Site-directed spin label EPR studies of the structure and membrane interactions of the bacterial phospholipase ExoU.

Site-directed spin labeling (SDSL) has been invaluable in the analysis of protein structure and dynamics, and has been particularly useful in the study of membrane proteins. ExoU, an important virulence factor in infections, is a bacterial phospholipase A2 that functions at the membrane - aqueous interface. Using SDSL methodology developed in the Hubbell lab, we find that the region surrounding the catalytic site of ExoU is buried within the tertiary structure of the protein in the soluble, apoenzyme state, but shows a significant increase in dynamics upon membrane binding and activation by ubiquitin.

cUMP elicits interendothelial gap formation during infection.

utilizes a type 3 secretion system to intoxicate host cells with the nucleotidyl cyclase ExoY. After activation by its host cell cofactor, filamentous actin, ExoY produces purine and pyrimidine cyclic nucleotides, including cAMP, cGMP, and cUMP. ExoY-generated cyclic nucleotides promote interendothelial gap formation, impair motility, and arrest cell growth.

Residues within the LptC transmembrane helix are critical for Escherichia coli LptB FG ATPase regulation.

Lipopolysaccharide (LPS) synthesis in Gram-negative bacteria is completed at the outer leaflet of the inner membrane (IM). Following synthesis, seven LPS transport (Lpt) proteins facilitate the movement of LPS to the outer membrane (OM), an essential process that if disrupted at any stage has lethal effects on bacterial viability. LptB FG, the IM component of the Lpt bridge system, is a type VI ABC transporter that provides the driving force for LPS extraction from the IM and subsequent transport across a stable protein bridge to the outer leaflet of the OM.

Binding and transport of LPS occurs through the coordinated combination of an array of sites across the entire Escherichia coli LPS transport protein LptA.

The outer leaflet of the outer membrane (OM) of bacteria such as Escherichia coli, Pseudomonas aeruginosa, and other important pathogens is largely composed of lipopolysaccharide (LPS), which is essential to nearly all Gram-negative bacteria. LPS is transported to the outer leaflet of the OM through a yet unknown mechanism by seven proteins that comprise the LPS transport system. LptA, the only entirely periplasmic Lpt protein, bridges the periplasmic space between the IM LptB FGC and the OM LptDE complexes.

Gamma secretase activating protein promotes end-organ dysfunction after bacterial pneumonia.

Pneumonia elicits the production of cytotoxic beta amyloid (Aβ) that contributes to end-organ dysfunction, yet the mechanism(s) linking infection to activation of the amyloidogenic pathway that produces cytotoxic Aβ is unknown. Here, we tested the hypothesis that gamma-secretase activating protein (GSAP), which contributes to the amyloidogenic pathway in the brain, promotes end-organ dysfunction following bacterial pneumonia. First-in-kind knockout rats were generated.

Identification of Virulence Factors Involved in a Murine Model of Severe Achromobacter xylosoxidans Infection.

Achromobacter xylosoxidans (Ax) is an opportunistic pathogen and causative agent of numerous infections particularly in immunocompromised individuals with increasing prevalence in cystic fibrosis (CF). To date, investigations have focused on the clinical epidemiology and genomic comparisons of Ax isolates, yet little is known about disease pathology or the role that specific virulence factors play in tissue invasion or damage. Here, we model an acute Ax lung infection in immunocompetent C57BL/6 mice and immunocompromised CF mice, revealing a link between cytotoxicity and disease in an intact host.

The Replication Cycle of Achromobacter xylosoxidans and Identification of Virulence Genes Associated with Cytotoxicity in Macrophages.

Achromobacter xylosoxidans is an opportunistic pathogen implicated in a wide variety of human infections including the ability to colonize the lungs of cystic fibrosis (CF) patients. The role of A. xylosoxidans in human pathology remains controversial due to the lack of optimized and model systems to identify and test bacterial gene products that promote a pathological response.

Perspectives on the Type III Secretion System Effector ExoU and Its Subversion of the Host Innate Immune Response to Infection.

is an opportunistic, Gram-negative pathogen and an important cause of hospital acquired infections, especially in immunocompromised patients. Highly virulent strains use a type III secretion system (T3SS) to inject exoenzyme effectors directly into the cytoplasm of a target host cell. strains that express the T3SS effector, ExoU, associate with adverse outcomes in critically ill patients with pneumonia, owing to the ability of ExoU to rapidly damage host cell membranes and subvert the innate immune response to infection.

