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 in vitro cytotoxicity and disease in an intact host. Mice were intratracheally challenged with sublethal doses of a cytotoxic (GN050) or invasive (GN008) strain of Ax. Bacterial burden, immune cell populations, and inflammatory markers in bronchoalveolar lavage fluid and lung homogenates were measured at different time points to assess disease severity. CF mice had a similar but delayed immune response toward both Ax strains compared to C57BL/6J mice. GN050 caused more severe disease and higher mortality which correlated with greater bacterial burden and increased proinflammatory responses in both mouse models. In agreement with the cytotoxicity of GN050 toward macrophages in vitro, mice challenged with GN050 had fewer macrophages. Mutants with transposon insertions in predicted virulence factors of GN050 showed that disease severity depended on the type III secretion system, Vi capsule, antisigma-E factor, and partially on the ArtA adhesin. The development of an acute infection model provides an essential tool to better understand the infectivity of diverse Ax isolates and enable improved identification of virulence factors important to bacterial persistence and disease.

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

Pneumonia elicits the production of cytotoxic beta amyloid (Aß) that contributes to end-organ dysfunction, yet the mechanism(s) linking infection to activation of the amyloidogenic pathway that produces cytotoxic Aß 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 Gsap knockout rats were generated. Wild-type and knockout rats possessed similar body weights, organ weights, circulating blood cell counts, arterial blood gases, and cardiac indices at baseline. Intratracheal Pseudomonas aeruginosa infection caused acute lung injury and a hyperdynamic circulatory state. Whereas infection led to arterial hypoxemia in wild-type rats, the alveolar-capillary barrier integrity was preserved in Gsap knockout rats. Infection potentiated myocardial infarction following ischemia-reperfusion injury, and this potentiation was abolished in knockout rats. In the hippocampus, GSAP contributed to both pre- and postsynaptic neurotransmission, increasing the presynaptic action potential recruitment, decreasing neurotransmitter release probability, decreasing the postsynaptic response, and preventing postsynaptic hyperexcitability, resulting in greater early long-term potentiation but reduced late long-term potentiation. Infection abolished early and late long-term potentiation in wild-type rats, whereas the late long-term potentiation was partially preserved in Gsap knockout rats. Furthermore, hippocampi from knockout rats, and both the wild-type and knockout rats following infection, exhibited a GSAP-dependent increase in neurotransmitter release probability and postsynaptic hyperexcitability. These results elucidate an unappreciated role for GSAP in innate immunity and highlight the contribution of GSAP to end-organ dysfunction during infection.NEW & NOTEWORTHY Pneumonia is a common cause of end-organ dysfunction, both during and in the aftermath of infection. In particular, pneumonia is a common cause of lung injury, increased risk of myocardial infarction, and neurocognitive dysfunction, although the mechanisms responsible for such increased risk are unknown. Here, we reveal that gamma-secretase activating protein, which contributes to the amyloidogenic pathway, is important for end-organ dysfunction following 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. We show that cellular peroxidised phospholipids enhance ExoU phospholipase activity, which drives necrosis of immune and non-immune cells. Conversely, both the endogenous lipid peroxidation regulator GPX4 and the pharmacological inhibition of lipid peroxidation delay ExoU-dependent cell necrosis and improve bacterial elimination in vitro and in vivo. Our findings also pertain to the ExoU-related phospholipase from the bacterial pathogen Burkholderia thailandensis, suggesting that exploitation of peroxidised phospholipids might be a conserved virulence mechanism among various microbial phospholipases. Overall, our results identify an original lipid peroxidation-based virulence mechanism as a strong contributor of microbial phospholipase-driven pathology.

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. T3SS effector intoxication was sufficient to generate cytotoxic amyloid release, yet intoxication with exoenzyme Y (ExoY) alone or together with exoenzymes S and T (ExoS/T/Y) generated the most virulent amyloids. Infection with lab and clinical strains engendered cytotoxic amyloids that were capable of being propagated in endothelial cell culture and passed to naïve cells, indicative of a prion strain. Conversely, T3SS-incompetent P aeruginosa infection produced non-cytotoxic amyloids with antimicrobial properties. These findings provide evidence that (1) endothelial intoxication with ExoY is sufficient to elicit self-propagating amyloid cytotoxins during infection, (2) pulmonary endothelium contributes to innate immunity by generating antimicrobial amyloids in response to bacterial infection, and (3) ExoY contributes to the virulence arsenal of P aeruginosa through the subversion of endothelial amyloid host-defense to promote a lung endothelial-derived cytotoxic proteinopathy.

