Endogenous Morphine Levels Are Increased in Sepsis: A Partial Implication of Neutrophils

BACKGROUND: Mammalian cells synthesize morphine and the respective biosynthetic pathway has been elucidated. Human neutrophils release this alkaloid into the media after exposure to morphine precursors. However, the exact role of endogenous morphine in inflammatory processes remains unclear. We postulate that morphine is released during infection and can be determined in the serum of patients with severe infection such as sepsis. METHODOLOGY: The presence and subcellular immunolocalization of endogenous morphine was investigated by ELISA, mass spectrometry analysis and laser confocal microscopy. Neutrophils were activated with Interleukin-8 (IL-8) or lipopolysaccharide (LPS). Morphine secretion was determined by a morphine-specific ELISA. mu opioid receptor expression was assessed with flow cytometry. Serum morphine concentrations of septic patients were determined with a morphine-specific ELISA and morphine identity was confirmed in human neutrophils and serum of septic patients by mass spectrometry analysis. The effects of the concentration of morphine found in serum of septic patients on LPS-induced release of IL-8 by human neutrophils were tested. PRINCIPAL FINDINGS: We confirmed the presence of morphine in human neutrophil extracts and showed its colocalisation with lactoferrin within the secondary granules of neutrophils. Morphine secretion was quantified in the supernatant of activated human polymorphonuclear neutrophils in the presence and absence of Ca(2+). LPS and IL-8 were able to induce a significant release of morphine only in presence of Ca(2+). LPS treatment increased mu opioid receptor expression on neutrophils. Low concentration of morphine (8 nM) significantly inhibited the release of IL-8 from neutrophils when coincubated with LPS. This effect was reversed by naloxone. Patients with sepsis, severe sepsis and septic shock had significant higher circulating morphine levels compared to patients with systemic inflammatory response syndrome and healthy controls. Mass spectrometry analysis showed that endogenous morphine from serum of patient with sepsis was identical to poppy-derived morphine. CONCLUSIONS: Our results indicate that morphine concentrations are increased significantly in the serum of patients with systemic infection and that morphine is, at least in part, secreted from neutrophils during sepsis. Morphine concentrations equivalent to those found in the serum of septic patients significantly inhibited LPS-induced IL-8 secretion in neutrophils

Two Chromogranin A-Derived Peptides Induce Calcium Entry in Human Neutrophils by Calmodulin-Regulated Calcium Independent Phospholipase A2

Background: Antimicrobial peptides derived from the natural processing of chromogranin A (CgA) are co-secreted with catecholamines upon stimulation of chromaffin cells. Since PMNs play a central role in innate immunity, we examine responses by PMNs following stimulation by two antimicrobial CgA-derived peptides. Methodology/Principal Findings: PMNs were treated with different concentrations of CgA-derived peptides in presence of several drugs. Calcium mobilization was observed by using flow cytometry and calcium imaging experiments. Immunocytochemistry and confocal microscopy have shown the intracellular localization of the peptides. The calmodulin-binding and iPLA2 activating properties of the peptides were shown by Surface Plasmon Resonance and iPLA2 activity assays. Finally, a proteomic analysis of the material released after PMNs treatment with CgA-derived peptides was performed by using HPLC and Nano-LC MS-MS. By using flow cytometry we first observed that after 15 s, in presence of extracellular calcium, Chromofungin (CHR) or Catestatin (CAT) induce a concentration-dependent transient increase of intracellular calcium. In contrast, in absence of extra cellular calcium the peptides are unable to induce calcium depletion from the stores after 10 minutes exposure. Treatment with 2-APB (2-aminoethoxydiphenyl borate), a store operated channels (SOCs) blocker, inhibits completely the calcium entry, as shown by calcium imaging. We also showed that they activate iPLA2 as the two CaM-binding factors (W7 and CMZ) and that the two sequences can be aligned with the two CaMbinding domains reported for iPLA2. We finally analyzed by HPLC and Nano-LC MS-MS the material released by PMNs following stimulation by CHR and CAT. We characterized several factors important for inflammation and innate immunity. Conclusions/Significance: For the first time, we demonstrate that CHR and CAT, penetrate into PMNs, inducing extracellular calcium entry by a CaM-regulated iPLA2 pathway. Our study highlights the role of two CgA-derived peptides in the active communication between neuroendocrine and immune systems

Surface Sensing and Adaptation in Bacteria

Bacteria thrive both in liquids and attached to surfaces. The concentration of bacteria on surfaces is generally much higher than in the surrounding environment, offering bacteria ample opportunity for mutualistic, symbiotic, and pathogenic interactions. To efficiently populate surfaces, they have evolved mechanisms to sense mechanical or chemical cues upon contact with solid substrata. This is of particular importance for pathogens that interact with host tissue surfaces. In this review we discuss how bacteria are able to sense surfaces and how they use this information to adapt their physiology and behavior to this new environment. We first survey mechanosensing and chemosensing mechanisms and outline how specific macromolecular structures can inform bacteria about surfaces. We then discuss how mechanical cues are converted to biochemical signals to activate specific cellular processes in a defined chronological order and describe the role of two key second messengers, c-di-GMP and cAMP, in this process