Host phospholipid peroxidation fuels ExoU-dependent cell necrosis and supports Pseudomonas aeruginosa-driven pathology.

Regulated cell necrosis supports immune and anti-infectious strategies of the body; however, dysregulation of these processes drives pathological organ damage. Pseudomonas aeruginosa expresses a phospholipase, ExoU that triggers pathological host cell necrosis through a poorly characterized pathway. Here, we investigated the molecular and cellular mechanisms of ExoU-mediated necrosis.

Characterization of the ExoU activation mechanism using EPR and integrative modeling.

ExoU, a type III secreted phospholipase effector of Pseudomonas aeruginosa, serves as a prototype to model large, dynamic, membrane-associated proteins. ExoU is synergistically activated by interactions with membrane lipids and ubiquitin. To dissect the activation mechanism, structural homology was used to identify an unstructured loop of approximately 20 residues in the ExoU amino acid sequence.

Exoenzyme Y induces extracellular active caspase-7 accumulation independent from apoptosis: modulation of transmissible cytotoxicity.

Caspase-3 and -7 are executioner caspases whose enzymatic activity is necessary to complete apoptotic cell death. Here, we questioned whether endothelial cell infection leads to caspase-3/7-mediated cell death. Pulmonary microvascular endothelial cells (PMVECs) were infected with (PA103).

Virulent Pseudomonas aeruginosa infection converts antimicrobial amyloids into cytotoxic prions.

Pseudomonas aeruginosa infection elicits the production of cytotoxic amyloids from lung endothelium, yet molecular mechanisms of host-pathogen interaction that underlie the amyloid production are not well understood. We examined the importance of type III secretion system (T3SS) effectors in the production of cytotoxic amyloids. P aeruginosa possessing a functional T3SS and effectors induced the production and release of cytotoxic amyloids from lung endothelium, including beta amyloid, and tau.

Achromobacter xylosoxidans Cellular Pathology Is Correlated with Activation of a Type III Secretion System.

is increasingly recognized as a colonizer of cystic fibrosis (CF) patients, but the role that plays in pathology remains unknown. This knowledge gap is largely due to the lack of model systems available to study the toxic potential of this bacterium. Recently, a phospholipase A (PLA) encoded by a majority of genomes, termed AxoU, was identified.

Identification and Verification of Ubiquitin-Activated Bacterial Phospholipases.

ExoU is a potent type III secretion system effector that is injected directly into mammalian cells by the opportunistic pathogen As a ubiquitin-activated phospholipase A (PLA), ExoU exhibits cytotoxicity by cleaving membrane phospholipids, resulting in lysis of the host cells and inhibition of the innate immune response. Recently, ExoU has been established as a model protein for a group of ubiquitin-activated PLA enzymes encoded by a variety of bacteria. Bioinformatic analyses of homologous proteins is a powerful approach that can complement and enhance the overall understanding of protein structure and function.

Conformational Changes and Membrane Interaction of the Bacterial Phospholipase, ExoU: Characterization by Site-Directed Spin Labeling.

Numerous pathogenic bacteria produce proteins evolved to facilitate their survival and dissemination by modifying the host environment. These proteins, termed effectors, often play a significant role in determining the virulence of the infection. Consequently, bacterial effectors constitute an important class of targets for the development of novel antibiotics.

Identification of a ubiquitin-binding interface using Rosetta and DEER.

ExoU is a type III-secreted cytotoxin expressing A phospholipase activity when injected into eukaryotic target cells by the bacterium The enzymatic activity of ExoU is undetectable in vitro unless ubiquitin, a required cofactor, is added to the reaction. The role of ubiquitin in facilitating ExoU enzymatic activity is poorly understood but of significance for designing inhibitors to prevent tissue injury during infections with strains of producing this toxin. Most ubiquitin-binding proteins, including ExoU, demonstrate a low (micromolar) affinity for monoubiquitin (monoUb).

Structure and Dynamics of Type III Secretion Effector Protein ExoU As determined by SDSL-EPR Spectroscopy in Conjunction with De Novo Protein Folding.

