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

Transcriptome of Dickeya dadantii Infecting Acyrthosiphon pisum Reveals a Strong Defense against Antimicrobial Peptides

The plant pathogenic bacterium Dickeya dadantii has recently been shown to be able to kill the aphid Acyrthosiphon pisum. While the factors required to cause plant disease are now well characterized, those required for insect pathogeny remain mostly unknown. To identify these factors, we analyzed the transcriptome of the bacteria isolated from infected aphids. More than 150 genes were upregulated and 300 downregulated more than 5-fold at 3 days post infection. No homologue to known toxin genes could be identified in the upregulated genes. The upregulated genes reflect the response of the bacteria to the conditions encountered inside aphids. While only a few genes involved in the response to oxidative stress were induced, a strong defense against antimicrobial peptides (AMP) was induced. Expression of a great number of efflux proteins and transporters was increased. Besides the genes involved in LPS modification by addition of 4-aminoarabinose (the arnBCADTEF operon) and phosphoethanolamine (pmrC, eptB) usually induced in Gram negative bacteria in response to AMPs, dltBAC and pbpG genes, which confer Gram positive bacteria resistance to AMPs by adding alanine to teichoic acids, were also induced. Both types of modification confer D. dadantii resistance to the AMP polymyxin. A. pisum harbors symbiotic bacteria and it is thought that it has a very limited immune system to maintain these populations and do not synthesize AMPs. The arnB mutant was less pathogenic to A. pisum, which suggests that, in contrast to what has bee

Is the apocrine milk secretion process observed in the goat species rooted in the perturbation of the intracellular transport mechanism induced by defective alleles at the αs1\alpha_{{\rm s}1}-Cn locus?

The structural and quantitative variability of caprine $\alpha_{{\rm s}1}$-casein induced by the extensive polymorphism recorded at the corresponding locus strongly influences the composition (proteins as well as lipids) and the technological behaviour of milk. Immuno-histo-chemistry studies coupled with electron microscopy analysis have shown that a dysfunction exists in the intracellular transport of caseins when $\alpha_{{\rm s}1}$-casein is lacking. Casein accumulation in the endoplasmic reticulum leads to a dilation of the cisternae that could disturb the whole secretion process (including lipids). Despite a long controversy, goat milk secretion is still considered to occur through an apocrine process contrary to the merocrine process described for cow's milk. We suggest that the apocrine pathway of secretion described in the goat could be the consequence of the dysfunction observed in the intracellular transport of caseins when $\alpha_{{\rm s}1}$-casein is lacking. To obtain further clues in the favour of such a hypothesis, we compared the protein and lipid fractions of milks from goats homozygous for different $\alpha_{{\rm s}1}$-casein alleles.Le processus de sécrétion apocrine du lait décrit chez la chèvre ne trouverait-il pas son origine dans la perturbation du transport intracellulaire induit par les allèles défectifs détectés au locus $\alpha_{{\rm s}1}$-Cn ? La variabilité structurale et quantitative de la caséine $\alpha_{{\rm s}1}$ caprine consécutive au fort polymorphisme décrit au locus correspondant exerce une forte influence sur la composition (protéique mais aussi lipidique) et le comportement technologique du lait. Des études d'immuno-histo-chimie couplée à la microscopie électronique ont révélé l'existence d'un dysfonctionnement du transport intracellulaire des caséines en cas de déficience en caséine $\alpha_{{\rm s}1}$. L'accumulation de caséines dans le réticulum endoplasmique entraîne une dilatation des citernes qui pourrait perturber l'ensemble des mécanismes sécrétoires (lipides notamment). Malgré une longue controverse, on attribue aujourd'hui encore à la chèvre un processus de sécrétion du lait de type apocrine contrairement à la sécrétion de type mérocrine décrite chez la vache. Nous suggérons que la sécrétion apocrine décrite chez la chèvre pourrait découler du dysfonctionnement des mécanismes de transport intracellulaire des caséines observé en cas de déficience en caséine $\alpha_{{\rm s}1}$. Afin d'obtenir des indices supplémentaires en faveur d'une telle hypothèse les fractions protéiques et lipidiques de laits d'individus homozygotes pour différents allèles de caséine $\alpha_{{\rm s}1}$ ont été comparées

Le processus de sécrétion apocrine du lait décrit chez la chèvre ne trouverait-il pas son origine dans la perturbation du transport intracellulaire induit par les allèles défectifs détectés au locus alphaS1-Cn ?

"Chantier qualité spécifique "Auteurs Externes" département de Génétique animale : uniquement liaison auteur au référentiel HR-Access "International audienc

Lactic acid bacteria and proteomics: current knowledge and perspectives

International audienceLactic acid bacteria (LAB) are widely used in the agro-food industry. Some of the LAB also participate in the natural flora in humans and animals. We review here proteomic studies concerning LAB. Two methods of research can be distinguished. In the first one, a systematic mapping of proteins is attempted, which will be useful for taxonomy and to function assignment of proteins. The second one focuses particularly on proteins whose synthesis is induced by various environmental situations or stresses. However, both approaches are complementary and will give new insights for the use of bacteria in industry, in human health and in the struggle against bacterial pathogens. Interest in LAB is growing, showing thus an increasing concern of their rational use and one can foresee in the near future an increasing use of proteomics as well as genomics

Glycosyl hydrolases de Vitis vinifera : rôle des glucanases GH17 dans la défense inductible contre le mildiou

National audienc

Survival of various mutants to polymyxin.

<p>Wild type and various mutants in genes involved in resistance to AMP (<i>phoP</i>, <i>pmrA</i>, <i>dltB</i>, <i>arnB</i>) were incubated in the presence of 1 µg ml⁻¹ polymyxin for 1 h. Samples were diluted and plated on LB agar plates to assess bacterial viability. Survival values are relative to the original inoculum. Data correspond to mean values of three independent experiments.</p

A subset of <i>D. dadantii</i> genes whose expression varies in aphid.

a<p>Positive values represent genes upregulated in aphids, whereas negative values represent genes downregulated.</p

Survival of pea aphids after oral infection by wt and mutants of <i>D. dadantii</i> 3937.

<p>Survival is shown for aphids treated with wt bacteria (red), <i>arnB</i> (green), <i>dltB</i> (blue), and <i>dltB arnB</i> (brown) mutants. Results were obtained with 2×30 third instar aphid nymphs per treatment, including a diet-treated control (no mortality, not shown). The experiment was repeated twice with very similar results (p<0.06 in all comparisons of wt with <i>arnB</i> mutants). Median survival times (LT50s) were calculated with a Weibull fit (inlet), and give the following series [95% confidence intervals]: wt, 2.92 [2.18–3.92]; <i>arnB</i>, 4.74 [3.42–6.55]; <i>dltB</i> 3.58 [2.62–4.88] and <i>arnB</i>-<i>dltB</i> double mutant, 4.46 [3.23–6.18].</p