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

ExoU is a type III-secreted cytotoxin expressing A2 phospholipase activity when injected into eukaryotic target cells by the bacterium Pseudomonas aeruginosa 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 P. aeruginosa producing this toxin. Most ubiquitin-binding proteins, including ExoU, demonstrate a low (micromolar) affinity for monoubiquitin (monoUb). Additionally, ExoU is a large and dynamic protein, limiting the applicability of traditional structural techniques such as NMR and X-ray crystallography to define this protein-protein interaction. Recent advancements in computational methods, however, have allowed high-resolution protein modeling using sparse data. In this study, we combine double electron-electron resonance (DEER) spectroscopy and Rosetta modeling to identify potential binding interfaces of ExoU and monoUb. The lowest-energy scoring model was tested using biochemical, biophysical, and biological techniques. To verify the binding interface, Rosetta was used to design a panel of mutations to modulate binding, including one variant with enhanced binding affinity. Our analyses show the utility of computational modeling when combined with sensitive biological assays and biophysical approaches that are exquisitely suited for large dynamic proteins.

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

Achromobacter xylosoxidans is increasingly recognized as a colonizer of cystic fibrosis (CF) patients, but the role that A. xylosoxidans 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 A2 (PLA2) encoded by a majority of A. xylosoxidans genomes, termed AxoU, was identified. Here, we show that AxoU is a type III secretion system (T3SS) substrate that induces cytotoxicity to mammalian cells. A tissue culture model was developed showing that a subset of A. xylosoxidans isolates from CF patients induce cytotoxicity in macrophages, suggestive of a pathogenic or inflammatory role in the CF lung. In a toxic strain, cytotoxicity is correlated with transcriptional activation of axoU and T3SS genes, demonstrating that this model can be used as a tool to identify and track expression of virulence determinants produced by this poorly understood bacterium.

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 Pseudomonas aeruginosa (PA103). PA103 caused cell swelling with a granular appearance, paralleled by intracellular caspase-3/7 activation and cell death. In contrast, PMVEC infection with ExoY+ (PA103 ΔexoUexoT::Tc pUCPexoY) caused cell rounding, but it did not activate intracellular caspase-3/7 and it did not cause cell death. However, ExoY+ led to a time-dependent accumulation of active caspase-7, but not caspase-3, in the supernatant, independent of apoptosis. To study the function of extracellular caspase-7, caspase-7- and caspase-3-deficient PMVECs were generated using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology. Caspase-7 activity was significantly reduced in supernatants from infected caspase-7-deficient cells but was unchanged in supernatants from infected caspase-3 deficient cells, indicating an uncoupling in the mechanism of activation of these two enzymes. Because ExoY+ leads to the release of heat stable amyloid cytotoxins that are responsible for transmissible cytotoxicity, we next questioned whether caspase-7 contributes to the severity of this process. Supernatants obtained from infected caspase-7-deficient cells displayed significantly reduced transmissible cytotoxicity when compared with supernatants from infected wild-type controls, illustrating an essential role for caspase-7 in promoting the potency of transmissible cytotoxicity. Thus, we report a mechanism whereby ExoY+ infection induces active caspase-7 accumulation in the extracellular space, independent of both caspase-3 and cell death, where it modulates ExoY+-induced transmissible cytotoxicity.

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 Pseudomonas aeruginosa As a ubiquitin-activated phospholipase A2 (PLA2), 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 PLA2 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. To conduct homology studies, it is important to have efficient and effective tools to screen and to validate the putative homologs of interest. Here we make use of an Escherichia coli-based dual expression system to screen putative ubiquitin-activated PLA2 enzymes from a variety of bacteria that are known to colonize humans and to cause human infections. The screen effectively identified multiple ubiquitin-activated phospholipases, which were validated using both biological and biochemical techniques. In this study, two new ExoU orthologs were identified and the ubiquitin activation of the rickettsial enzyme RP534 was verified. Conversely, ubiquitin was not found to regulate the activity of several other tested enzymes. Based on structural homology analyses, functional properties were predicted for AxoU, a unique member of the group expressed by Achromobacter xylosoxidansIMPORTANCE Bacterial phospholipases act as intracellular and extracellular enzymes promoting the destruction of phospholipid barriers and inflammation during infections. Identifying enzymes with a common mechanism of activation is an initial step in understanding structural and functional properties. These properties serve as critical information for the design of specific inhibitors to reduce enzymatic activity and ameliorate host cell death. In this study, we identify and verify cytotoxic PLA2 enzymes from several bacterial pathogens. Similar to the founding member of the group, ExoU, these enzymes share the property of ubiquitin-mediated activation. The identification and validation of potential toxins from multiple bacterial species provide additional proteins from which to derive structural insights that could lead to paninhibitors useful for treating a variety of infections.