Etude des mécanismes d'action précoces de la leucocidine de Panton et Valentine et développement d'inhibiteurs des leucotoxines de staphylococcus aureus

Les leucotoxines sont des facteurs de virulence majeurs sécrétés par la bactérie Staphylococcus aureus. Ces toxines formant des pores sont constituées de l association de 2 protéines (S + F) qui s oligomérisent en octamères sur les membranes des leucocytes, puis se reconfigurent en un pore transmembranaire sélectif des cations monovalents, et finalement conduit à la lyse de la cellule. Des substitutions en Alanine par mutagénèse dirigée ont permis de caractériser un cluster d acides aminés essentiels pour la fixation de LukS-PV, localisé sur 2 boucles du domaine Rim. Nous avons également apporté de nouveaux éléments sur les voies de signalisation impliquées dans l augmentation de la concentration intracellulaire en Ca2+ provoquée par les leucotoxines. Une pharmacologie des canaux calciques a montré une implication des récepteurs de l IP3 et des Store Operated Channels (SOCs). Les para-sulfonato-calix[n]arènes (SCn) neutralisent la lyse des neutrophiles avec des IC50 2 50 M. Les SCn empêchent la fixation de toutes les leucotoxines aux cellules ciblés, en formant des complexes 1:1 avec les protéines de classe S. Les SCn ont également montré leur efficacité dans un modèle d endophtalmie chez le lapin. Les anticorps à chaine lourde humanisés (HCAbs) anti-LPV ont également montré leur capacité à inhiber la fixation de LukS-PV et/ou LukF-PV aux membranes des cellules, empêchant ainsi leur activation et leur lyse. Le même modèle in vivo a montré que les HCAbs inhibent la réponse inflammatoire et la destruction des tissus.Leucotoxins are major virulence factors secreted by the bacteria Staphylococcus aureus. These pore forming toxins are formed by the association of 2 b-stranded proteins (class S and class F). Bi-partite leucotoxins oligomerize into hetero-octamers on leukocytes membranes, leading to cell activation and degranulation. Then they reconfigure into a monovalent cation selective transmembrane pore, and finally cause cell lysis.Alanine-scanning mutagenesis (19 mutants) allowed the characterization of a cluster of amino acids localized on two loops of the Rim domain essential for LukS-PV binding. We also aimed to characterize the pathways involved in intracellular Ca2+ concentration increase following leucotoxin addition onto neutrophils. Pharmacological targeting of calcium channels showed that IP3 receptors and Store Operated Channels (SOCs) were involved. These results illustrate that leucotoxins have complex direct and indirect impacts on immune system, that their mode of action is much more than simply forming pores.Para-sulfonato-calix[n]arenes (SCn) have IC50 values between 6 22 M for neutrophil lysis inhibition. They form 1:1 complex with class S proteins of all leucotoxins, which prevent further binding to cell membrane. The inhibitory properties of SCn were also observed in vivo in a toxin-induced rabbit endophthalmitis model. Anti-PVL Humanized Heavy Chain only Antibodies (HCAb) also prevent binding to cell membranes, and thus pore formation. The same in vivo model showed that HCAbs inhibit inflammatory reactions and tissue destruction.STRASBOURG-Sc. et Techniques (674822102) / SudocSudocFranceF

Pull-Down with a c-di-GMP-Specific Capture Compound Coupled to Mass Spectrometry as a Powerful Tool to Identify Novel Effector Proteins

Capture compound technology coupled to mass spectrometry (CCMS) allows to biochemically identify ligand receptors. Using a c-di-GMP-specific Capture Compound, we adapted this method for the identification and characterization of c-di-GMP binding proteins in any bacterial species. Because in silico analysis often fails to predict novel c-di-GMP effectors, this universal method aims at better defining the cellular c-di-GMP network in a wide range of bacteria. CCMS was successfully applied in several bacterial species (Nesper et al., J Proteom 75:4874-4878, 2012; Steiner et al., EMBO J 32:354-368, 2013; Tschowri et al., Cell 158:1136-1147, 2014; Trampari et al., J Biol Chem 290:24470-24483, 2015; Rotem et al., J Bacteriol 198:127-137, 2015). To outline the detailed protocol and to illustrate its power, we use Pseudomonas aeruginosa, an opportunistic pathogen in which c-di-GMP plays a critical role in virulence and biofilm control, as an example. CCMS identified 74% (38/51) of the known or predicted components of the c-di-GMP network

Catalytic carbene transfer allows the direct customization of cyclic purine dinucleotides

We describe a simple method for the direct modification of nucleobases in cyclic purine dinucleotides, important signalling molecules in both prokaryotes and eukaryotes. The method tolerates all members of the cyclic dinucleotide family and could be used to modulate their function or introduce useful side-chains such as fluorophores and photo-crosslinking groups

Capture compound mass spectrometry - a powerful tool to identify novel c-di-GMP effector proteins

