU and ExoT [denoted PA103 (ΔU/ΔT)], also caused sepsis and modest distal organ injury whereas an isogenic PA103 strain lacking the T3SS needle tip complex assembly protein [denoted PA103 (ΔPcrV)] did not. PA103 (ΔU/ΔT) infection caused neutrophil influx into the lung parenchyma, lung endothelial injury, and distal organ injury (reminiscent of sepsis). In contrast, PA103 (ΔPcrV) infection caused nominal neutrophil infiltration and lung endothelial injury, but no distal organ injury. We further examined pathogenic mechanisms of the T3SS needle tip complex using cultured rat pulmonary microvascular endothelial cells (PMVECs) and revealed a two-phase, temporal nature of infection. At 5-hours post-inoculation (early phase infection), PA103 (ΔU/ΔT) elicited PMVEC barrier disruption via perturbation of the actin cytoskeleton and did so in a cell death-independent manner. Conversely, PA103 (ΔPcrV) infection did not elicit early phase PMVEC barrier disruption. At 24-hours post-inoculation (late phase infection), PA103 (ΔU/ΔT) induced PMVEC damage and death that displayed an apoptotic component. Although PA103 (ΔPcrV) infection induced late phase PMVEC damage and death, it did so to an attenuated extent. The PA103 (ΔU/ΔT) and PA103 (ΔPcrV) mutants grew at similar rates and were able to adhere equally to PMVECs post-inoculation indicating that the observed differences in damage and barrier disruption are likely attributable to T3SS needle tip complex-mediated pathogenic differences post host cell attachment. Together, these infection data suggest that the T3SS needle tip complex and/or another undefined secreted effector(s) are important determinants of P. aeruginosa pneumonia-induced lung endothelial barrier disruption.

The Rickettsia prowazekii ExoU homologue possesses phospholipase A1 (PLA1), PLA2, and lyso-PLA2 activities and can function in the absence of any eukaryotic cofactors in vitro.

Here we have characterized the Rickettsia prowazekii RP534 protein, a homologue of the Pseudomonas aeruginosa ExoU phospholipase A (PLA) secreted cytotoxin. Our studies showed that purified recombinant RP534 PLA possessed the predicted PLA(2) and lyso-PLA(2) activities based on what has been published for P. aeruginosa ExoU. RP534 also displayed PLA(1) activity under the conditions tested, whereas ExoU did not. In addition, recombinant RP534 displayed a basal PLA activity that could hydrolyze phosphatidylcholine in the absence of any eukaryotic cofactors. Interestingly, the addition of bovine liver superoxide dismutase 1 (SOD1), a known activator of P. aeruginosa ExoU, resulted in an increased rate of RP534-catalyzed phospholipid hydrolysis, indicating that mechanisms of activation of the ExoU family of PLAs may be evolutionarily conserved. The mechanism of SOD1-dependent stimulation of RP534 was further examined using active site mutants and a fluorogenic phospholipid substrate whose hydrolysis by RP534 over a short time course is measureable only in the presence of SOD1. These studies suggest a mechanism by which SOD1 stimulates RP534 activity once it has bound to the substrate. We also show that antibody raised against RP534 was useful for immunoprecipitating active RP534 from R. prowazekii lysed cell extracts, thus verifying that this protein is expressed and active in rickettsiae isolated from embryonated hen egg yolk sacs.

Rickettsia prowazekii uses an sn-glycerol-3-phosphate dehydrogenase and a novel dihydroxyacetone phosphate transport system to supply triose phosphate for phospholipid biosynthesis.

Rickettsia prowazekii is an obligate intracellular pathogen that possesses a small genome and a highly refined repertoire of biochemical pathways compared to those of free-living bacteria. Here we describe a novel biochemical pathway that relies on rickettsial transport of host cytosolic dihydroxyacetone phosphate (DHAP) and its subsequent conversion to sn-glycerol-3-phosphate (G3P) for synthesis of phospholipids. This rickettsial pathway compensates for the evolutionary loss of rickettsial glycolysis/gluconeogenesis, the typical endogenous source of G3P. One of the components of this pathway is R. prowazekii open reading frame RP442, which is annotated GpsA, a G3P dehydrogenase (G3PDH). Purified recombinant rickettsial GpsA was shown to specifically catalyze the conversion of DHAP to G3P in vitro. The products of the GpsA assay were monitored spectrophotometrically, and the identity of the reaction product was verified by paper chromatography. In addition, heterologous expression of the R. prowazekii gpsA gene functioned to complement an Escherichia coli gpsA mutant. Furthermore, gpsA mRNA was detected in R. prowazekii purified from hen egg yolk sacs, and G3PDH activity was assayable in R. prowazekii lysed-cell extracts. Together, these data strongly suggested that R. prowazekii encodes and synthesizes a functional GpsA enzyme, yet R. prowazekii is unable to synthesize DHAP as a substrate for the GpsA enzymatic reaction. On the basis of the fact that intracellular organisms often avail themselves of resources in the host cell cytosol via the activity of novel carrier-mediated transport systems, we reasoned that R. prowazekii transports DHAP to supply substrate for GpsA. In support of this hypothesis, we show that purified R. prowazekii transported and incorporated DHAP into phospholipids, thus implicating a role for GpsA in vivo as part of a novel rickettsial G3P acquisition pathway for phospholipid biosynthesis.

Ligand-linked stability of mutants of the C-domain of calmodulin.

There is a necessary energetic linkage between ligand binding and stability in biological molecules. The critical glutamate in Site 4 was mutated to create two mutants of the C-domain of calmodulin yielding E140D and E140Q. These proteins were stably folded in the absence of calcium, but had dramatically impaired binding of calcium. We determined the stability of the mutant proteins in the absence and presence of calcium using urea-induced unfolding monitored by circular dichroism (CD) spectroscopy. These calcium-dependent unfolding curves were fit to models that allowed for linkage of stability to binding of a single calcium ion to the native and unfolded states. Simultaneous analysis of the unfolding profiles for each mutant yielded estimates for calcium-binding constants that were consistent with results from direct titrations monitored by fluorescence. Binding to the unfolded state was not an important energetic contributor to the ligand-linked stability of these mutants.

Thermodynamic stability of domain 2 of epithelial cadherin.

Cadherin is a cell adhesion molecule that participates in ordered calcium-dependent self-association interactions both between molecules on the same cell surface (cis-interactions) and on neighboring cell surfaces (trans-interactions). Cadherin is a transmembrane protein that has 3-7 independently folded beta-barrel extracellular domains. Both types of self-association interactions are mediated through the most N-terminal domain (Domain 1). Although the structural nature of the trans-interactions is clear, the nature of the cis-interactions is ambiguous despite several high-resolution structural studies. From earlier studies, it is understood that for the trans-interactions to happen, cis-interactions are mandatory. Hence, our first steps are to study the energetic driving forces for the cis-interactions. We have simplified the approach by first examining participating extracellular domains individually. We report here our initial experiments into the stability of Domain 2 of E-cadherin (ECAD2). ECAD2 appears monomeric, according to results from mass spectrometry and sedimentation equilibrium studies. We report denaturation data from differential scanning calorimetric experiments, and temperature and denaturant-induced unfolding experiments monitored by circular dichroism. These studies give a unified picture of the energetics of ECAD2-folding and stability, for which DeltaG degrees is 6.6 kcal/mol, T(m) is 54 degrees C, DeltaH(m) is 90 kcal/mol, and DeltaC(p) is 1300 cal/Kmol. These parameters are independent of calcium up to 5 mM, indicating that ECAD2 does not bind calcium at physiological calcium levels.