Cooperative Substrate-Cofactor Interactions and Membrane Localization of the Bacterial Phospholipase A2 (PLA2) Enzyme, ExoU.

The ExoU type III secretion enzyme is a potent phospholipase A2 secreted by the Gram-negative opportunistic pathogen, Pseudomonas aeruginosa 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. In the absence of ubiquitin or substrate liposomes, the overall structure of the C-terminal domain is in good agreement with crystallographic models derived from ExoU in complex with its chaperone, SpcU. Significant conformational changes are observed throughout the domain in the presence of ubiquitin and liposomes combined that are not observed with either liposomes or ubiquitin alone. In the presence of ubiquitin, two interhelical loops of the C-terminal four-helix bundle appear to penetrate the membrane bilayer, stabilizing ExoU-membrane association. Thus, ubiquitin and the substrate lipid bilayer act synergistically to induce a conformational rearrangement in the C-terminal domain of ExoU.

Pseudomonas aeruginosa 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 Pseudomonas aeruginosa, a common cause of hospital-acquired pneumonia, as a model for investigating this phenomenon. We show that infection of pulmonary endothelial cells by P. aeruginosa induces production and release of a cytotoxic amyloid molecule with prion characteristics, including resistance to various nucleases and proteases. This cytotoxin was self-propagating, was neutralized by anti-amyloid Abs, and induced death of endothelial cells and neurons. Moreover, the cytotoxin induced edema in isolated lungs. Endothelial cells and isolated lungs were protected from cytotoxin-induced death by stimulation of signal transduction pathways that are linked to prion protein. Analysis of bronchoalveolar lavage fluid collected from human patients with P. aeruginosa pneumonia demonstrated cytotoxic activity, and lavage fluid contained amyloid molecules, including oligomeric τ and Aß. Demonstration of long-lived cytotoxic agents after Pseudomonas infection may establish a molecular link to the high rates of death as a result of end-organ damage in the months after recovery from pneumonia, and modulation of signal transduction pathways that have been linked to prion protein may provide a mechanism for intervention.-Balczon, R., Morrow, K. A., Zhou, C., Edmonds, B., Alexeyev, M., Pittet, J.-F., Wagener, B. M., Moser, S. A., Leavesley, S., Zha, X., Frank, D. W., Stevens, T. Pseudomonas aeruginosa infection liberates transmissible, cytotoxic prion amyloids.

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. However, recent evidence indicates that ExoY is not just an adenylyl cyclase; rather, it is a promiscuous cyclase capable of generating purine and pyrimidine cyclic nucleotide monophosphates. ExoY's enzymatic activity causes a characteristic rounding of mammalian cells, due to microtubule breakdown. In endothelium, this cell rounding disrupts cell-to-cell junctions, leading to loss of barrier integrity and an increase in tissue edema. Microtubule breakdown seems to depend upon tau phosphorylation, where the elevation of cyclic nucleotide monophosphates activates protein kinases A and G and causes phosphorylation of endothelial microtubule associated protein tau. Phosphorylation is a stimulus for tau release from microtubules, leading to microtubule instability. Phosphorylated tau accumulates inside endothelium as a high molecular weight, oligomeric form, and is then released from the cell. Extracellular high molecular weight tau causes a transmissible cytotoxicity that significantly hinders cellular repair following infection. Thus, ExoY may contribute to bacterial virulence in at least two ways; first, by microtubule breakdown leading to loss of endothelial cell barrier integrity, and second, by promoting release of a high molecular weight tau cytotoxin that impairs cellular recovery following infection.

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. Tau oligomer monoclonal antibodies captured monomeric tau from filtered supernatant but did not retrieve higher-molecular-weight endothelial tau and did not rescue the injurious effects of tau. Enrichment and transfer of high-molecular-weight tau to naïve cells was sufficient to cause injury. Thus we provide the first evidence for a pathophysiological stimulus that induces release and transmissibility of high-molecular-weight endothelial tau characteristic of an endothelial proteinopathy.