Considerable progress has been made during the last decade towards the identification and characterization of enzymes involved in the synthesis (diguanylate cyclases) and degradation (phosphodiesterases) of the second messenger c-di-GMP. In contrast, little information is available regarding the molecular mechanisms and cellular components through which this signaling molecule regulates a diverse range of cellular processes. Most of the known effector proteins belong to the PilZ family or are degenerated diguanylate cyclases or phosphodiesterases that have given up on catalysis and have adopted effector function. Thus, to better define the cellular c-di-GMP network in a wide range of bacteria experimental methods are required to identify and validate novel effectors for which reliable in silico predictions fail. We have recently developed a novel Capture Compound Mass Spectrometry (CCMS) based technology as a powerful tool to biochemically identify and characterize c-di-GMP binding proteins. This technique has previously been reported to be applicable to a wide range of organisms(1). Here we give a detailed description of the protocol that we utilize to probe such signaling components. As an example, we use Pseudomonas aeruginosa, an opportunistic pathogen in which c-di-GMP plays a critical role in virulence and biofilm control. CCMS identified 74% (38/51) of the known or predicted components of the c-di-GMP network. This study explains the CCMS procedure in detail, and establishes it as a powerful and versatile tool to identify novel components involved in small molecule signaling

Dynamics of HsbD and FhlF cellular (co-)localization.

<p><b>(A)</b> Localization of HsbD-YFP (green) and FhlF-RFP (red) in a <i>P</i>. <i>aeruginosa</i> wild-type strain. Scale bar = 2 μm. Bottom panel: three representative patterns (1:1, 1:2 foci and 2:2 YFP:RFP foci) are illustrated in a closer view. Scale bar = 1 μm. Cell boundaries are delineated in white. <b>(B)</b> HsbD and FhlF polar localization frequency and their colocalization. More than 200 cells were analyzed from different fields. At least three independent experiments were performed. <b>(C)</b> Dynamics of HsbD-YFP (green) and FhlF-RFP (red). An overlay of the fluorescence channel(s) and the phase contrast image illustrate HsbD-YFP and FlhF-RFP subcellular localization pattern. Fluorescence images are shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006354#pgen.1006354.s006" target="_blank">S6 Fig</a>. Scale bar = 1 μm. Bottom panel shows the quantification of the HsbD-YFP (green) and FhlF-RFP (red) fluorescence intensity across the cell length. Cartoon: representation of coordinates reported in the graph.</p

Working model for the HptB signaling pathway in <i>P</i>. <i>aeruginosa</i>.

<p><b>(A)</b> HptB is activated (<i>i</i>.<i>e</i>. phosphorylated) via either one of three orphan sensor kinase hybrids: PA1611, ErcS’ (PA1976) or SagS (PA2824) [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006354#pgen.1006354.ref026" target="_blank">26</a>–<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006354#pgen.1006354.ref028" target="_blank">28</a>]. When phosphorylated, HptB transfers a phosphoryl group to the HsbR receiver domain (HsbR-P), thus repressing HsbR kinase activity while activating its phosphatase activity. HsbR-P dephosphorylates the anti-anti-sigma factor HsbA. Dephosphorylated HsbA leads to dissociation of the HsbA-HsbR complex and a subsequent stable sequestration of the anti-sigma factor FlgM by HsbA [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006354#pgen.1006354.ref020" target="_blank">20</a>]. The interaction HsbA-FlgM induces flagellar genes expression, by allowing the release of the FliA sigma factor (σ²⁸) [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006354#pgen.1006354.ref051" target="_blank">51</a>]. When HptB is inactive (dephosphorylated or in an <i>hptB</i> mutant), swimming and swarming are no longer supported. Instead, the HsbR kinase phosphorylates HsbA (HsbA-P) and HsbA-P interaction with HsbD leads to an increase of c-di-GMP and RsmY levels. The activation of HsbD strengthens swarming repression and results in hyper-biofilm and hyper-twitching phenotypes. This cascade of events is in agreement with the waves of regulatory actions that cause bacteria progression from an early surface attachment and colonization (swimming and swarming) to the development of mature sessile biofilms [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006354#pgen.1006354.ref015" target="_blank">15</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006354#pgen.1006354.ref026" target="_blank">26</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006354#pgen.1006354.ref056" target="_blank">56</a>]. The symbol ➔ indicates positive regulation, while -¦ repression and de–P (de)phosphorylation. Dashed lines suggest a probable indirect regulation. Components of the HptB signaling pathway are colored in green. <b>(B)</b> Localization dynamics of HsbD (green square) during cell division. FhlF (red square) dynamics is reported as commented previously by Burrows LL [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006354#pgen.1006354.ref049" target="_blank">49</a>]. Green gradient in the cell undergoing division illustrates bimodal distribution of c-di-GMP levels as reported by Christen and colleagues [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006354#pgen.1006354.ref014" target="_blank">14</a>].? Question mark represents possible scenarios on the asymmetric partitioning of HsbD during cell division in respect to flagellum biogenesis and DipA (blue square) localization. For details see text.</p