ExoU is a 74 kDa cytotoxin that undergoes substantial conformational changes as part of its function, that is, it has multiple thermodynamically stable conformations that interchange depending on its environment. Such flexible proteins pose unique challenges to structural biology: (1) not only is it often difficult to determine structures by X-ray crystallography for all biologically relevant conformations because of the flat energy landscape (2) but also experimental conditions can easily perturb the biologically relevant conformation. The first challenge can be overcome by applying orthogonal structural biology techniques that are capable of observing alternative, biologically relevant conformations.

infection liberates transmissible, cytotoxic prion amyloids.

Patients who recover from pneumonia subsequently have elevated rates of death after hospital discharge as a result of secondary organ damage, the causes of which are unknown. We used the bacterium , a common cause of hospital-acquired pneumonia, as a model for investigating this phenomenon. We show that infection of pulmonary endothelial cells by induces production and release of a cytotoxic amyloid molecule with prion characteristics, including resistance to various nucleases and proteases.

The Pseudomonas aeruginosa Exoenzyme Y: A Promiscuous Nucleotidyl Cyclase Edema Factor and Virulence Determinant.

Exoenzyme Y (ExoY) was identified as a component of the Pseudomonas aeruginosa type 3 secretion system secretome in 1998. It is a common contributor to the arsenal of type 3 secretion system effectors, as it is present in approximately 90% of Pseudomonas isolates. ExoY has adenylyl cyclase activity that is dependent upon its association with a host cell cofactor.

Cooperative Substrate-Cofactor Interactions and Membrane Localization of the Bacterial Phospholipase A (PLA) Enzyme, ExoU.

The ExoU type III secretion enzyme is a potent phospholipase A secreted by the Gram-negative opportunistic pathogen, Activation of phospholipase activity is induced by protein-protein interactions with ubiquitin in the cytosol of a targeted eukaryotic cell, leading to destruction of host cell membranes. Previous work in our laboratory suggested that conformational changes within a C-terminal domain of the toxin might be involved in the activation mechanism. In this study, we use site-directed spin-labeling electron paramagnetic resonance spectroscopy to investigate conformational changes in a C-terminal four-helical bundle region of ExoU as it interacts with lipid substrates and ubiquitin, and to examine the localization of this domain with respect to the lipid bilayer.

Pseudomonas aeruginosa exoenzymes U and Y induce a transmissible endothelial proteinopathy.

We tested the hypothesis that Pseudomonas aeruginosa type 3 secretion system effectors exoenzymes Y and U (ExoY and ExoU) induce release of a high-molecular-weight endothelial tau, causing transmissible cell injury characteristic of an infectious proteinopathy. Both the bacterial delivery of ExoY and ExoU and the conditional expression of an activity-attenuated ExoU induced time-dependent pulmonary microvascular endothelial cell gap formation that was paralleled by the loss of intracellular tau and the concomitant appearance of high-molecular-weight extracellular tau. Transfer of the high-molecular-weight tau in filtered supernatant to naïve endothelial cells resulted in intracellular accumulation of tau clusters, which was accompanied by cell injury, interendothelial gap formation, decreased endothelial network stability in Matrigel, and increased lung permeability.

cCMP and cUMP occur in vivo.

Mammalian cells contain the cyclic pyrimidine nucleotides cCMP and cUMP. It is unknown whether these tentative new second messenger molecules occur in vivo. We used high performance liquid chromatography quadrupole tandem mass spectrometry to quantitate nucleoside 3',5'-cyclic monophosphates.

Ubiquitin activates patatin-like phospholipases from multiple bacterial species.

Phospholipase A2 enzymes are ubiquitously distributed throughout the prokaryotic and eukaryotic kingdoms and are utilized in a wide array of cellular processes and physiological and immunological responses. Several patatin-like phospholipase homologs of ExoU from Pseudomonas aeruginosa were selected on the premise that ubiquitin activation of this class of bacterial enzymes was a conserved process. We found that ubiquitin activated all phospholipases tested in both in vitro and in vivo assays via a conserved serine-aspartate catalytic dyad.

Reproducible and quantitative model of infection of Dermacentor variabilis with the live vaccine strain of Francisella tularensis.

Pathogen life cycles in mammalian hosts have been studied extensively, but studies with arthropod vectors represent considerable challenges. In part this is due to the difficulty of delivering a reproducible dose of bacteria to follow arthropod-associated replication. We have established reproducible techniques to introduce known numbers of Francisella tularensis strain LVS from mice into Dermacentor variabilis nymphs.