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. Ubiquitin chains versus monomeric ubiquitin were superior in inducing catalysis, and ubiquitin-like proteins failed to activate phospholipase activity. Toxicity studies in a prokaryotic dual-expression system grouped the enzymes into high- and low-toxicity classes. Toxicity measured in eukaryotic cells also suggested a two-tiered classification but was not predictive of the severity of cellular damage, suggesting that each enzyme may correspond to unique properties perhaps based on its specific biological function. Additional studies on lipid binding preference suggest that some enzymes in this family may be differentially sensitive to phosphatidyl-4,5-bisphosphate in terms of catalytic activation enhancement and binding affinity. Further analysis of the function and amino acid sequences of this enzyme family may lead to a useful approach to formulating a unifying model of how these phospholipases behave after delivery into the cytoplasmic compartment.

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. cCMP was detected in all organs studied, most notably pancreas, spleen and the female reproductive system. cUMP was not detected in organs, probably due to the intrinsically low sensitivity of mass spectrometry to detect this molecule and organ matrix effects. Intratracheal infection of mice with recombinant Pseudomonas aeruginosa harboring the nucleotidyl cyclase toxin ExoY massively increased cUMP in lung. The identity of cCMP and cUMP in organs was confirmed by high performance liquid chromatography quadrupole time of flight mass spectrometry. cUMP also appeared in serum, urine and faeces following infection. Taken together, this report unequivocally shows for the first time that cCMP and cUMP occur in vivo.

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. Using this model infection system, we performed dose-response infection experiments and followed bacterial replication through the molt to adults and at later time points. During development to adults, bacteria replicate to high numbers and can be found associated with the gut tissues, salivary glands, and hemolymph of adult ticks. Further, we can transmit a mutant of LVS (LVS ΔpurMCD) that cannot replicate in macrophages in vitro or in mice to nymphs. Our data show that the LVS ΔpurMCD mutant cannot be transstadially transmitted from nymphs to adult ticks. We then show that a plasmid-complemented strain of this mutant is recoverable in adult ticks and necessary for bacterial replication during the molt. In a mixed-infection assay (ΔpurMCD mutant versus ΔpurMCD complement), 98% of the recovered bacteria retained the plasmid marker. These data suggest that the ΔpurMCD mutation cannot be rescued by the presence a complemented strain in a mixed infection. Importantly, our infection model provides a platform to test specific mutants for their replication in ticks, perform competition studies, and use other genetic techniques to identify F. tularensis genes that are expressed or required in this unique environment.

Identification of the major ubiquitin-binding domain of the Pseudomonas aeruginosa ExoU A2 phospholipase.

Numerous Gram-negative bacterial pathogens use type III secretion systems to deliver effector molecules into the cytoplasm of a host cell. Many of these effectors have evolved to manipulate the host ubiquitin system to alter host cell physiology or the location, stability, or function of the effector itself. ExoU is a potent A2 phospholipase used by Pseudomonas aeruginosa to destroy membranes of infected cells. The enzyme is held in an inactive state inside of the bacterium due to the absence of a required eukaryotic activator, which was recently identified as ubiquitin. This study sought to identify the region of ExoU required to mediate this interaction and determine the properties of ubiquitin important for binding, ExoU activation, or both. Biochemical and biophysical approaches were used to map the ubiquitin-binding domain to a C-terminal four-helix bundle of ExoU. The hydrophobic patch of ubiquitin is required for full binding affinity and activation. Binding and activation were uncoupled by introducing an L8R substitution in ubiquitin. Purified L8R demonstrated a parental binding phenotype to ExoU but did not activate the phospholipase in vitro. Utilizing these new biochemical data and intermolecular distance measurements by double electron-electron resonance, we propose a model for an ExoU-monoubiquitin complex.

The Pseudomonas aeruginosa exoenzyme Y impairs endothelial cell proliferation and vascular repair following lung injury.

Exoenzyme Y (ExoY) is a Pseudomonas aeruginosa toxin that is introduced into host cells through the type 3 secretion system (T3SS). Once inside the host cell cytoplasm, ExoY generates cyclic nucleotides that cause tau phosphorylation and microtubule breakdown. Microtubule breakdown causes interendothelial cell gap formation and tissue edema. Although ExoY transiently induces interendothelial cell gap formation, it remains unclear whether ExoY prevents repair of the endothelial cell barrier. Here, we test the hypothesis that ExoY intoxication impairs recovery of the endothelial cell barrier following gap formation, decreasing migration, proliferation, and lung repair. Pulmonary microvascular endothelial cells (PMVECs) were infected with P. aeruginosa strains for 6 h, including one possessing an active ExoY (PA103 exoUexoT::Tc pUCPexoY; ExoY(+)), one with an inactive ExoY (PA103ΔexoUexoT::Tc pUCPexoY(K81M); ExoY(K81M)), and one that lacks PcrV required for a functional T3SS (ΔPcrV). ExoY(+) induced interendothelial cell gaps, whereas ExoY(K81M) and ΔPcrV did not promote gap formation. Following gap formation, bacteria were removed and endothelial cell repair was examined. PMVECs were unable to repair gaps even 3-5 days after infection. Serum-stimulated growth was greatly diminished following ExoY intoxication. Intratracheal inoculation of ExoY(+) and ExoY(K81M) caused severe pneumonia and acute lung injury. However, whereas the pulmonary endothelial cell barrier was functionally improved 1 wk following ExoY(K81M) infection, pulmonary endothelium was unable to restrict the hyperpermeability response to elevated hydrostatic pressure following ExoY(+) infection. In conclusion, ExoY is an edema factor that chronically impairs endothelial cell barrier integrity following lung injury.

ExoY from Pseudomonas aeruginosa is a nucleotidyl cyclase with preference for cGMP and cUMP formation.

In addition to the well known second messengers cAMP and cGMP, mammalian cells contain the cyclic pyrimidine nucleotides cCMP and cUMP. Soluble guanylyl cyclase and soluble adenylyl cyclase produce all four cNMPs. Several bacterial toxins exploit mammalian cyclic nucleotide signaling. The type III secretion protein ExoY from Pseudomonas aeruginosa induces severe lung damage and effectively produces cGMP. Here, we show that transfection of mammalian cells with ExoY or infection with ExoY-expressing P. aeruginosa not only massively increases cGMP but also cUMP levels. In contrast, the structurally related CyaA from Bordetella pertussis and edema factor from Bacillus anthracis exhibit a striking preference for cAMP increases. Thus, ExoY is a nucleotidyl cyclase with preference for cGMP and cUMP production. The differential effects of bacterial toxins on cNMP levels suggest that cUMP plays a distinct second messenger role.

Residues within the LptC transmembrane helix are critical for Escherichia coli LptB2 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. LptB2 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. LptC is a periplasmic protein anchored to the IM by a single transmembrane (TM) helix intercalating within the lateral gate formed by LptF TM5 and LptG TM1. LptC facilitates the hand-off of LPS from LptB2 FG to the periplasmic protein LptA and has been shown to regulate the ATPase activity of LptB2 FG. Here, using an engineered chromosomal knockout system in Escherichia coli to assess the effects of LptC mutations in vivo, we identified six partial loss of function LptC mutations in the first unbiased alanine screen of this essential protein. To investigate the functional effects of these mutations, nanoDSF (differential scanning fluorimetry) and site-directed spin labeling (SDSL) electron paramagnetic resonance (EPR) spectroscopy in combination with an in vitro ATPase assay show that specific residues in the TM helix of LptC destabilize the LptB2 FGC complex and regulate the ATPase activity of LptB.

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 LptB2 FGC and the OM LptDE complexes. LptA is postulated to protect the hydrophobic acyl chains of LPS as it crosses the hydrophilic periplasm, is essential to cell viability, and contains many conserved residues distributed across the protein. To identify which side chains are required for function of E. coli LptA in vivo, we performed a systematic, unbiased, high-throughput screen of the effect of 172 single alanine substitutions on cell viability utilizing an engineered BL21 derivative with a chromosomal knockout of the lptA gene. Remarkably, LptA is highly tolerant to amino acid substitution with alanine. Only four alanine mutants could not complement the chromosomal knockout; CD spectroscopy showed that these substitutions resulted in proteins with significantly altered secondary structure. In addition, 29 partial loss-of-function mutants were identified that led to OM permeability defects; interestingly, these sites were solely located within ß-strands of the central core of the protein and each resulted in misfolding of the protein. Therefore, no single residue within LptA is responsible for LPS binding, supporting previous EPR spectroscopy data indicating that sites across the entire protein work in concert to bind and transport LPS.